Understanding Helicases as a Means of Virus Control

Current Pharmaceutical Design, 2006, 12, 1315-1338 1315

1381-6128/06 $50.00+.00 © 2006 Bentham Science Publishers Ltd.

Understanding Helicases as a Means of Virus Control

D. N. Frick* and A. M. I. Lam

Department of Biochemistry & Molecular Biology, New York Medical College, Valhalla, NY 10595, USA

Abstract: Helicases are promising antiviral drug targets because their enzymatic activities are essential for viral genomereplication, transcription, and translation. Numerous potent inhibitors of helicases encoded by herpes simplex virus, se-vere acute respiratory syndrome coronavirus, hepatitis C virus, Japanese encephalitis virus, West Nile virus, and humanpapillomavirus have been recently reported in the scientific literature. Some inhibitors have also been shown to decreaseviral replication in cell culture and animal models. This review discusses recent progress in understanding the structureand function of viral helicases to help clarify how these potential antiviral compounds function and to facilitate the designof better inhibitors. The above helicases and all related viral proteins are classified here based on their evolutionary andfunctional similarities, and the key mechanistic features of each group are noted. All helicases share a common motorfunction fueled by ATP hydrolysis, but differ in exactly how the motor moves the protein and its cargo on a nucleic acidchain. The helicase inhibitors discussed here influence rates of helicase-catalyzed DNA (or RNA) unwinding by prevent-ing ATP hydrolysis, nucleic acid binding, nucleic acid release, or by disrupting the interaction of a helicase with a re-quired cofactor.

Key Words: ATPase, DExD/H-box proteins, motor proteins, enzyme mechanism.

INTRODUCTION

Viruses evolve rapidly to evade not only the human im-mune system but also antiviral drugs. Although it is some-times possible to use only a single antiviral drug to controlDNA viruses that proofread and repair their genomes, singleagents are rarely effective for viruses that evolve more rap-idly. The most successful method to control such a virus is tosimultaneously target multiple loci with several agents. Suchcombination therapies successfully control human immu-nodeficiency virus (HIV) replication to delay the develop-ment of AIDS, and are presently the standard of care for HIVpatients. Similar approaches could be used to combat otherviruses, but the technique is limited because most presentantiviral drugs function by inhibiting either viral proteases,which are needed to process viral proteins, or nucleic acidpolymerases, which are required to copy the viral genome.Although a few other antiviral drugs function by blockingviral entry or by modulating the host immune system, moredrug targets are clearly needed. This review focuses on thedevelopment of another critical protein synthesized by manyviruses, an enzyme called helicase, as a potential antiviraldrug target.

Helicases are motor proteins that use energy derivedfrom ATP hydrolysis to separate nucleic acid strands. Suchan activity is necessary at several points during genome rep-lication. In viruses with duplex nucleic acid genomes, thedouble helix must be separated for copying. Conversely,viruses with single-stranded genomes must separate duplexesthat form after genome replication. Helicases have also beenshown to be required in transcription of viral mRNAs,

*Address correspondence to this author at the Department of Biochemistryand Molecular Biology, New York Medical College, Valhalla, NY 10595,USA; Tel: 914-594-4190; Fax: 914-594-4058;E-mail: [email protected]

translation, disruption of RNA-protein complexes, and pack-aging of nucleic acids into virions. Most importantly, theirvalidity as antiviral drug targets was recently confirmedwhen compounds that inhibit a helicase encoded by herpessimplex virus (HSV) were shown to block viral replicationand disease progression in animal models [1, 2].

Most of the best-studied viral helicases are encoded bylarge DNA viruses or bacteriophage. In fact, some of the firsthelicases identified over two decades ago were purified fromthese organisms [3-5]. Similar proteins are encoded by sev-eral important human pathogens like HSV and human pa-pillomavirus (HPV). About 10 years after the discovery ofDNA helicases, similar proteins were found to be made byRNA viruses [6]. Most species of positive sense single-stranded RNA ((+)ssRNA) viruses encode a helicase, andseveral of these proteins have now been extensively studied.The most detailed structural and mechanistic studies havebeen performed using the RNA helicase encoded by hepatitisC virus (HCV). A few other helicases from pathogenic hu-man RNA viruses have also been studied in detail includingthose from severe acute respiratory syndrome coronavirus(SARS-CoV), Dengue fever virus (DFV), Japanese en-cephalitis virus (JEV), and West Nile virus (WNV). Curi-ously, no retroviruses or negative sense single-stranded RNA((-)ssRNA) viruses have been reported to encode the synthe-sis of a helicase. Since the members of these classes replicatein the nucleus, they might simply utilize helicases encodedby the host instead of their own proteins. Indeed, HIV repli-cation has recently been shown to require the human DDX3DEAD-box RNA helicase [7]. Large DNA viruses that in-vade the nucleus, like HSV, frequently bring with them a fullcomplement of replication proteins, including at least onehelicase. Viruses that replicate outside the nucleus almostalways encode a helicase, without which they cannot sur-vive.

1316 Current Pharmaceutical Design, 2006, Vol. 12, No. 11 Frick and Lam

Developing non-toxic helicase inhibitors is considerablymore difficult than developing drugs designed to inhibitother viral enzymes. Problems arise from several areas. First,compared with proteases and polymerases, the mechanismsof the reactions catalyzed by helicases are still not as clearlyunderstood. Second, the helicase ATP-binding site is con-served not only in all classes of helicases, but also in motorproteins, small GTPases, kinases, the AAA+ family (AT-Pases associated with various cellular activities), and eventhe mitochondrial ATP synthase (F1 ATPase) (Fig. (1)).Thus, compounds that inhibit helicases via their ATP-binding sites could be quite toxic. Third, the role of helicasesin the viral lifecycle is still not well-defined. Although, invitro all helicases are able to separate a nucleic acid strandfrom its complement, their movements could also rearrangesecondary structures or dislodge nucleic acid binding pro-teins. Finally, the traditional assays measuring helicase-catalyzed unwinding are tedious, making inhibitor screeningtime-consuming. In the past few years, considerable progresshas been made in the area of assay development (see refer-ence [8] for a review) and it is now possible to identify po-tent helicase inhibitors using high throughput screening. Thechallenge now is to understand how these compounds inter-act with helicases so that they can be developed into actualdrugs.

This review will summarize the various viral helicasesthat have been characterized to date, their evolutionary rela-tionships, mechanisms of action, and any inhibitors that havebeen reported in the scientific literature. The helicase litera-ture is rapidly expanding but fortunately the field is fre-quently reviewed, and the reader will be directed to relevantreviews in appropriate sections. It should also be noted atthis point that Delagoutte & von Hippel have recently re-viewed the entire helicase field in an extensive two-part re-view that is highly recommended [9, 10].

VIRAL HELICASE CLASSIFICATION

The best way to understand the bewildering amount ofviral helicase information is to realize that all helicases, fromboth viruses and cellular organisms, share many commonproperties. Understanding these features will provide thebasis for understanding the mechanism of action of thesecomplex enzymes. It should also be recognized that naturehas used the basic building blocks shared by all helicases tomanipulate nucleic acids in many different ways and formany different purposes. As a result, different helicase fami-lies have evolved that share little resemblance, at least super-ficially. Thus, to really understand viral helicases, one mustunderstand the common properties shared by all helicases,and the distinctive properties that characterize the various

Fig. (1). Evolutionary relationship of viral helicases. All known viral helicases belong either to one of three helicase superfamilies or to theRecA/F1-ATPase superfamily. The five families of viral helicases from DNA viruses and three families of viral helicases from RNA virusesare highlighted with grey bars. Prototypes of each family are listed in parentheses. SF3 and DnaB-like helicases are fundamentally differentfrom SF1 and SF2 helicases in that they contain only one RecA-like domain per subunit and must form rings and/or filaments to catalyzeATP hydrolysis. All helicases are in the ASCE subdivision of P-loop NTPases and share many basic features with the numerous other pro-tein families listed. The diagram is based on information found in references [234, 235].

Understanding Helicases as a Means of Virus Control Current Pharmaceutical Design, 2006, Vol. 12, No. 11 1317

helicase families. If a new viral pathogen is discovered, itsgenome sequence can be used to predict not only if the virusencodes a helicase, but also exactly which helicase family inwhich the putative helicase belongs. If the properties of thatparticular family are understood, it is likely that the helicaseof interest shares many of the same features.

The evolutionary relationship of all known viral helicasesis outlined in Fig. (1). Based only on protein sequence analy-sis, Koonin and his colleagues have shown that all helicasescan be placed in one of several genetic families [11]. All buttwo of the helicase families can be grouped into one of threelarger “superfamilies, ” designated as superfamily 1 (SF1),superfamily 2 (SF2) [12], and superfamily 3 (SF3) [13]. Theremaining 2 families are more similar to the RecA proteinand the F1 ATP synthase than helicases in the three helicasesuperfamilies. One family is similar to the DnaB helicase ofE. coli [14] and the other is similar to the E. coli Rho heli-case that is used in transcriptional termination [15]. Of thesetwo “odd” families, only the DnaB-like family contains viral(i.e. bacteriophage) proteins. The DnaB-like helicase familyis sometimes called family 4 (F4) [16], some authors refer toit as superfamily 4 (SF4), and others mistakenly combine theDnaB-like helicases with those in SF3. Sequence analysisreveals the DnaB-like family and the SF3 families are evo-lutionarily distinct [14], and as we will see below, the SF3and DnaB-like helicases appear to function somewhat differ-ently. The various helicase families are distinguished by thepresence of specific conserved signature sequences, some ofwhich are conserved in all helicase families, some of whichare slightly different between families, and some of whichare unique (see reference [16] for a review of these helicasemotifs).

Helicases are also classified based on their quaternarystructures and their direction of movement. Some helicasesoligomerize to form rings (usually hexamers) that appear inelectron micrographs to encircle one nucleic acid strand (seereference [17] for a review). Other helicases do not formsuch rings. All helicases can also be classified according totheir movement relative to the nucleic acid strand to whichthey are primarily associated. In most present models forhelicase action, the helicase is depicted as moving along onestrand of a double helix while displacing the other strand.Since the strands in a double helix are oriented in an anti-parallel configuration, such movement could either occur ina 5'-3' direction or in a 3'-5' direction. The direction of heli-case movement can be easily diagnosed by analyzing theability of a helicase to unwind duplex substrates possessingssDNA (or ssRNA) tails. 5'-3' helicases only unwind sub-strates with a 5'-tail, while 3'-5' helicases need a 3'-tail toinitiate unwinding. Thus, a helicase can be classified basedon each of the three above schemes. For example the heli-case encoded by HCV is a SF2, non-ring, 3'-5' RNA heli-case. HPV helicase is a SF3, ring, 3'-5' DNA helicase.

KNOWN VIRAL HELICASES AND THEIR PROPER-TIES

Viruses encode helicases that are members of SF1, SF2,SF3, and the DnaB-like family. In Table 1, the known viralhelicases are grouped according to virus genome structure,order, family, and species. Below, key viral helicases are

discussed based on their functional similarities rather thantheir phylogenic relationships. We begin with the DnaB-likehelicase family, then discuss SF1 DNA helicases, SF1 RNAhelicases, SF2 DNA helicases, SF2 RNA helicases, SF3DNA helicases, and finally SF3 RNA helicases. The discus-sion is focused on helicases from model organisms and thoseencoded by important human pathogens, which are high-lighted with subheadings.

Two of the best studied viral helicases are the DnaB-likeproteins needed for replication of E. coli phages T7 and T4.These helicases are believed to unwind DNA by encirclingone strand while displacing the other. While unwinding thedouble helix, DnaB-like helicases also coordinate leadingand lagging strand DNA synthesis. Replication fork coordi-nation is accomplished by interactions with the primase thatsynthesizes the RNA oligonucleotides needed to initiate newOkazaki fragments (for a review see reference [18]).

Phage T4 is still perhaps the best model system to studythe replication of large DNA viruses. The 168,930 base pairT4 genome with its approximately 300 genes (of which 62are essential) encodes analogs of almost all proteins neededfor DNA replication [19]. The T4 helicase responsible forcoordinating leading and lagging strand replication is theproduct of T4 gene 41 (gp41). The gene 41 protein forms ahexamer [20] that moves in a 5' to 3' direction along the lag-ging strand DNA template. The gene 59 protein loads thehelicase onto the DNA [21], and once loaded onto DNA,gp41 forms a tight complex with the T4 gene 61 primase[22]. T4 encodes two additional proteins that have also beenshown to possess helicase activity but which are not abso-lutely essential for viral replication under ideal conditions.Both proteins apparently unwind DNA as monomers not asrings like gp41. One of these is the SF1 helicase Dda [23],and the second is the SF2 helicase UvsW [24].

The T7 DNA replication system is simpler than that ofT4 in that the primase and helicase are covalently tethered aspart of the same polypeptide, which is encoded by phagegene 4. T7 gene 4 encodes two different proteins from twoseparate in-frame start codons. The larger, 63-kDa gene 4protein, sometimes called gene 4A protein or gp4A, pos-sesses a zinc finger domain that is missing from the shorter56-kDa protein, which is also referred to as the 4B protein orgp4B. Both gp4A and gp4B combine to form a hexamer thatpossesses both helicase and primase activities. The N-terminal portion of the gene 4 protein provides primasefunction [25], and the C-terminal portion provides helicasefunction [26]. The zinc finger domain missing from the 56-kDa gp4B helps guide T7 primase to certain sequences onthe lagging strand template where primers are synthesized[27, 28]. As a result, hexamers comprised of only the 56-kDagene 4 protein lack primase activity but retain helicase activ-ity [29].

There are no known viruses that infect animals (or plants)that express a helicase in the DnaB-like family. Neverthe-less, other viral helicases are functionally similar. For exam-ple, as discussed below, the SF3 DNA helicases form ringslike those made by the DnaB-like proteins. Similarly, one ofthe helicases produced by HSV forms a tight complex withthat virus’ primase and is thought to coordinate activities atthe DNA replication fork like the DnaB-like proteins. It is


Table 1. Viral Helicases

Genome Host Family (genus) Species Protein Activity [Reference] Family

dsDNA bacteria Myoviridae (T4-like) Enterobacteria phage T4 gp41 5'-3' DNA helicase [208] DnaB-like

Dda 5'-3' DNA helicase [209] SF1

UvsW RNA-DNA helicase [210] SF2

(P2-like) Bacteriophage P4 Gene α 3'-5' DNA helicase [211] DnaB-like

Bacteriophage P1 Ban DNA helicase [212] DnaB-like

Podoviridae Enterobacteria phage T7Gene 4A,4B protein

5'-3' DNA helicase [5, 213] DnaB-like

Siphoviridae (λ-like) Bacteriophage SPP1 G40P 5'-3' DNA helicase [214] DnaB-like

animals BaculoviridaeAutographa californica nuclear

polyhedrosis virusp143 DNA binding [215] SF1

Herpesviridae Human herpesvirus I UL5 5'-3' DNA helicase [32] SF1

UL9 3'-5' DNA helicase [40, 41] SF2

Polyomaviridae Simian virus 40 Tag 3'-5' DNA/RNA helicase [216] SF3

Papillomaviridae Human papillomavirus E1 DNA helicase [217] SF3

Poxviridae Vaccinia virus NPH-I DNA-dependent ATPase [81] SF2

NPH-II 3'-5' RNA helicase [85, 218] SF2

A18R 3'-5' DNA helicase [83] SF2

VETF ATPase, DNA binding [84] SF2

ssDNA plants Geminiviridae Coconut foliar decay virus Rep ATPase, DNA binding [219] SF3

animals Parvoviridae Adeno-associated virus 2 Rep 3'-5' DNA helicase [105] SF3

Minute Virus of Mice NS-1 DNA helicase [220] SF3

dsRNA Viruses bacteria Cystoviridae Bacteriophage φ6 P4 5'-3' RNA helicase [221] SF3

animals Reoviridae Bluetongue virus VP6 5'-3' and 3'-5' RNA helicase [222] SF2

(+)sense RNA plantsBromoviridae (Bromovirus)

Brome mosaic virus 1a Predicted [223] SF1

Closteroviridae (Closterovirus)

beet yellows closterovirus hel RNA binding [224] SF1

Flexiviridae (Potexvirus)

bamboo mosaic virus Orf1 RTPase [55] SF1

Potato virus X TGNBp1 5'-3' and 3'-5' RNA helicase [225] SF1

Potyviridae (Potyvirus)

plum pox virus C1 RNA helicase [6] SF2

Tamarillo mosaic virus C1 RNA helicase [226] SF2

Tymoviridae (Tymovirus)

turnip yellow mosaic virus p206 NTPase, RNA binding [227] SF1

No assigned family (Hordeivirus)

poa semilatent virus TGNBp1 5'-3' and 3'-5' RNA helicase [225] SF1

barley stripe mosaic virus TGNBp1 5'-3' and 3'-5' RNA helicase [225] SF1

(Tobamovirus) Tobacco mosaic virus Rep RNA helicase [159] SF1


(Table 1) contd….

Genome Host Family (genus) Species Protein Activity [Reference] Family

animals Arteriviridae Equine arteritis virus nsp10 5'-3' RNA/DNA helicase [57] SF1

Caliciviridae (Norovirus)

Southampton virus p41 NTPase [110] SF3

Coronaviridae human coronavirus 229E nsp135'-3' RNA/DNA helicase [56]

RTPase [52]SF1

SARS coronavirus nsp135'-3' RNA/DNA helicase [53]

RTPase [54]SF1

Flaviviridae (Flavivirus)

Yellow fever virus NS3 RNA stimulated NTPase [69] SF2

West Nile Virus NS3RTPase [78]

3'-5' helicase [72]SF2

Dengue fever virus NS33'-5' RNA helicase [70]

RTPase [71, 77]SF2

Japanese encephalitis virus NS3 3'-5' RNA helicase [73] SF2

Powassan virus NS3 RNA-stimulated ATPase [75] SF2

(Pestivirus) Bovine viral diarrhea virus NS3 3'-5' RNA helicase [228, 229] SF2

(Hepacivirus) Hepatitis C virus NS3 3'-5' RNA/DNA helicase[65] SF2

(unclassified) GB virus B NS3 3'-5' RNA helicase [230] SF2

Hepatitis G virus NS3 3'-5' RNA/DNA helicase [231] SF2

Picornaviridae (Enterovirus)

Poliovirus 2C NTPase [106] SF3

Echovirus 9 strain barty 2C NTPase [108] SF3

Togaviridae (Alphavirus)

Semliki Forest virus nsP2 RNA helicase [232] SF1

(Rubivirus) Rubella virus P70 RNA stimulated NTPase [233] SF1

the HSV helicase-primase complex that is the target of thenew anti-HSV drug candidates.

Herpes Simplex Virus Type UL5:UL52:UL8 Helicase

There are nine different human herpesviruses (HHV),which can be grouped into three sub-families based on theirgenome sequence and biological characteristics. The α typeincludes the herpes simplex viruses HHV-1 and HHV-2, andVaricella zoster virus, which cause oral and genital herpessimplex and chicken pox, respectively. The β group includeshuman cytomegalovirus (HHV-5), HHV-6A, HHV-6B, andHHV-7, and the γ class includes Epstein-Barr virus (HHV-4)and Karposi’s sarcoma associated herpesvirus (HHV-8). Ofthese, HHV-1 (also called herpes simplex virus-1 (HSV-1))is the best-characterized. The 152,000 base pair HSV-1 ge-nome encodes the synthesis of all the key proteins requiredfor its replication: an origin binding protein (UL9), a ssDNAbinding protein (ICP8), a DNA polymerase (UL30), a po-lymerase processivity factor (UL42), and a helicase-primasecomplex consisting of the UL5, UL52, and UL8 proteins

(see references [30] and [31] for reviews). Within the HSVhelicase-primase complex, the NTPase activity is part of theUL5 protein [32, 33]. The UL52 protein contains the primaseactive site that is responsible for synthesizing RNA primerson the lagging strand DNA. The UL8 protein does not ex-hibit any enzymatic activity but serves as a co-factor tostimulate both the helicase and the primase activities. UL8also facilitates nuclear transport of the helicase-primasecomplex, and coordinates the helicase and primase activitieswith those of other HSV proteins involved in DNA replica-tion.

The HSV helicase-primase complex unwinds duplexDNA with a 5'-3' directionality and requires the presence ofICP8 and ATP, indicating that ATP fuels the process of un-winding, and that ssDNA binding proteins assist duplexseparation by stabilizing partially unwound products [32,34]. UL5 possesses all the motifs that are conserved amongSF1 proteins, and site-directed mutagenesis of the SF1 heli-case motifs leads to a complex that lacks an ability to un-wind DNA [35, 36]. When UL52 is separated from UL5,


ATP hydrolysis is not stimulated by DNA and the protein nolonger unwinds DNA [37], indicating that UL52 is an inte-gral part of the HSV helicase. Site-directed mutagenesis waslater used to confirm that UL52 interacts directly with DNAthrough a zinc finger domain [38] and that while UL52mainly contacts the ssDNA tail, UL5 interacts with DNAcloser to the replication fork [39]. Thus, UL5 and UL52work together at the replication fork to unwind the doublehelix and simultaneously synthesize primers on the laggingstrand DNA template.

In addition to the above helicase-primase complex, HSVexpresses a second helicase that is a member of SF2, notSF1. UL9 is a 851 amino acid (94-kDa) protein that wasoriginally isolated as a protein that specifically binds theHSV origin of replication [40]. Later, it was found that UL9unwinds DNA with a 3′ to 5′ directionality beginning atthese HSV origins [41-43].

T4 Dda is another well-characterized SF1 DNA helicase.Dda was one of the first helicases purified because it can berelatively easily separated from cellular ATPases usingchromatography [3]. Dda acts as a monomer [44] and trans-locates in a 5' to 3' direction [45]. Soon after its discovery, itwas noted that Dda allows replication to proceed past aDNA-bound RNA polymerase, suggesting that the Dda heli-case unwinds a double helix and can also displace proteinsbound to DNA [46]. Recently, a kinetic mechanism for suchprotein displacement by Dda has been proposed based onmeasurements of the rate of Dda catalyzed displacement ofstreptavidin from biotin labeled DNA [47].

Several SF1 helicases have also been cloned from RNAviruses. Most of the RNA virus SF1 helicases are encodedby plant viruses, few of which have been studied in greatdetail. A few SF1 helicases from animal RNA viruses havebeen purified and characterized, but in general, less is knownabout SF1 RNA helicases than the above SF1 DNA heli-cases. Some of the helicases in this family are made bypathogenic human viruses, the most noteworthy being thevirus that causes SARS.

SARS Coronavirus Helicase

Severe acute respiratory syndrome (SARS) is a life-threatening form of pneumonia caused by an RNA virusclassified in the genus Coronavirus. The finding that theSARS virus is a member of this group was surprising be-cause most other coronaviruses cause only relatively mildrespiratory illnesses (i.e. the common cold). The genusCoronavirus is part of family Coronaviridae that, along withthe families Arteriviridae and Roniviridae, belongs to theorder Nidovirales. Nidovirales contains viruses with thelargest known RNA genomes. The (+)ssRNA SARS-CoVgenome has about 29,700 ribonucleotides with fourteenopen reading frames that encode both structural and replica-tive proteins [48]. The two largest open reading frames(ORF1a and ORF1b) overlap at the 5′ end of the genome.ORF1a and ORF1b are translated into two large polyproteins(pp): pp1a (~490 kDa) and pp1ab (~790 kDa). The N-terminus of pp1ab is identical to pp1a; the C-terminal half ofpp1ab is generated by ribosomal slippage [49, 50]. TheSARS-CoV polyproteins are then processed by two viralproteases into mature viral peptides, which include both

structural and nonstructural proteins. The helicase is part ofnonstructural protein 13 (nsp13), which is processed fromthe C-terminal portion of pp1ab [51]. SARS-CoV helicase islocalized on the endoplasmic reticulum of SARS-CoV in-fected cells, where RNA replication is likely to take place[52].

Even though SARS-CoV is an RNA virus with no knownDNA stage in its lifecycle, SARS-CoV helicase unwindsboth RNA and DNA duplexes in a 5′ to 3 ′ direction. To fuelstrand separation, SARS-CoV helicase hydrolyzes ATP orany of the eight canonical NTPs [53]. SARS-CoV helicasealso has the ability to cleave the terminal phosphate from atriphosphate moiety linked to the 5′ end of a RNA molecule[52, 54]. Using this RNA 5′-triphosphatase activity (RTPase)activity, SARS-CoV helicase is able to prepare viral RNA toreceive a 5′ cap structure. The fact that ATP is a competitiveinhibitor of the RTPase reaction catalyzed by SARS helicasesuggests that the RNA is hydrolyzed at the same active siteused to fuel helicase movement [54]. A similar RTPase thatplays a role in CAP formation was also found in the SF1helicase encoded by bamboo mosaic virus [55]

In addition to the SF1 helicase motifs, SARS-CoV nsp13protein, and its relatives from human coronavirus 229E [56]and equine arteritis virus [57], all contain a cysteine-rich zincbinding domain located at their N-termini. Zinc binding do-mains have been found linked to other helicases and they arefrequently found in proteins that interact intimately with nu-cleic acids. For example, both the T7 and HSV helicaseshave zinc binding domains that help facilitate primer synthe-sis by their attached primases, and the HCV helicase has azinc ion bound to its N-terminal protease region. When thezinc binding domain attached to nidovirus helicases is al-tered using site-directed mutagenesis, the virus displays de-fective RNA synthesis in infected cells [58] and the helicaseis not fully active [59].

Like the proteins in SF1, SF2 helicases generally do notform rings. SF2 is almost as large as SF1 and likewise con-tains cellular proteins, and proteins encoded by both DNAand RNA viruses. Many are key proteins for the replicationof RNA viruses, and they unwind duplex RNA structuresbefore and/or after RNA genomes are copied by viral RNA-dependent RNA polymerases. Other SF2 helicases act duringtranscription, translation, or to dislodge RNA binding pro-teins. Several SF2 proteins have been isolated from a varietyof viral pathogens; the best studied being the helicase en-coded by HCV. Many of the viral RNA helicases in SF2belong to a large family of evolutionarily related proteinscalled DExD/H box proteins [60]. This family is named aftera shared Asp, Glu, and Asp (or His) containing sequence inthe second conserved SF2 helicase motif (see references [61]and [62] for reviews of DExD/H box proteins).

Hepatitis C Virus NS3 Helicase

HCV is the main agent responsible for non-A-non-B viralhepatitis, and the cause of a world-wide epidemic of chronicliver disease. Current treatments using pegylated interferonand ribavirin are costly, produce severe side effects, andeliminate detectable virus in less than 60% of HCV patients.HCV contains a (+)ssRNA genome with one main openreading frame that encodes a long polypeptide about 3000


amino acids long. The genome is translated and the polypep-tide is processed into structural and non-structural (NS) pro-teins by both cellular and viral proteases. While the struc-tural proteins generate the viral capsid and envelope proteins,the NS proteins are responsible for genome replication. RNAsynthesis is carried out by the NS5B RNA-dependent RNApolymerase. HCV helicase is part of the bi-functional NS3protein, which possesses helicase, NTPase, and serine prote-ase activities. The two N-terminal NS3 domains provideprotease function, and the remaining three C-terminal do-mains comprise the helicase activity. The NS3 helicase re-gion was first shown to have RNA stimulated ATPase [63],and was later shown to bind RNA [64], unwind RNA [65,66] and DNA [67, 68].

There are approximately 12 distinct types of HCV, calledgenotypes, which have nucleotide sequences that differ by asmuch as 30%. All of the various HCV genotypes aregrouped into the genus Hepacivirus, which is one of threegenera in Flaviviridae. The other two genera are Flavivirusand Pestivirus, both of which have a genome organizationsimilar to HCV, with the helicase formed by the C-terminusof the NS3 protein. The Flavivirus genus includes severalimportant pathogens such as the prototype yellow fever virus(YFV), Dengue fever virus (DFV), West Nile virus (WNV),and Japanese encephalitis virus (JEV). The Pestivirus genuscontains viruses infecting non-human animals includingsome that cause serious livestock infections, such as Bovineviral diarrhea virus (BVDV).

Flavivirus Helicases (YFV, JEV, DFV, and WNV Heli-cases)

The mosquito-born YFV is endemic in sub-Saharan Af-rica and tropical South America and, although rarely fatal,still poses a threat to unvaccinated travelers. Like YFV, thereis a vaccine for JEV, which is the leading cause of viral en-cephalitis in Asia. Dengue fever, with symptoms that rangefrom mild fever to a fatal hemorrhagic syndrome, is moreserious because no vaccine is available. Both yellow andDengue fever have seen resurgence since mosquito eradica-tion programs have been scaled back. WNV is a bird virusthat is also spread to humans by mosquitoes and has recentlyreceived much attention since its introduction to NorthAmerica. WNV normally causes only mild flu-like symp-toms but compromised WNV patients may suffer severe neu-rological damage.

Although the NS3 protein from YFV has not yet beenshown to unwind RNA, it does possess RNA stimulatedNTPase activity [69]. Many related viruses including DFV[70, 71], WNV [72], and JEV [73] have each been shown tounwind RNA in a 3′ to 5′ direction. Although nucleic acidstimulates ATP hydrolysis catalyzed by flavivirus, pestivi-rus, and hepacivirus helicases, different nucleic acids stimu-late the enzymes differently. Each enzyme possesses a dis-tinct nucleic acid stimulation profile, suggesting that theybind RNA (or DNA) in a sequence specific manner or un-wind certain sequences better than others [63, 74]. Someflavivirus helicases also contain an additional conserved nineamino acid sequence upstream from the SF2 helicase motif 1that is not present in HCV NS3 [75]. Called the “Q motif, ”this region likely interacts with the adenine base and could

explain the ability of certain nucleoside analogs to inhibitflavivirus helicases but not HCV helicase [76].

The flavivirus NS3 helicases also differ from HCV heli-case in that flavivirus helicases possess RTPase activity, ashas been shown with both DFV [71, 77] and WNV [78].Like the SARS-CoV helicase, the flavivirus helicases spe-cifically cleave the β-γ bond of a triphosphate linked to the 5′end of a RNA in the first step needed to generate a RNA 5′-terminal cap structure [71, 77, 78]. Although the next en-zyme in the capping pathway, which transfers a GMP to theRNA 5' diphosphate has not yet been identified, flavivirusesencode a RNA cap methyltransferase (the NS5 protein) thatmethylates the G5_ppp-5_N RNA cap at the 7 position of gua-nine [79]. Since HCV utilizes a 5′ internal ribosomal entrysite (IRES) instead of cap-dependent RNA translation, itsRNA likely lacks the 5′-cap structure present in flavivirusand coronavirus genomes.

DNA viruses also encode members of SF2. As mentionedabove, both HSV and phage T4 encode SF2 helicases, theUL9 protein and UvsW proteins, respectively (Table 1). Ingeneral, SF2 helicases encoded by DNA viruses are not di-rectly involved in coordinating proteins at the DNA replica-tion fork (like the DnaB-like and SF3 helicases). Rather, SF2proteins from DNA viruses are often found to be involved inDNA repair, recombination, transcription, or translation.Those needed for transcription are most similar to the RNAhelicases in the DExD/H-box family. Some of the proteins inthis group are required for virus replication, but some are notbecause host factors can substitute for their functions.

Four different proteins in the SF2 helicase family areencoded by the prototypic poxvirus vaccinia virus [80]. Vac-cinia virus (VV) has been the choice model system to studytranscription in part because the viral RNA polymerase andnumerous transcription factors are packaged into each virion.Vaccinia virus has a linear double-stranded DNA genome of192,000 base pairs, which it replicates in the cytoplasm. VVgenes are assigned letters and numbers based on a restrictionmap of the virus. The four VV SF2 helicases are the NTPphosphohydrolase I (NPH-I) encoded by gene D11L [81],NTP phosphohydrolase II (NPH-II) encoded by I8R [82], theA18R gene product [83], and the VV early transcriptionfactor (VETF) encoded by D6R [84]. Of the four, NPH-II isthe best characterized. As an RNA helicase, NPH-II can un-wind duplex RNA in a processive manner [85], and dislodgeRNA binding proteins as it moves in a 3'-5' direction [86,87].

Unlike the SF1 and SF2 helicases, SF3 helicases gener-ally form rings like the DnaB-like helicases. SF3 containsmany viral proteins. In fact, SF3 contains only viral proteins,which are derived from both DNA and RNA viruses. Al-though the best studied SF3 helicase is a protein synthesizedby SV40, which causes no known disease in humans, SF3also contains proteins from key human pathogens, such asHPV and poliovirus.

The best studied SF3 helicases are those encoded bysmall non-enveloped viruses with short, circular double-stranded DNA genomes. These viruses are grouped into thefamilies Polyomaviridae and Papillomaviridae, which untilrecently were subfamilies in a family called Papovavirdae.


They are now grouped as separate families because theirgenomes have been found to be clearly different. Polyomavi-ruses have smaller genomes (~5,000 base pairs), and papil-lomaviruses have larger genomes (~8,000).

The prototype polyomavirus is simian virus 40 (SV40).SV40 has been intensely studied since it was found contami-nating early polio vaccines. SV40 is pathogenic to monkeys,and causes tumors in hamsters, but fortunately seems tocause no ill effects to humans who were inadvertently in-oculated with SV40. Related polyomaviruses, such as the JCvirus and the BK virus, infect the majority of the world’spopulation causing no apparent harm except to severely im-muno-compromised patients, in whom JC virus can causethe fatal neurological disorder progressive multifocal leu-koencephalopathy [88, 89]. The small polyomavirus genomeencodes only six proteins, which are expressed from eitherearly or late promoters. Three of the four proteins expressedfrom the late promoter are capsid proteins. The mRNA tran-scribed from the early promoter is alternatively spliced toyield either small or large T antigens, so named because theycan be detected in animals bearing polyomavirus inducedtumors. The small t antigen (tag) and large T antigen (Tag)share the same N-terminus, but differ at their C-termini. Tag(79-kDa) is much larger than tag, which is only 20-kDa. Tagassembles as two hexamers that each surround DNA whenthey binds the SV40 origin of replication [90-92]. The duelTag hexamers both move in a 3' to 5' direction [93] with re-spect to the strand on which they are bound and act to open areplication bubble so that two replication forks can assembleand proceed in opposite directions. In the process, Tag inter-acts with and coordinates the action of numerous cellularDNA replication proteins (for review see [94]).

Human Papillomaviruses Helicase

The human papillomaviruses are similar to polyomavi-ruses except that they have slightly larger capsids and larger,somewhat more complex, genomes. There are over 100known human papillomaviruses, which are numbered asHPV-1 through HPV-96 (over 20 types are still unclassi-fied). All HPVs infect the epithelial tissues, and the vastmajority “low risk” types only lead to benign warts or le-sions on the pharynx, esophagus, or genitals. A few “highrisk” variants, such as HPV-16, HPV-18, and HPV-31, cancause cervical cancer [95]. The ~8,000 bp small closed-circular double-stranded DNA HPV genome contains severalopen reading frames that are translated and processed intoeight early (E1 to E8) and two late (L1, L2) gene products[96, 97]. L1 and L2 form the viral capsid, and the early geneproducts are responsible for viral replication. The HPV heli-case is the E1 protein (for review see [98]).

HPV E1 contains a nuclear localization signal at its N-terminus, a site-specific DNA binding domain in the centralregion, and an SF3 helicase domain within the C-terminalregion. While the E1 C-terminal domain alone is sufficientfor oligomerization, ATP hydrolysis and DNA unwinding,the entire E1 protein is needed for HPV replication in vivobecause the N-terminal domains specifically bind origins.Stable formation of an E1 complex at the HPV DNA originof replication requires the recruitment of E1 monomers bythe HPV E2 protein, which also binds to the HPV DNA ori-

gins through its own DNA binding domain [99]. By inter-acting with E1, E2 loads the E1 helicase on the HPV DNAorigin. When E1 and E2 first interact, E2 blocks the E1 oli-gomerization interface, ensuring the proper assembly of theE1 proteins at the HPV DNA origin [100]. E2 associationwith the E1-DNA origin complex is then destabilized uponbinding of ATP to the E1 protein [100, 101]. The E1 mono-mers then oligomerize, forming a double hexamer around theDNA substrate [102]. As in the SV40 system, the hexamersmove away from each other because they are associated withcomplementary strands that have opposite polarities, andtheir movement forms two replication forks at opposite endsof a replication bubble [102].

SF3 DNA helicases are also encoded by viruses withssDNA genomes, such as the human parvovirus adeno-associated virus (AAV), a small, apparently harmless virusused for gene therapy [103]. The 4700 nt ssDNA AAV ge-nome has only two open reading frames, one of which en-codes four Rep proteins that make a SF3 DNA helicaseneeded for integration into the human genome. The Repopen reading frame encodes 4 different proteins, calledRep78, Rep68, Rep52, and Rep40, by alternate RNA splic-ing. All four Rep proteins, including the smallest Rep40,share a central motor domain needed for integration. Thelargest version, Rep78, forms a hexameric ring when boundto the AAV origin of replication [104]. Rep68 and Rep40lack a C-terminal zinc finger domain that is present in Rep78and Rep 52. Rep40 and Rep52 lack the 224-amino acid N-terminal domain common to Rep78 and Rep68. Rep52 re-tains an ability to unwind DNA in a 3' to 5' direction, butdoes not apparently need to form rings to catalyze the reac-tion [105].

Surprisingly little work has been done with the SF3 heli-cases encoded by RNA viruses. One might suspect that theyare similar to SF3 helicases encoded by the small DNA vi-ruses in form and function, but there is as yet no direct evi-dence to support this contention. RNA viruses that infectboth plants (families Comoviridae and Sequiviridae) andanimals (families Picornaviridae and Caliciviridae) havebeen predicted to encode SF3 helicases but none of the pro-teins have been shown to unwind duplex RNA. Nevertheless,the poliovirus 2C protein [106, 107], the polio-like virusechovirus 9 strain barty NS protein 2C [108, 109], and aNorwalk-like virus 2c-like protein, called p41 [110] have theabilities to hydrolyze NTPs. Perhaps the conserved motorfunction in these proteins is used for something other thanRNA unwinding.

HELICASE STRUCTURE AND FUNCTION

Over 20 different helicases have been studied at anatomic resolution using x-ray crystallography and/or nuclearmagnetic resonance (NMR). Six of these helicases are pro-teins from viruses, and they include two non-ring helicasesfrom HCV and phage T4, and four ring helicases from phageT7, AAV, HPV, and SV40. The structures reveal that eventhough all helicases share a common fold surrounding theATP-binding site, ring (DnaB-like and SF3) and non-ringhelicases (SF1 and SF2) harness the energy liberated fromATP hydrolysis to unwind DNA differently. ATP hydrolysisby non-ring helicases causes a shift in two domains cova-


lently linked on the same polypeptide, while in ring helicasesATP hydrolysis leads to movement of the various subunits inthe ring relative to each other. The structures of several SF1,SF2, SF3, and DnaB-like helicases have been reported andthe structures within each superfamily appear quite similar.How these structures relate to the function of each class ofhelicases is discussed below. Correlating structure and func-tion not only clarifies how helicases move along nucleic ac-ids but also facilitates the rational design of helicase inhibi-tors.

The first viral helicase structures reported were of theHCV RNA helicase (Fig. (2a)). The first studies utilizedrecombinant proteins containing only the helicase portion ofthe HCV NS3 protein, which was isolated from genotype 1a(PDB 1HEI) [111] or genotype 1b (PDB 8OHM) [112]. TheHCV genotype 1a NS3 helicase fragment was also crystal-lized with a bound DNA oligonucleotide (PDB 1A1V) [113].Later, a crystal structure of the full-length NS3 protein withits NS4 cofactor covalently tethered to the recombinant pro-tein’s N-terminus was reported (PDB 1CU1) [114]. Theatomic structure of an isolated domain of the 3-domain HCVhelicase has also been determined in solution using NMR(PDB 1JR6 and 1ONB) [115]. The structure of another SF2viral helicase, the UvsW protein from phage T4 (PDB 1RIF),has also been recently reported [24].

The first ring helicase crystallized was the DnaB-likehelicase of phage T7 (Fig. (2b)). The first studies utilized ashort fragment of the T7 gene 4 primase/helicase proteinlacking the primase and the linker region connecting thehelicase to the primase. Because the linker region is neces-sary for hexamer formation, this fragment, called the 4Epeptide, exists as a monomer in solution and is unable tounwind DNA [26] but crystallized as a filament, which whenviewed down the helical axis resembles a six-member ring[116]. Structures are available for the T7 4E peptide alone(PDB 1CR0), with dTTP (PDB 1CR1), with dATP (PDB1CR2), and with dTDP (PDB 1CR4) [116]. Later, a structurewas reported for a larger fragment of the gene 4 protein,called the 4D peptide, that had been previously crystallized[117]. The 4D fragment contains the primase linker, formshexamers and unwinds DNA. Structures were determined forT7 4D both alone (PDB 1E0K) and with the ATP analog 5'-adenylyl-β−γ-imidodiphosphate (ADPNP) (PDB 1E0J)[118]. Most recently, a structure of the full-length 56-kDagene 4 protein (4B protein) was reported (PDB 1Q57) [119].

The three SF3 viral helicases that have been examinedusing x-ray crystallography all have similar overall folds thatare somewhat different from the DnaB-like T7 helicase. TheAAV helicase Rep40 has been crystallized without ligands(PDB 1S9H) [120] and as a complex with ADP (PDB 1U0J )[121]. SV40 Tag (Fig. (2c)) structure has been determined inthe absence of ligands (PDB 1N25 [122] and PDB 1SVO[123]), with ATP bound (PDB 1SVM) [123] and with ADPbound (PDB 1SVL) [123]. The helicase portion of the HPVE1 protein (residues 428-629) has also been crystallizedalong with the E2 viral protein, which aids its assembly atthe origin (PDB 1TUE) [100].

All helicase structures share several common features.First, they share a common fold that was first described inthe E. coli protein RecA [124]. This “RecA-like” domain

consists of a series of beta-sheet sandwiched between twosets of alpha-helices and is so critical to helicase functionthat all helicases contain at least two such domain. In thenon-ring helicases each protein monomer contains twoRecA-like domains covalently attached to one another by ashort linker (Fig. (2a)). In ring helicases each subunit of thering contains one RecA-like domain (Fig. (2b, 2c)).

The configuration of residues at the ATP-binding site isalso similar for all helicases. ATP and a required metal ioncofactor (usually Mg2+) bind to a helicase in the cleft thatseparates two adjacent RecA-like domains as diagramed inFig. (3). The ATP-binding cleft is lined by the conservedresidues from the helicase signature sequences. Although theexact composition of the signature sequences varies amongthe helicase families, all helicases share two amino acid pat-terns that were first described by John Walker [125], whichare honorably referred to as the Walker A and Walker B mo-tifs. The Walker A motif forms a phosphate binding loop (P-loop) with a conserved Lys that contacts the γ phosphate ofATP. The Walker B motif contains acidic residues that coor-dinate the positively charged divalent metal cation, which inturn contacts the phosphates of ATP. The ATP-binding siteof a helicase is completed by an Arg “finger” and a catalyticbase, which accepts a proton from the attacking water mole-cule. In related proteins this catalytic base has been demon-strated to be a conserved Glu near the Walker B motif [126,127]. The Arg-finger points from a second RecA-like do-main that is adjacent to the domain containing the P-loop andprovides a positive charge that stabilizes the transition state[128, 129]. In ring helicases the Arg-finger and P-loop arepart of different polypeptide chains in the hexamer [130],explaining why ring helicases need to oligomerize in order tocatalyze a reaction. In non-ring helicases the Arg-finger ispart of the C-terminal RecA-like domain while the P-loop ison the N-terminal RecA-like domain.

All theories explaining helicase function suggest thatchanges in the orientations of the RecA-like domains occurupon ATP binding and hydrolysis, and that these changessubsequently allow the protein to move along a nucleic acidtemplate. The RecA-like domains are thus analogous to amolecular motor and are therefore commonly referred to as“motor” domains. The motor domains move the helicase andany attached cargo along a track of DNA or RNA.

Mechanism of Action of Non-Ring Helicases (SF1, SF2Helicases)

In addition to contacting ATP, the RecA-like domains inhelicases also contact nucleic acids. When the first structuresof the HCV helicase were published, the authors speculatedthat nucleic acid would bind in the cleft that separates thetwo RecA-like domains [111, 112]. The N-terminal RecA-like domain of HCV helicase (domain 1) contains the P-loopand the C-terminal RecA-like domain (domain 2) containsseveral conserved arginines that form a basic patch that linesthe inter-domain cleft. Although one of these arginines islikely the Arg finger described above (Fig. (3)), they were atthe time believed to form an RNA binding motif. Becausethe cleft that separates the third HCV helicase domain (do-main 3) from domains 1 and 2 is lined with negativelycharged residues that should repel RNA, molecular modeling


Fig. (2). Helicase Structures. A. HCV helicase structures [113, 114]. The N-terminal RecA-like domain (domain 1) is colored yellow, the C-terminal RecA-like domain is purple, domain 3 is pink, the protease is green, and the NS3 protease cofactor NS4A is blue. DNA and sulfateions (which occupy the ATP binding site) are noted as spheres. B. T7 helicase structures [116, 118, 119]. The 4D protein lacks the primasedomain of the T7 gene 4B protein (56-kDa gene 4 protein). The 4E protein is like the 4D protein but lacks a linker region necessary for hex-amer formation. In the 4D and 4B structures, the different subunits are colored differently. The 4E filament structure is colored based on sec-ondary structure. C. SV40 Tag helicase structures [123]. The different subunits in the SV40 hexamer are noted with different colors. In bothB and C nucleotides are displayed as spheres. The noted PDB coordinate files were used to generate the structures using Pymol (DeLanoScientific LLC, San Francisco, CA).


Fig. (3). Mechanism of helicase-catalyzed ATP hydrolysis. All known helicases and related proteins coordinate an ATP, Mg2+, and a watermolecule using a conserved Lys and Asp in the Walker A and B motifs on one RecA-like domain and an Arg on an adjacent RecA-like do-main. A nearby Glu likely acts as a catalytic base by accepting a proton from the attacking water molecule.

logically predicted RNA to bind in the other cleft, the onewhich separates the RecA-like domains [111, 112]. It wastherefore somewhat surprising when a co-structure of a shortoligonucleotide bound to HCV helicase showed the DNAbound to the cleft separating domain 3 from the RecA-likedomains (Fig (2a)) [113]. Extensive site-directed mutagene-sis of HCV helicase has since confirmed the validity of theHCV helicase-DNA structure and demonstrated that the ob-served protein-nucleic acid contacts are needed for efficientunwinding [131-136]. Furthermore, a similar DNA bindingcleft, which is perpendicular to the ATP binding cleft, hassince been seen in co-structures of two other non-ring SF1helicases with DNA [137, 138]. Thus, in both SF1 and SF2helicases, it appears that the single-stranded nucleic acid isbound in a cleft that is perpendicular to the ATP-bindingsite.

How ATP hydrolysis is translated into helicase move-ment on nucleic acid is the key question that is presentlybeing addressed in several laboratories. Many of the leadingtheories propose that movement of the RecA-like domainsupon ATP binding and hydrolysis allow the protein to movelike an inchworm on one strand, while simultaneously dis-placing the complementary strand or nucleic acid bindingproteins. The most convincing evidence for such a modelcomes from studies of a non-viral SF1 helicase encoded byBacillus stearothermophilus called PcrA. PcrA has beencrystallized alone [139], in the presence of an ATP analog[140], in the presence of DNA, and in the presence of DNAand an ATP analog [138]. These structures show movementsof the non-RecA-like domains upon DNA binding, and aclosure of the cleft between the RecA-like domains whenATP binds, which coincides with a distortion of the DNAduplex. Based on the rigorous analysis of numerous PcrAsite-directed mutants, the interactions seen in the PcrA

structures appear mechanistically relevant [141, 142], andsuggest that DNA passes through one end of the helicasewhen the protein is in the open conformation and the otherwhen it is closed. When ATP is bound, the protein is closedand the cleft opens again after hydrolysis and ADP release.Such movement would permit the protein to move like aninchworm.

The PcrA structures have also provided informationabout the nature of the forces that move the helicase alongDNA. The PcrA-DNA structure differs from that of thePcrA-DNA-NTP structure significantly in the DNA bindingsite. In both structures, several bases are flipped out of thehelix to interact with certain amino acids, but this flippingpattern is different in the two structures. Velenkar et al. pro-pose that sequential ATP hydrolysis events lead to a wavelike motion in the side chains that acts to move the proteinalong ssDNA in a 3' to 5' direction [138]. Soultanas & Wig-ley call this the “Mexican wave mechanism” because themotion of the side chains is reminiscent of the “waves” thatpropagate through stadium crowds, which (though commonin the USA for decades) were first recognized internationallyat the Mexican world cup [143]. This modified inchwormmechanism implies that PcrA helicase would move only onenucleotide for each molecule of ATP hydrolyzed, and thishypothesis is supported by an analysis of the kinetics of oli-gonucleotide-stimulated PcrA-catalyzed ATP hydrolysis[144]. It is likely that SF1 viral helicases, such as the SARSand HSV helicases, utilize such a method to move on DNAor RNA.

While this wave-like action might work well for non-ringSF1 helicases, SF2 helicases likely function somewhat dif-ferently. Although similar inchworm models have been pro-posed to explain the movement of SF2 helicases, the struc-ture of HCV helicase reveals that the protein makes fewer

N N

N

N O

OO

O P

O

O

O P O

O

OP O

O

O

NH3+

N

N

O

H

H

O

O

NH3+ Mg2+

O

O

Lys

Glu

Arginine Finger

Walker A

Walker B

Catalytic E

Asp


direct contacts with nucleic acid bases than does PcrA [113].Instead, most contacts are made with the phosphodiesterbackbone. The importance of several residues that contactthe backbone in the HCV-DNA structure has been verifiedusing site-directed mutagenesis. Critical nucleic acid back-bone-binding HCV residues include Thr269 [131], Thr411[131], Thr450 [74], Arg461[132] and Arg393 [136]. Onlyone HCV helicase amino acid side chain, that of Trp501, isknown to contact the bases [113]. Trp501 stacks against thebase at the 3'-end of the HCV bound oligonucleotide, and isnot in a RecA-like domain. Site-directed mutants with analanine substituted at position 501 unwind RNA poorly [131-135]. Because it stacks with a base, Trp501 is believed toprevent DNA (or RNA) from sliding through the helicase inthe absence of ATP. Kim et al. [113] proposed a “ratcheting”inchworm model suggesting that when ATP binds, the cleftbetween the RecA-like domains closes, and Trp501 slidespast one or two nucleotides. When ATP is hydrolyzed, thecleft opens, and Trp501 acts as a bookend so that the proteinmoves towards the 5'-end of the nucleic acid. Supporting thismodel are the observations by Porter et al. that only a fewbase pairs of fluorescently-labeled DNA are unwound byHCV helicase in a single turnover event and that the helicasebinds DNA weaker in the presence of ATP [145]. Othershave since confirmed that ATP binding weakens the affinityof HCV helicase for DNA and RNA [136, 146]. Howeverthere is yet no general agreement on the number of base pairsunwound in a single event (called “step size”). Levin et al.[147] have calculated a step size of 9 base pairs using unla-beled DNA, and using a long RNA substrate Serebrov &Pyle have determined that 18 base pairs are unwound byHCV helicase in a single step [148].

Insights into the force which propels the nucleic acidthrough HCV helicase were recently made when Lam et al.

observed that the binding of nucleic acid to a HCV helicase-ATP complex is pH dependent [149]. Thus, electrostaticforces may be as critical for helicase action as the mechani-cal forces described by the Mexican wave model. Lam et al.proposed that since negatively charged nucleic acid is held ina negatively charged protein cleft, the potential energybuildup could be utilized to propel the helicase along a nu-cleic acid track. In this “propulsion-by-repulsion” model,ATP binding leads to a conformational change such that thenucleic acid bases can clear the Trp501 bookend. As dia-gramed in Fig. (4), in the absence of ATP, RNA cannot exitthe enzyme because it is blocked by Trp501 and clamped inthe cleft by an Arg “clamp” on domain 2, which has beenshown to be critical for binding and translocation [136].When ATP binds, domain 2 rotates bringing with it the posi-tively charged Arg-clamp. The Arg-clamp attracts the nega-tively charged phosphodiester backbone so that the RNAmoves free from the bookend. The negatively charged RNAis then repelled by the negatively charged binding cleft, so itmoves through the protein until ATP is hydrolyzed, and theprotein clamps tightly again. Thus, the step size of the pro-tein would depend on the nature of the nucleic acid on whichthe protein is translocating. For example, since RNA is morepolar than DNA, one would predict a larger step size onRNA than DNA as has been observed [147, 148]. Also, astronger repulsion of RNA would cause the enzyme to fallfrom that substrate before it would fall from DNA. Such alower processivity for HCV helicase-catalyzed RNA un-winding than DNA unwinding has been frequently reported[136, 150, 151].

The above inchworm models all predict that a SF1 orSF2 helicase can function as a monomer but do not accountfor all observed behaviors of non-ring helicases. For exam-ple, with HCV helicase, unwinding rates are not linearly

Fig. (4). The electrostatic inchworm model for HCV helicase movement. In the model, ssRNA is held in a negatively charged cleft separat-ing the RecA-like domains from a third domain. A bookend residue (Trp501 in HCV helicase) prevents ssRNA from sliding through thiscleft. Upon ATP binding, the RecA-like domains rotate, moving positively charged RNA-bound residues (e.g. Arg393 in HCV helicase),which in turn move the RNA so that it clears the bookend. Charge repulsion between the RNA and the negatively charged cleft causes theprotein to slide along the helix. Based on information from references [136, 149, 236].


dependent on the amount of protein present in the reactionbut rather accelerate greatly once a critical protein concen-tration is reached [136, 151]. Yeast two-hybrid assays alsosuggest that HCV helicase forms a dimer [152, 153], and thatresidues Thr266, Tyr267 and Met288 in domain 1 are criticalfor dimerization [153]. Kinetic models explaining this coop-erativity have recently been presented [147, 154], and it re-mains possible that HCV and other non-ring (SF1/2) viralhelicases could function as dimers as has been proposed forother helicases like the E. coli Rep [155] and UvrD helicases[156, 157] (for a review of the “rolling dimer” helicasemechanism see [158]). There is also the possibility that allhelicases form rings or filaments on nucleic acid, and wehave yet to find the proper conditions to observe these ringswith the SF1 and SF2 helicases discussed above. For exam-ple, a purified recombinant form of a SF1 helicase encodedby tobacco mosaic virus is capable of forming hexamericrings that can be observed using both gel filtration chroma-tography and electron microscopy [159].

Mechanism of Action of Ring Helicases (DnaB-like, SF3Helicases)

ATP binding and the mechanism of its hydrolysis by ringhelicases is basically identical to that outlined above for non-ring helicases (Fig. (3)). The main difference between ringand non-ring helicases is that the Arg finger is on a separatepolypeptide chain. Since in ring helicases, there is only oneRecA-like domain, each subunit has both a P-loop and anArg-finger. The subunits are arranged in a head to tail man-ner, with the head being the P-loop, and the Arg-finger beingthe tail. The ATP is bound between the head and tail, andthere is the same number of binding sites as there aresubunits in the oligomer (Figs. (2) and (5)). Even thoughthere can be up to six ATP molecules bound per hexamerichelicase ring, fewer sites are often observed. The DnaB-likehelicases, like the F1 ATPase, bind ATP with negative coop-erativity. This means that the binding of the first ligandscause a decrease in the affinity for subsequent ligands. As aresult, only 2-4 NTPs bind per hexamer [160]. The kineticsof NTP hydrolysis catalyzed by T7 helicase are also re-markably similar to those seen with F1 ATPase [161]. In bothproteins, it appears that ATP is hydrolyzed sequentially bydifferent subunits. In the case of the F1 ATPase, this leads toa rotation of the γ subunit, which in turn contacts the mem-brane embedded Fo proton channel [162]. When protons passthrough the Fo complex, the reverse rotation leads to ATPsynthesis [163]. In the T7 helicase, the DNA is proposed tobe analogous to the γ subunit of F1 ATPase, and the rotationleads to the helix moving through the oligomer.

The above “rotational” model predicts that one or bothstrands of DNA pass through the center of the hexamer, asdiagramed in Fig. (5). Although ring helicases have not yetbeen crystallized in the presence of DNA, this notion is sup-ported by electron micrographs and model building. In thepresence of DNA, extra density appears in the central chan-nels of hexamers formed by both T7 helicase [164] andSV40 Tag [165]. Also, electrostatic analysis of hexamerichelicase structures reveals in all cases that the central holesare lined with positive charges [116, 118, 120, 122, 123],which would attract negatively charged DNA. In crystalstructures of the T7 helicase portion of the gene 4 protein

(Fig. (2b)) the channel is only large enough to accommodateone DNA strand, suggesting that the other must wind outsidethe ring as shown in Fig. (5a) [118].

If DNA does, in fact, pass through the center of T7 heli-case, it would make contact with loops that contain residuesthat were previously shown using mutagenesis to be in-volved in DNA binding, like Arg487 [166, 167]. These resi-dues are part of the loops that extend from the RecA-likedomains into the central channel. The DNA would thereforebe perpendicular to the ATP binding cleft, but at an anglesomewhat different from that seen in the SF1 and SF2 heli-case structures. It is also interesting to note that the subunitsof the T7 helicase do not form a symmetrical hexamer.Rather, each subunit of each half hexamer is rotated 15ºrelative to each other. This means that each DNA bindingloop is also rotated relative to the loops in adjacent subunits(Fig. (5c)). Singleton et al. [118] have proposed that thissubunit rotation is modulated by the binding and hydrolysisof ATP. Thus, as the binding loops make sequential contactswith the DNA backbone, they rotate and push the ssDNAthrough the hexamer. In support of this model, when NTPsare added to T7 helicase crystals, not all the potential sitesare occupied, and the side chain configurations are differentsurrounding each of the three binding site types seen in theasymmetric hexamer [118].

The ring structure of SF3 and DnaB-like helicases raisesthe question of how ring helicases are loaded onto DNA toinitiate unwinding. In vitro, ring helicases require a forkedmolecule with ssDNA tails to initiate unwinding, but in vivo,unwinding begins within a duplex at replication origins.Some viruses express AAA+ proteins that use ATP to loadthe helicase onto DNA. An example is the gene 59 protein ofT4 [21]. Other helicases, like T7 helicase, appear to be ableto break open the ring to bind DNA. For example, not allcrystal structures show the T7 helicase as a ring. In the firststructure the subunits packed like a filament (Fig. (2b))[116], and such a filament could be the conformation usedby the protein to slip on an off DNA.

In the latest structure of the full length T7 gene 4 protein,the protein is seen as a seven subunit ring instead of a hex-amer (Fig. (2b)) [119]. In the T7 heptamer structure the cen-tral channel is large enough to accommodate a duplex. Asimilar large channel has been reported in SV40 Tag struc-tures (Fig. (2c)) [122, 123]. A larger channel suggests that adouble helix could pass through the protein, and such anarrangement is diagramed for Tag in Fig (5b). SV40 Tagwas recently crystallized alone, with ADP, and with ATPwith interesting results that support a fundamentally differentmodel for translocation than the rotational model describingT7 helicase action. First, all six potential SV40 Tag NTPbinding sites bind nucleotides. This evidence would supportthe more traditional Monod-Wyman-Changeux “concerted”model of ligand binding [168]. Second, upon NTP binding,changes in subunit rotation create an “iris-like” motion sothat the diameter of the channel changes and six β hairpins,suspected to bind DNA, change orientation within the chan-nel. The motion of the hairpins could push DNA through thecenter of the protein as has been proposed with T7 helicase,but all binding loops move together rather than in pairs.Thus, SF3 helicases could be fundamentally different from


DnaB-like helicases in that ATP hydrolysis occurs via a con-certed rather than a sequential mechanism. Both the sequen-tial model (Fig. (5a)) and concerted model (Fig. (5b)) predictthat DNA movements are controlled by a binding loop (orhairpin) in the center of the ring that remains connected tothe same point on the DNA backbone during a single cycleof ATP binding and hydrolysis. As diagramed in Fig. (5c), asthe hairpin moves, so does the protein relative to the DNA.

Fig. (5). The sequential and concerted models for ring helicaseaction. A. In the sequential “rotational” model for T7 helicasemovement, the various subunits hydrolyze ATP in turn to cause theprotein to rotate along one strand of DNA while displacing thecomplementary strand [118]. B. In the concerted model for SV40helicase action, all subunits simultaneously bind and hydrolyzeATP [123]. C. Both models predict that ATP hydrolysis leads to arotation of DNA binding loops that contact DNA in the center ofthe hexamer. If the binding loops remain attached to the same pointon DNA during an entire ATP hydrolysis cycle, then the proteinwill move along DNA as diagramed.

Outside the RecA-like domains helicase structures differconsiderably. The non-RecA-like domains act to conferunique properties to the helicase, which might involve addi-tional nucleic acid binding motifs, or motifs needed to inter-

act with other proteins. Helicases are usually intimately as-sociated with other proteins that travel with them along anucleic acid chain. Many helicases form a tight complexwith one or more other proteins and sometime the helicase iscovalently tethered to a separate independent functional do-main with an entirely different biological activity. For exam-ple in many RNA viruses, including HCV and SARS-CoV,the helicase is part of a protein that also possesses a proteasefunction (Fig. (2a)). No relationship between helicase andprotease has been clearly defined, but the helicase might, forexample, allow the protease to travel with cellular translationmachinery to help coordinate viral polyprotein processing. Ina similar example, helicases that coordinate semi-disconti-nuous DNA replication, such as T7 and HSV helicases, arefound coupled to a primase that synthesizes primers on thelagging strand (Fig. (2b)).

POTENTIAL ANTIVIRAL AGENTS

SF1 Helicase Inhibitors

In addition to the HSV helicase-primase inhibitors thatare being developed as antiviral drugs [1, 2], inhibitors ofother SF1 RNA helicases such as the SARS-CoV helicase,have been reported [169]. Both classes of compounds couldserve as templates to develop inhibitors of any SF1 helicase(Fig. (6)).

Current HSV therapies using nucleoside analogs such asacyclovir are quite effective but frequently lead to the evolu-tion of resistant viruses. Since chain terminating nucleosideanalogs must be activated before incorporation by HSV po-lymerase, the virus can develop resistance by not expressinga functional kinase or by evolving a polymerase with ahigher fidelity. Because the new helicase-primase inhibitorsdo not require metabolic activation, the virus has fewerpathways through which it can develop resistance. The newseries of HSV helicase-primase inhibitors, currently in pre-clinical development at Boehringer Ingelheim [1] and Bayer[2], offer a new option for treating acyclovir-resistant infec-tions, and appear more effective than current drugs fortreating latent HSV infections. Representatives of the twoseries of compounds are shown in Fig. (6a). The BoehringerIngelheim series (e.g. BILS 179 BS) was first identified byscreening inhibitors of DNA unwinding, while the Bayerseries (e.g. BAY 57-1293) was discovered using a cell-basedviral replication assay.

The aminothiazolylphenyl compound BILS 179 BS wasidentified at Boehringer Ingelheim as a HSV UL5/UL52/UL8 inhibitor using a high throughput helicase assay [1]. Alater derivative called BILS 45 BS, which is absorbed morerapidly into the blood, differs only by the lack of a methylgroup [170]. In the presence of the inhibitor, HSV primaseactivity is inhibited with an IC50 of 0.15 µM, while the DNAunwinding activity of the HSV helicase is inhibited with anIC50 of 1.3 µM. BILS 179 BS also inhibits DNA-stimulatedATP hydrolysis (IC50 ~0.43 µM) but does not affect the ATPhydrolysis in the absence of DNA (IC50 >100 µM). DNAbinds more tightly to an UL5/UL52/UL8 complex in thepresence of BILS 179 BS. Thus, BILS 179 BS likely inhibitsHSV helicase-primase activity by preventing DNA releaseduring translocation [1].


As measured by plaque formation, BILS 179 BS de-creases HSV DNA replication in BHK cells infected witheither wild type HSV strains or acyclovir-resistant mutants(EC50 ~100 nM). HSV infected mice fed BILS 179 BS, or itsderivative BILS 45 BS, have fewer and smaller cutaneousand genital lesions than mice fed a placebo. HSV viral titersalso show a dose dependent reduction following administra-tion of the BILS compounds [1, 170]. Mice infected withacyclovir-resistant HSV also respond to oral administrationof BILS 45 BS [170]. Thus, these aminothiazolphenyl-basedmolecules are a promising class of compounds for the treat-ment of nucleoside-resistant HSV disease in humans.

In order to determine which protein component of theHSV helicase-primase complex is targeted by the BILScompounds, HSV was incubated with cells in the presence ofBILS 22 BS (an analog of BILS 45 BS). Three mutant vi-ruses were found to have mutations in their UL5 gene [171].One had Gly352 changed to Val, one had Gly352 changed toCys, and the third had Lys356 changed to Asn, indicatingthat the UL5 subunit of the HSV helicase-primase is likelythe target of the BILS inhibitors. Gly352 and Lys356 arenear SF1 conserved motif IV, with Gly352 only 2 aminoacids downstream from SF1 motif IV. DNA-stimulated ATPhydrolysis catalyzed by purified UL5/UL52/UL8 complexeswith these mutations is less sensitive to the inhibitors. BothGly352 and Lys356 are completely conserved in all humanherpesviruses [172], but the mutant viruses are viable andstill cause disease [171]. The frequency with which BILSresistant mutants arise is significantly less than that seen foracyclovir-resistant mutants [171].

Unlike the Boehringer Ingelheim compounds, whichwere identified using high throughput enzyme assays, thedevelopment of BAY 57-1293 (Fig. (6a)), was initiated us-ing a cell-based high throughput assay [2]. In this assay,which measures HSV induced cytopathogenicity, BAY 57-1293 (IC50 = 20 nM) is 50-fold more potent than acyclovir(IC50 = 1 µM). In addition, BAY 57-1293 inhibits acyclovir-resistant HSV-induced cell lysis with the same potency aswild type HSV-induced cell damage.

The first indication that the HSV helicase-primase wasthe target for the BAY compounds came when resistant mu-tants were sequenced. Seven amino acid substitutions wereidentified, six of them clustered near the N-terminal of theUL5 gene and one mutant had a substitution in the UL52gene [2]. As with the BILS compounds, BAY 57-1293-resistant mutants contain mutations (G352V, M355T,K356Q) near SF1 motif IV. Remarkably, two of these are atthe same positions as the BILS-resistant mutants. All threepositions are completely conserved in all human herpesvi-ruses. BAY 57-1293 resistance occurs about 10 times lessfrequently than acyclovir resistance [2].

Biochemical assays using purified HSV helicase-primasealso show that BAY 57-1293 inhibits the DNA-stimulatedATPase activity in a dose-dependent manner with an IC50 of~30 nM. Orally administered BAY 57-1293 has potent anti-viral efficiency in mice, rats and guinea pigs infected withHSV-1 or HSV-2. BAY 57-1293 is 40 times more effectivethan acyclovir in treating mice infected with cutaneous HSV-1, suppresses intravaginal lesions in HSV-2 infected guineapigs, and exhibits a potent antiviral effect in rats infected

with HSV-1 [2, 173]. In addition, BAY 57-1293 reduceshealing time and is effective even when treatment is delayed,as opposed to the nucleoside analogs which require earlyapplication upon HSV infection [2].

A cell-based assay similar to that used to discover theBAY series of HSV helicase inhibitors was also used to findcompounds that inhibit cell death induced by SARS-CoV.Kao et al. found 104 compounds that protect cells fromSARS-CoV induced cytopathic effects [169]. Of these, sevenwere potent inhibitors of recombinant purified SARS-CoVhelicase-catalyzed DNA unwinding and ATP hydrolysis.The structure of one, which also suppresses viral plaqueformation in SARS-infected tissue culture cells with an EC50

of 6 µM, is shown in Fig. (6b)). HE602 inhibits nucleic acid-stimulated ATP hydrolysis by SARS-CoV helicase with anIC50 of 6.9 µM, but has no effect on its basal rate of ATPhydrolysis.

Fig. (6). SF1 helicase Inhibitors. A. HSV helicase-primase inhibi-tors [1, 173]. B. SARS-CoV helicase inhibitor [169].


Numerous compounds have been reported that inhibit theHCV, WNV, and JEV helicases. These inhibitors can beclassified into three basic groups: (1) nucleoside analogs andrelated small molecules that modulate ATP hydrolysis and/ornucleic acid unwinding, (2) nucleic acids and modified de-rivatives that specifically target the RNA-binding site, and(3) antibodies.

Small molecule inhibitors of HCV helicase and relatedhelicases were recently reviewed by Borowski and his col-leagues in references [174] and [175]. One of the most

N

O

N

HN

O OCl

N

O

CH3

S

NS

O

O

NH2

N

CH3

N

S

HN

H2N NO

N

O

CH3

A

B

HE602

BAY 57-1293

BILS 179 BS


promising of these early inhibitors is 1-(2'-O-methyl-beta-D-ribofuranosyl)imidazo[4, 5-d]pyridazine-4, 7(5H, 6H)-dione(HMC-HO4) (Fig. (7)), which inhibits WNV helicase andproduces a potent antiviral effect (IC50 = 25-30 µM) inWNV-infected cells [176]. Interestingly, a lower concentra-tion of HMC-HO4 is needed to inhibit viral RNA synthesisin cells than is needed to inhibit DNA (and presumablyRNA) unwinding by purified WNV helicase. At the lowerHMC-HO4 concentrations that produce an antiviral effect(i.e. below 20 µM), DNA unwinding rates catalyzed by thepurified enzyme are actually stimulated. At higher HMC-HO4 concentrations, unwinding is inhibited as is viral RNAsynthesis. At all concentrations of HMC-HO4, ATP hydroly-sis is stimulated, suggesting that the inhibitor somehow un-couples the ATPase and helicase functions [176].

Another explanation for the effect of HMC-HO4 onWNV helicase is that the compound binds to a second NTPbinding site other than the one made by the cleft between theRecA-like domains. Some evidence for such a site has comefrom studies of the related HCV helicase. The HCV helicasehydrolyzes all eight canonical nucleoside triphosphates [74,177, 178], but some reports suggest that not all NTPs fuelequal rates of unwinding. For example, Locatelli et al. foundthat only some NTPs fuel unwinding with an efficiencycomparable to that seen with ATP whereas other (d)NTPs,particularly dATP, were found to be poor substrates and po-tent inhibitors of unwinding [179]. Although other studieshave not confirmed their observations [178, 180], Locatelliet al.’s report [179] suggests that NTPs could bind at twosites, one used to fuel helicase movement, and a second thatallosterically modulates the enzyme’s activity.

Other evidence for a second site arises from product in-hibition studies. The products of ATP hydrolysis, ADP andPi, do not inhibit the HCV NS3 helicase. However, in thepresence of NaF and PolyU RNA, ADP inhibits ATP hy-drolysis with about two moles of ADP binding per proteinmonomer [181]. When beryllium fluoride is added to thereaction, ADP inhibits ATP hydrolysis more potently with aKi of about 8.5 µM [136, 146]. In structures of other en-zymes, BeF3 is often found to occupy a position analogous tothe γ phosphate of ATP when bound with ADP and Mg2+

[182]. Thus, ADP(BeF3) is likely a non-hydrolyzable ATPanalog, but unlike other non-hydrolyzable ATP analogs suchas ADPNP [177, 183], ADP(BeF3) binds HCV helicasetightly. ADP(BeF3) binds with a stoichiometry of one nu-cleotide per protein monomer and mimics ATP in that it re-duces the affinity of the helicase for nucleic acids in thesame manner as ATP [136, 146, 149, 184]. ADP(BeF3) alsobinds other P-loop ATPases as a ground state ATP analog[182], explaining the cellular toxicity of beryllium fluoride.ADP fluoride complexes, on the other hand, likely do notresemble the substrate as closely, so they may bind both ac-tive and allosteric sites. Other evidence of a second ATPbinding site was presented in a second report by Locatelli etal. that demonstrated that nucleotides bind HCV helicasecooperatively [185].

Thus, it is possible that the interaction between HCVhelicase and nucleotides could be effectively exploited fordrug design by finding compounds that bind the second pu-tative binding site. Such compounds would not competitively

inhibit ATP hydrolysis, but would modulate ATP hydrolysisand/or unwinding by binding an allosteric site. Several suchcompounds recently have been reported [186-188]. Repre-sentatives are shown along with HMC-HO4 in Fig. (7).

A series of halogenated benzimidazoles and benzotria-zoles, which were originally reported as HCV inhibitors inpatents by Viropharma Inc., were recently examined for theirability to inhibit HCV, WNV, and JEV helicases [186]. Di-chloro(ribofuranosyl)benzotriazole (DRBT) and di-chloro(ribofuranosyl)benzimidazole (DRB) are nucleosideanalogs, while tetrachlorobenzotriazole (TCBT) and tetrab-romobenzotriazole (TBBT) are base analogs. DRBT, DRB,TCBT, and TBBT inhibit WNV helicase catalyzed RNAunwinding with IC50’s in the low micromolar range, but onlyTBBT (Fig. (7)) inhibits RNA unwinding by HCV NS3 heli-case (IC50 ~60 µM). Interestingly, none of these inhibit theJEV helicase, even though it is closely related to WNV heli-case. Under the conditions used, none of the compoundsinhibited helicase-catalyzed ATP hydrolysis, suggesting thatthey may bind to another NTP-binding site or interact withthe nucleic acid substrates [186].

Fig. (7). SF2 helicase inhibitors. A. imidazo[4, 5-d]pyridazine nu-cleoside inhibitor of WNV helicase [176]. B. Halogenated benzotri-azole inhibitor of WNV helicase and HCV helicase [186]. C. Ring-expanded nucleoside inhibitor of WNV helicase [187].

Several ring-expanded “fat” nucleosides (RENs), whichare currently being investigated as antiviral and anticanceragents (see ref. [189] for a review), have also recently beenexamined as potential inhibitors of the WNV, JEV, and HCVhelicases [187, 188]. At least five different RENs inhibitWNV, one inhibits JEV, but none inhibit HCV helicasecatalyzed DNA or RNA unwinding. Many of the RENs, suchas compound 17 (Fig. (7)), are more potent inhibitors of ei-ther WNV helicase-catalyzed RNA or DNA unwinding, sug-gesting that they exert their effects by binding the major orminor groove of the duplex nucleic acid. Only one REN wasfound to inhibit both DNA and RNA unwinding by WNV

N

N

OHO

HO

NH

NH

O

O

OMe

N

NH

N

Br

Br

Br

Br

O

HO

OH

OH

N

N

HN

O

N

O

NH

(CH2)13CH3

HMC-HO4

REN Compound 17

TBBT


helicase. Again, as seen with HMC-HO4 and TBBT, inhibi-tion of the unwinding activity by RENs does not correlatewith a reduction in ATP hydrolysis (unless REN triphos-phates are used in the assays). To see any inhibition of ATPhydrolysis ATP must be used at a concentration well belowits apparent Km, and under such conditions only two RENsshow any inhibition. At high concentrations (> 0.5 mM), allof the RENs enhanced ATP hydrolysis. None of the RENswere reportedly tested in cell-based assays for a true antiviraleffect although antiviral effects for RENs have been reportedfor other viruses [189].

Another method to inhibit a helicase is to directly targetthe nucleic acid-binding site rather than the NTP-bindingsite. The HCV NS3 helicase nucleic acid-binding site is lo-cated in the cleft that separates domains 1 and 2 from do-main 3 (Fig. (2)), and represents a target site for antiviraldrugs that compete with HCV helicase for its viral RNA sub-strate.

SELEX (systematic evolution of ligands by exponentialamplification) has been used to find RNA sequences thattightly bind HCV helicase. Such sequences, called “aptam-ers, ” are oligonucleotides that exhibit molecular recognition,and could conceivably be directly used as antivirals, or couldprovide templates for the synthesis of antiviral drugs. In theSELEX procedure (see [190] for a review), an RNA libraryis screened for sequences that bind a macromolecule using abinding assay. Only those sequences that bind tightly areamplified to create a new library, and the selection process isrepeated with the new library. Nishikawa’s group first usedSELEX to uncover a series of aptamers with specific affinityto the NS3 protease region. The protease-binding aptamersall share the conserved sequence GA(A/U)UGGGAC [191].These aptamers bind to the NS3 protease with high affinity(Kd ~10 nM) and are effective at inhibiting the HCV NS3protease [191, 192]. The conserved part of the RNA aptam-ers likely interacts with a cluster of basic amino acid (resi-dues Arg130, Arg161 and Lys165), which is located outsideof the serine protease catalytic pocket in the region that linksthe protease to the helicase [193]. When a 14-mer uridine tailis added to one of the most effective HCV protease-bindingRNA aptamers, the new, longer aptamer interacts with boththe protease and helicase domains of the full-length NS3protein [194]. This dual-function RNA aptamer binds to theHCV NS3 helicase domain with high affinity (Kd ~4 nM)[194] and also inhibits the NS3 helicase activity with anEC50 of ~500 nM.

An aptamer that bind HCV helicase have also been di-rectly selected using the helicase domain of the NS3 proteinas the bait in the SELEX procedure [195]. This aptamer(called SE RNA) folds to form four stem loops with GCpairs that are similar to the stem loop located at the 3'-terminal of the negative strand HCV RNA. Since previousstudies have shown that HCV helicase specifically binds tothis region of the HCV genome [196], the SE aptamer mightcompete for this binding site. SE RNA binds the HCV heli-case with a high affinity (Kd ~990 pM), efficiently competeswith poly(U), stimulates ATP hydrolysis, but severely inhib-its RNA unwinding (IC50 ~12.5 nM). Most importantly, hu-man liver cells (Huh 7) infected with HCV replicons showless HCV RNA synthesis in the presence of SE RNA.

Although directly using RNA as an antiviral drug will bechallenging because of its cellular instability, the informationderived from aptamer studies could be used to make morestable derivatives. For example, the same aptamer sequencescould be used to generate peptide nucleic acids (PNA),which are modified nucleic acids composed of natural purineand pyrimidine bases linked together via an uncharged 2-aminoethyl-glycine backbone. Both HCV helicase [197] andHSV UL9 helicase [198] have been shown to be inhibited byPNA. PNA most likely inhibits helicase activity by forminga duplex that is more stable than those seen with naturalDNA or RNA: The PNA-DNA substrates are unwound moreslowly (up to 80-fold) when compared to DNA duplexescontaining the same sequence, and only minimal PNA-RNAduplex is unwound by the HCV NS3 helicase [197].

Delivery of RNA aptamers to infected cells also presentsa challenging problem. One logical way to introduce an ap-tamer is to introduce a gene, encoding its transcription, intocells using an appropriate vector. Using such a gene therapyapproach, the aptamer would be expressed inside cells (forreview see [199]).

Gene therapy can also be used to introduce protein mole-cules into cells. The most practical proteins to introduce areantibodies, which could conceivably act inside a cell to in-hibit any enzyme. Antibodies are the first line of defense thatthe body has against viruses, and have long been recognizedas powerful antiviral agents. Natural biological diversityallows for the selection of antibodies that tightly bind virtu-ally any epitope of any enzyme. Once antibodies are isolatedwith a high avidity for a desired antigen, a gene encodingthat antibody can be cloned and introduced into infectedcells. This process, called “cellular immunization, ” has beenused successfully with HIV [200], and such an approach hasbeen used to target the HCV helicase [201-203].

The first anti-HCV helicase antibodies designed for in-tracellular immunization were single chain fragment (ScF)antibodies. A ScF is composed of the immunoglobulin vari-able domains, of both the heavy and light chains, connectedby a polypeptide linker, and can be constructed using PCR.Several high affinity ScFs that specifically interact withHCV NS3 helicase have been cloned [201, 204] and shownto inhibit HCV helicase catalyzed DNA unwinding [202,204]. One ScF has been expressed in HCV infected hepato-cytes and shown to reduce HCV RNA synthesis [204]. Thisparticular ScF consists of the variable regions of the humanmonoclonal antibody CM3.B6, which recognizes an epitopethat spans conserved SF2 motifs IV and V of the NS3 heli-case [205]. Immunoblots show that an intracellularly ex-pressed ScF derived from CM3.B6 interacts with the HCVNS3 protein, and HCV RNA synthesis within primary he-patocytes infected with HCV is reduced by 10-fold when thecells contain an adenoviral vector carrying the ScF gene[204].

Another approach to achieve intracellular immunizationuses an antibody fragment (Fab). A Fab is larger and usuallymore stable than a ScF because in addition to regions presentin ScFs, it contains the complete light chain and the firstconstant domain of the heavy chain. Prabhu et al. has iso-lated a human Fab, termed HFab-aNS3, with effective HCVantiviral activity [203]. Derived from a HCV-infected pa-


tient, HFab-aNS3 binds HCV helicase and recognizes anepitope that spans motifs I to V of the protein. HCV NS3helicase pre-incubated with the HFab-aNS3 fails to unwindDNA duplexes. Intracellular expression of HFab-aNS3within replicon-transfected Huh 7 cells suppresses NS3 pro-tein expression and significantly inhibits positive strandHCV RNA synthesis of both subgenomic and full-lengthHCV replicons [203].


Among the viral helicases from SF3, the E1 helicasefrom HPV is currently the target of antiviral molecules forthe treatment of HPV infection. Through screening a com-pound library, two classes of small molecules have beenidentified and are being developed at Boehringer Ingelheim.The first group involves agents that disrupt the interactionbetween the HPV E1 helicase and its co-activator HPV E2protein. Because HPV DNA replication is dependent onproper assembly of E1 proteins at the HPV origin of replica-tion, which requires initial complex formation between E1and E2, compounds that interrupt this interaction can poten-tially serve as promising antiviral drugs. The second groupconsists of inhibitors that specifically target HPV E1 helicaseDNA unwinding.

Using a high throughput assay that analyzes the bindingbetween E1 and E2 proteins, a class of small molecules(HPV E1-E2 inhibitors 1 to 3) that disrupt the E1-E2 com-plex has been identified [206]. The most potent, inhibitor 3,is shown in Fig. (8). These inhibitors do not significantlyaffect E1 helicase activity, nor do they block DNA bindingto either E1 or E2. These molecules only target the E2 pro-tein [206]. These molecules bind to the region of E2 thatinteracts with E1 and prevent it from associating with the E1helicase. In Chinese hamster ovary cells transfected withplasmids expressing HPV E1 and E2 together with a plasmidcontaining the HPV DNA origin of replication, HPV DNAreplication is inhibited in a dose-dependent manner in thepresence of inhibitor 3 (EC50 ~1 µM). Currently, this class ofinhibitors is effective against low risk HPV (types 6 and 11),but unfortunately not the high risk types.

Inhibitors that are specific for the E1 helicase are alsobeing developed [207]. High throughput screening of a li-brary of compounds has identified a lead molecule bearing abiphenyl-4-sulfonylacetic acid moiety, which specificallyinhibits the ATPase activity of the HPV-6 E1 helicase.Among the derivatives of this parent molecule, compound 6gdemonstrates potent inhibition against the ATPase activity ofthe E1 protein (IC50 ~4.3 nM) (Fig. (8b)). However, it haslimited in vivo application because of poor cell permeabilityand is relatively unstable. These compounds are currentlybeing improved in their chemical structures so they can besuitable for further cell-based evaluation [207].

CONCLUSION

All viral helicases can be classified as DnaB-like, SF1,SF2, or SF3 helicases. All helicases share a common mecha-nism of ATP hydrolysis to produce conformational changesallowing the protein to move on DNA (or RNA). Althoughprecisely how chemical energy is converted to mechanicalmovements is unknown at this time, there is solid evidence

that DnaB-like and SF3 helicases encircle DNA as rings.ATP binding to ring helicases causes the movement of loopsimplicated in DNA binding that could push the protein eitherby moving in a sequential or concerted manner. The activesites appear to work in concert in SF3 helicases, and sequen-tially in DnaB-like helicases. There is little evidence thatSF1 and SF2 helicases function as rings. Rather, most evi-dence points to an inchworm mechanism in which the func-tional unit is a monomer. In non-ring helicases, ATP binds atan interface between two RecA-like domains in a cleft thatcloses upon ATP binding. The main difference between SF1and SF2 proteins appears to be that the SF1 helicases moveby interacting with the nucleic acid bases whereas SF2 pro-teins interact primarily with the phosphodiester backbone.

Fig. (8). SF3 helicase inhibitors. A. HPV E1-E2 complex inhibitor[206]. B. HPV E1 inhibitor [207].

Potent inhibitors have been isolated for members of eachsuperfamily of viral helicases. The best characterized arethose that inhibit the SF1 HSV helicase-primase complex.The anti-HSV compounds function by binding near con-served SF1 motif IV, preventing dissociation of the proteinfrom viral DNA, slowing helicase movement, and conse-quently the activity of the associated primase necessary forviral genome synthesis. The inhibitors of HCV helicase andrelated SF2 proteins that have been isolated are small mole-cules, RNA aptamers, or antibodies. Developing the HCVinhibitors will likely be slow but several helicase inhibitorshave shown clear efficacy in treating West Nile virus infec-tion. SF3 helicase inhibitors targeting HPV also have beenisolated and work by either directly reducing HPV helicase-catalyzed ATP hydrolysis or its interaction with a proteinthat loads the helicase on replication origins.

Clearly, helicase inhibitors are in an early stage of devel-opment and much more work will need to be done until thesecompounds will be regularly used as antiviral agents. Nev-ertheless, the critical first steps have been taken, and if newinsights into helicase mechanisms continue at the current

S

HOOC

OO

HN

O

O

O

O

Br

OO- O

N

Br

N

OE1-E2 Inhibitor 3

Compound 6g

A

B


pace, helicase inhibitors could one day be prescribed ascommonly as protease and polymerase inhibitors.

ACKNOWLEDGEMENT

This work was supported by National Institutes of Healthgrant AI052395.

ABBREVIATIONS

(+)ssRNA = Positive sense single-stranded RNA

(-)ssRNA = Negative sense single-stranded RNA

AAA+ = ATPases associated with various cellularactivities

AAV = Adeno-associated virus

ADPNP = 5'-adenylyl-β−γ-imidodiphosphate

BVDV = Bovine viral diarrhea virus

CoV = Coronavirus

ds = Double-stranded

DFV = Dengue fever virus

DRB = Dichloro(ribofuranosyl)benzimidazole

DRBT = Dichloro(ribofuranosyl)benzotriazole

IRES = Internal ribosome entry site

HCV = Hepatitis C virus

HHV = Human herpesviruses

HIV = Human immunodeficiency virus

HSV = Herpes simplex virus

HPV = Human papillomavirus

JEV = Japanese encephalitis virus

NMR = Nuclear magnetic resonance

NPH = NTP phosphohydrolase

NS = Nonstructural

nsp = Nonstructural protein

NTP = Nucleoside triphosphate

ORF = Open reading frame

PNA = Peptide nucleic acids

pp = Polyprotein

RTPase = RNA 5′-triphosphatase activity

RENs = Ring-expanded “fat” nucleosides

SARS = Severe acute respiratory syndrome

tag = Small T (tumor) antigen

Tag = Large T (tumor) antigen

SELEX = Systematic evolution of ligands by exponentialamplification

SF = Superfamily

SV40 = Simian virus 40

TBBT = Tetrabromobenzotriazole

TCBT = Tetrachlorobenzotriazole

ss = Single-stranded

VV = Vaccinia virus

YFV = Yellow fever virus

WNV = West Nile virus

REFERENCES

References 237-239 are related articles recently published inCurrent Pharmaceutical Design.

[1] Crute JJ, Grygon CA, Hargrave KD, Simoneau B, Faucher AM,Bolger G, et al. Herpes simplex virus helicase-primase inhibitorsare active in animal models of human disease. Nat Med 2002; 8:386-91.

[2] Kleymann G, Fischer R, Betz UA, Hendrix M, Bender W,Schneider U, et al. New helicase-primase inhibitors as drug candi-dates for the treatment of herpes simplex disease. Nat Med 2002; 8:392-8.

[3] Krell H, Durwald H, Hoffmann-Berling H. A DNA-unwindingenzyme induced in bacteriophage-T4-infected Escherichia colicells. Eur J Biochem 1979; 93: 387-95.

[4] Tjian R, Robbins A. Enzymatic activities associated with a purifiedsimian virus 40 T antigen-related protein. Proc Natl Acad Sci USA1979; 76: 610-4.

[5] Matson SW, Tabor S, Richardson CC. The gene 4 protein of bacte-riophage T7. Characterization of helicase activity. J Biol Chem1983; 258: 14017-24.

[6] Lain S, Riechmann JL, Garcia JA. RNA helicase: a novel activityassociated with a protein encoded by a positive strand RNA virus.Nucleic Acids Res 1990; 18: 7003-6.

[7] Yedavalli VS, Neuveut C, Chi YH, Kleiman L, Jeang KT. Re-quirement of DDX3 DEAD box RNA helicase for HIV-1 Rev-RREexport function. Cell 2004; 119: 381-92.

[8] Frick DN. Helicases as antiviral drug targets. Drug News Perspect2003; 16: 355-62.

[9] Delagoutte E, von Hippel PH. Helicase mechanisms and the cou-pling of helicases within macromolecular machines. Part I: Struc-tures and properties of isolated helicases. Q Rev Biophys 2002; 35:431-78.

[10] Delagoutte E, von Hippel PH. Helicase mechanisms and the cou-pling of helicases within macromolecular machines. Part II: Inte-gration of helicases into cellular processes. Q Rev Biophys 2003;36: 1-69.

[11] Gorbalenya AE, Koonin EV. Helicases: amino acid sequence com-parisons and structure-function relationships. Curr Opin Struct Biol1993; 3: 419-429.

[12] Gorbalenya AE, Koonin EV, Donchenko AP, Blinov VM. Tworelated superfamilies of putative helicases involved in replication,recombination, repair and expression of DNA and RNA genomes.Nucleic Acids Res 1989; 17: 4713-30.

[13] Gorbalenya AE, Koonin EV, Wolf YI. A new superfamily of puta-tive NTP-binding domains encoded by genomes of small DNA andRNA viruses. FEBS Lett 1990; 262: 145-8.

[14] Ilyina TV, Gorbalenya AE, Koonin EV. Organization and evolu-tion of bacterial and bacteriophage primase-helicase systems. J MolEvol 1992; 34: 351-7.

[15] Richardson JP. Loading Rho to terminate transcription. Cell 2003;114: 157-9.

[16] Hall MC, Matson SW. Helicase motifs: the engine that powersDNA unwinding. Mol Microbiol 1999; 34: 867-77.

[17] Patel SS, Picha KM. Structure and function of hexameric helicases.Annu Rev Biochem 2000; 69: 651-97.

[18] Frick DN, Richardson CC. DNA primases. Annu Rev Biochem2001; 70: 39-80.

[19] Miller ES, Kutter E, Mosig G, Arisaka F, Kunisawa T, Ruger W.Bacteriophage T4 genome. Microbiol Mol Biol Rev 2003; 67: 86-156, table of contents.

[20] Dong F, Gogol EP, von Hippel PH. The phage T4-coded DNAreplication helicase (gp41) forms a hexamer upon activation by nu-cleoside triphosphate. J Biol Chem 1995; 270: 7462-73.


[21] Morrical SW, Hempstead K, Morrical MD. The gene 59 protein ofbacteriophage T4 modulates the intrinsic and single-stranded DNA-stimulated ATPase activities of gene 41 protein, the T4 replicativeDNA helicase. J Biol Chem 1994; 269: 33069-81.

[22] Dong F, von Hippel PH. The ATP-activated hexameric helicase ofbacteriophage T4 (gp41) forms a stable primosome with a singlesubunit of T4-coded primase (gp61). J Biol Chem 1996; 271:19625-31.

[23] Jongeneel CV, Formosa T, Alberts BM. Purification and charac-terization of the bacteriophage T4 dda protein. A DNA helicasethat associates with the viral helix-destabilizing protein. J BiolChem 1984; 259: 12925-32.

[24] Sickmier EA, Kreuzer KN, White SW. The crystal structure of theUvsW helicase from bacteriophage T4. Structure (Camb) 2004; 12:583-92.

[25] Frick DN, Baradaran K, Richardson CC. An N-terminal fragmentof the gene 4 helicase/primase of bacteriophage T7 retains primaseactivity in the absence of helicase activity. Proc Natl Acad Sci USA1998; 95: 7957-62.

[26] Guo S, Tabor S, Richardson CC. The linker region between thehelicase and primase domains of the bacteriophage T7 gene 4 pro-tein is critical for hexamer formation. J Biol Chem 1999; 274:30303-9.

[27] Kusakabe T, Hine AV, Hyberts SG, Richardson CC. The Cys4 zincfinger of bacteriophage T7 primase in sequence-specific single-stranded DNA recognition. Proc Natl Acad Sci USA 1999; 96:4295-300.

[28] Frick DN, Richardson CC. Interaction of bacteriophage T7 gene 4primase with its template recognition site. J Biol Chem 1999; 274:35889-98.

[29] Bernstein JA, Richardson CC. A 7-kDa region of the bacteriophageT7 gene 4 protein is required for primase but not for helicase activ-ity. Proc Natl Acad Sci USA 1988; 85: 396-400.

[30] Boehmer PE, Lehman IR. Herpes simplex virus DNA replication.Annu Rev Biochem 1997; 66: 347-84.

[31] Marintcheva B, Weller SK. A tale of two HSV-1 helicases: roles ofphage and animal virus helicases in DNA replication and recombi-nation. Prog Nucleic Acid Res Mol Biol 2001; 70: 77-118.

[32] Crute JJ, Mocarski ES, Lehman IR. A DNA helicase induced byherpes simplex virus type 1. Nucleic Acids Res 1988; 16: 6585-96.

[33] Dodson MS, Crute JJ, Bruckner RC, Lehman IR. Overexpressionand assembly of the herpes simplex virus type 1 helicase-primasein insect cells. J Biol Chem 1989; 264: 20835-8.

[34] Crute JJ, Lehman IR. Herpes simplex virus-1 helicase-primase.Physical and catalytic properties. J Biol Chem 1991; 266: 4484-8.

[35] Zhu LA, Weller SK. The six conserved helicase motifs of the UL5gene product, a component of the herpes simplex virus type 1 heli-case-primase, are essential for its function. J Virol 1992; 66: 469-79.

[36] Graves-Woodward KL, Gottlieb J, Challberg MD, Weller SK.Biochemical analyses of mutations in the HSV-1 helicase-primasethat alter ATP hydrolysis, DNA unwinding, and coupling betweenhydrolysis and unwinding. J Biol Chem 1997; 272: 4623-30.

[37] Dodson MS, Lehman IR. Association of DNA helicase and primaseactivities with a subassembly of the herpes simplex virus 1 heli-case-primase composed of the UL5 and UL52 gene products. ProcNatl Acad Sci USA 1991; 88: 1105-9.

[38] Biswas N, Weller SK. A mutation in the C-terminal putative Zn2+

finger motif of UL52 severely affects the biochemical activities ofthe HSV-1 helicase- primase subcomplex. J Biol Chem 1999; 274:8068-76.

[39] Biswas N, Weller SK. The UL5 and UL52 subunits of the herpessimplex virus type 1 helicase-primase subcomplex exhibit a com-plex interdependence for DNA binding. J Biol Chem 2001; 276:17610-9.

[40] Fierer DS, Challberg MD. Purification and characterization ofUL9, the herpes simplex virus type 1 origin-binding protein. J Virol1992; 66: 3986-95.

[41] Boehmer PE, Dodson MS, Lehman IR. The herpes simplex virustype-1 origin binding protein. DNA helicase activity. J Biol Chem1993; 268: 1220-5.

[42] He X, Lehman IR. Unwinding of a herpes simplex virus type 1origin of replication (Ori(S)) by a complex of the viral originbinding protein and the single-stranded DNA binding protein. J Vi-rol 2000; 74: 5726-8.

[43] Makhov AM, Lee SS, Lehman IR, Griffith JD. Origin-specificunwinding of herpes simplex virus 1 DNA by the viral UL9 andICP8 proteins: Visualization of a specific preunwinding complex.Proc Natl Acad Sci USA 2003; 24: 24.

[44] Nanduri B, Byrd AK, Eoff RL, Tackett AJ, Raney KD. Pre-steady-state DNA unwinding by bacteriophage T4 Dda helicase reveals amonomeric molecular motor. Proc Natl Acad Sci USA 2002; 99:14722-7.

[45] Morris PD, Raney KD. DNA helicases displace streptavidin frombiotin-labeled oligonucleotides. Biochemistry 1999; 38: 5164-71.

[46] Bedinger P, Hochstrasser M, Jongeneel CV, Alberts BM. Proper-ties of the T4 bacteriophage DNA replication apparatus: the T4 ddaDNA helicase is required to pass a bound RNA polymerase mole-cule. Cell 1983; 34: 115-23.

[47] Byrd AK, Raney KD. Protein displacement by an assembly ofhelicase molecules aligned along single-stranded DNA. Nat StructMol Biol 2004; 11: 531-8.

[48] Ziebuhr J. Molecular biology of severe acute respiratory syndromecoronavirus. Curr Opin Microbiol 2004; 7: 412-9.

[49] Marra MA, Jones SJ, Astell CR, Holt RA, Brooks-Wilson A, But-terfield YS, et al. The Genome sequence of the SARS-associatedcoronavirus. Science 2003; 300: 1399-404.

[50] Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R,Icenogle JP , et al. Characterization of a novel coronavirus associ-ated with severe acute respiratory syndrome. Science 2003; 300:1394-9.

[51] Thiel V, Ivanov KA, Putics A, Hertzig T, Schelle B, Bayer S, et al.Mechanisms and enzymes involved in SARS coronavirus genomeexpression. J Gen Virol 2003; 84: 2305-15.

[52] Ivanov KA, Ziebuhr J. Human coronavirus 229E nonstructuralprotein 13: characterization of duplex-unwinding, nucleosidetriphosphatase, and RNA 5'-triphosphatase activities. J Virol 2004;78: 7833-8.

[53] Tanner JA, Watt RM, Chai YB, Lu LY, Lin MC, Peiris JS, et al.The severe acute respiratory syndrome (SARS) coronavirusNTPase/helicase belongs to a distinct class of 5' to 3' viral heli-cases. J Biol Chem 2003; 278: 39578-82.

[54] Ivanov KA, Thiel V, Dobbe JC, van der Meer Y, Snijder EJ, Zie-buhr J. Multiple enzymatic activities associated with severe acuterespiratory syndrome coronavirus helicase. J Virol 2004; 78: 5619-32.

[55] Li YI, Shih TW, Hsu YH, Han YT, Huang YL, Meng M. The heli-case-like domain of plant potexvirus replicase participates in for-mation of RNA 5' cap structure by exhibiting RNA 5'-triphosphatase activity. J Virol 2001; 75: 12114-20.

[56] Seybert A, Hegyi A, Siddell SG, Ziebuhr J. The human coronavirus229E superfamily 1 helicase has RNA and DNA duplex-unwindingactivities with 5'-to-3' polarity. RNA 2000; 6: 1056-68.

[57] Seybert A, van Dinten LC, Snijder EJ, Ziebuhr J. Biochemicalcharacterization of the equine arteritis virus helicase suggests aclose functional relationship between arterivirus and coronavirushelicases. J Virol 2000; 74: 9586-93.

[58] van Dinten LC, van Tol H, Gorbalenya AE, Snijder EJ. The pre-dicted metal-binding region of the arterivirus helicase protein is in-volved in subgenomic mRNA synthesis, genome replication, andvirion biogenesis. J Virol 2000; 74: 5213-23.

[59] Seybert A, Posthuma CC, van Dinten LC, Snijder EJ, GorbalenyaAE, Ziebuhr J. A complex zinc finger controls the enzymatic ac-tivities of nidovirus helicases. J Virol 2005; 79: 696-704.

[60] Bork P, Koonin EV. An expanding family of helicases within the'DEAD/H' superfamily. Nucleic Acids Res 1993; 21: 751-2.

[61] Tanner NK, Linder P. DExD/H box RNA helicases: from genericmotors to specific dissociation functions. Mol Cell 2001; 8: 251-62.

[62] Silverman E, Edwalds-Gilbert G, Lin RJ. DExD/H-box proteinsand their partners: helping RNA helicases unwind. Gene 2003; 312:1-16.

[63] Suzich JA, Tamura JK, Palmer-Hill F, Warrener P, Grakoui A,Rice CM, et al. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the re-lated pestivirus and flavivirus enzymes. J Virol 1993; 67: 6152-8.

[64] Kanai A, Tanabe K, Kohara M. Poly(U) binding activity of hepati-tis C virus NS3 protein, a putative RNA helicase. FEBS Lett 1995;376: 221-4.

[65] Kim DW, Gwack Y, Han JH, Choe J. C-terminal domain of thehepatitis C virus NS3 protein contains an RNA helicase activity.Biochem Biophys Res Commun 1995; 215: 160-6.


[66] Gwack Y, Kim DW, Han JH, Choe J. Characterization of RNAbinding activity and RNA helicase activity of the hepatitis C virusNS3 protein. Biochem Biophys Res Commun 1996; 225: 654-9.

[67] Tai CL, Chi WK, Chen DS, Hwang LH. The helicase activity asso-ciated with hepatitis C virus nonstructural protein 3 (NS3). J Virol1996; 70: 8477-84.

[68] Gwack Y, Kim DW, Han JH, Choe J. DNA helicase activity of thehepatitis C virus nonstructural protein 3. Eur J Biochem 1997; 250:47-54.

[69] Warrener P, Tamura JK, Collett MS. RNA-stimulated NTPaseactivity associated with yellow fever virus NS3 protein expressedin bacteria. J Virol 1993; 67: 989-96.

[70] Li H, Clum S, You S, Ebner KE, Padmanabhan R. The serine pro-tease and RNA-stimulated nucleoside triphosphatase and RNAhelicase functional domains of dengue virus type 2 NS3 convergewithin a region of 20 amino acids. J Virol 1999; 73: 3108-16.

[71] Benarroch D, Selisko B, Locatelli GA, Maga G, Romette JL, Ca-nard B. The RNA helicase, nucleotide 5'-triphosphatase, and RNA5'-triphosphatase activities of Dengue virus protein NS3 are Mg2+-dependent and require a functional Walker B motif in the helicasecatalytic core. Virology 2004; 328: 208-18.

[72] Borowski P, Niebuhr A, Mueller O, Bretner M, Felczak K,Kulikowski T, et al. Purification and characterization of West Nilevirus nucleoside triphosphatase (NTPase)/helicase: evidence fordissociation of the NTPase and helicase activities of the enzyme. JVirol 2001; 75: 3220-9.

[73] Utama A, Shimizu H, Morikawa S, Hasebe F, Morita K, IgarashiA, et al. Identification and characterization of the RNA helicaseactivity of Japanese encephalitis virus NS3 protein. FEBS Lett2000; 465: 74-8.

[74] Lam AMI, Keeney D, Eckert PQ, Frick DN. Hepatitis C virus NS3ATPases/helicases from different genotypes exhibit variations inenzymatic properties. J Virol 2003; 77: 3950-3961.

[75] Gallivan JP, McGarvey MJ. The importance of the Q motif in theATPase activity of a viral helicase. FEBS Lett 2003; 554: 485-8.

[76] Tanner NK, Cordin O, Banroques J, Doere M, Linder P. The Qmotif: a newly identified motif in DEAD box helicases may regu-late ATP binding and hydrolysis. Mol Cell 2003; 11: 127-38.

[77] Bartelma G, Padmanabhan R. Expression, purification, and char-acterization of the RNA 5'-triphosphatase activity of dengue virustype 2 nonstructural protein 3. Virology 2002; 299: 122-32.

[78] Wengler G. The NS 3 nonstructural protein of flaviviruses containsan RNA triphosphatase activity. Virology 1993; 197: 265-73.

[79] Egloff MP, Benarroch D, Selisko B, Romette JL, Canard B. AnRNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirusRNA polymerase NS5: crystal structure and functional characteri-zation. Embo J 2002; 21: 2757-68.

[80] Koonin EV, Senkevich TG. Vaccinia virus encodes four putativeDNA and/or RNA helicases distantly related to each other. J GenVirol 1992; 73 ( Pt 4): 989-93.

[81] Deng L, Shuman S. Vaccinia NPH-I, a DExH-box ATPase, is theenergy coupling factor for mRNA transcription termination. GenesDev 1998; 12: 538-46.

[82] Shuman S. Vaccinia virus RNA helicase: an essential enzymerelated to the DE-H family of RNA-dependent NTPases. Proc NatlAcad Sci USA 1992; 89: 10935-9.

[83] Simpson DA, Condit RC. Vaccinia virus gene A18R encodes anessential DNA helicase. J Virol 1995; 69: 6131-9.

[84] Li J, Broyles SS. The DNA binding domain of the vaccinia virusearly transcription factor small subunit is an extended helicase-likemotif. Nucleic Acids Res 1995; 23: 1590-6.

[85] Jankowsky E, Gross CH, Shuman S, Pyle AM. The DExH proteinNPH-II is a processive and directional motor for unwinding RNA.Nature 2000; 403: 447-51.

[86] Jankowsky E, Gross CH, Shuman S, Pyle AM. Active Disruptionof an RNA-Protein Interaction by a DExH/D RNA Helicase. Sci-ence 2001; 291: 121-125.

[87] Kawaoka J, Jankowsky E, Pyle AM. Backbone tracking by the SF2helicase NPH-II. Nat Struct Mol Biol 2004; 11: 526-30.

[88] Shitrit D, Lev N, Bar-Gil-Shitrit A, Kramer MR. Progressive multi-focal leukoencephalopathy in transplant recipients. Transpl Int2004.

[89] Berger JR, Pall L, Lanska D, Whiteman M. Progressive multifocalleukoencephalopathy in patients with HIV infection. J Neurovirol1998; 4: 59-68.

[90] Valle M, Gruss C, Halmer L, Carazo JM, Donate LE. Large T-antigen double hexamers imaged at the simian virus 40 origin ofreplication. Mol Cell Biol 2000; 20: 34-41.

[91] Alexandrov AI, Botchan MR, Cozzarelli NR. Characterization ofsimian virus 40 T-antigen double hexamers bound to a replicationfork. The active form of the helicase. J Biol Chem 2002; 277:44886-97.

[92] Gomez-Lorenzo MG, Valle M, Frank J, Gruss C, Sorzano CO,Chen XS , et al. Large T antigen on the simian virus 40 origin ofreplication: a 3D snapshot prior to DNA replication. Embo J 2003;22: 6205-13.

[93] Morris PD, Byrd AK, Tackett AJ, Cameron CE, Tanega P, Ott R, etal. Hepatitis C virus NS3 and simian virus 40 T antigen helicasesdisplace streptavidin from 5'-biotinylated oligonucleotides but notfrom 3'- biotinylated oligonucleotides: evidence for directional biasin translocation on single-stranded DNA. Biochemistry 2002; 41:2372-8.

[94] Fanning E, Knippers R. Structure and function of simian virus 40large tumor antigen. Annu Rev Biochem 1992; 61: 55-85.

[95] Walboomers JM, Jacobs MV, Manos MM, Bosch FX, Kummer JA,Shah KV , et al. Human papillomavirus is a necessary cause of in-vasive cervical cancer worldwide. J Pathol 1999; 189: 12-9.

[96] Chen EY, Howley PM, Levinson AD, Seeburg PH. The primarystructure and genetic organization of the bovine papillomavirustype 1 genome. Nature 1982; 299: 529-34.

[97] Danos O, Katinka M, Yaniv M. Human papillomavirus 1a com-plete DNA sequence: a novel type of genome organization amongpapovaviridae. Embo J 1982; 1: 231-6.

[98] Wilson VG, West M, Woytek K, Rangasamy D. Papillomavirus E1proteins: form, function, and features. Virus Genes 2002; 24: 275-90.

[99] Frattini MG, Laimins LA. Binding of the human papillomavirus E1origin-recognition protein is regulated through complex formationwith the E2 enhancer-binding protein. Proc Natl Acad Sci USA1994; 91: 12398-402.

[100] Abbate EA, Berger JM, Botchan MR. The X-ray structure of thepapillomavirus helicase in complex with its molecular matchmakerE2. Genes Dev 2004; 18: 1981-96.

[101] White PW, Pelletier A, Brault K, Titolo S, Welchner E, ThauvetteL, et al. Characterization of recombinant HPV6 and 11 E1 heli-cases: effect of ATP on the interaction of E1 with E2 and mappingof a minimal helicase domain. J Biol Chem 2001; 276: 22426-38.

[102] Fouts ET, Yu X, Egelman EH, Botchan MR. Biochemical andelectron microscopic image analysis of the hexameric E1 helicase.J Biol Chem 1999; 274: 4447-58.

[103] McCarty DM, Young Jr SM, Samulski RJ. Integration of Adeno-Associated Virus (AAV) and Recombinant AAV Vectors. AnnuRev Genet 2004.

[104] Smith RH, Spano AJ, Kotin RM. The Rep78 gene product ofadeno-associated virus (AAV) self-associates to form a hexamericcomplex in the presence of AAV ori sequences. J Virol 1997; 71:4461-71.

[105] Smith RH, Kotin RM. The Rep52 gene product of adeno-associatedvirus is a DNA helicase with 3'-to-5' polarity. J Virol 1998; 72:4874-81.

[106] Rodriguez PL, Carrasco L. Poliovirus protein 2C has ATPase andGTPase activities. J Biol Chem 1993; 268: 8105-10.

[107] Mirzayan C, Wimmer E. Biochemical studies on poliovirus poly-peptide 2C: evidence for ATPase activity. Virology 1994; 199:176-87.

[108] Klein M, Eggers HJ, Nelsen-Salz B. Echovirus 9 strain barty non-structural protein 2C has NTPase activity. Virus Res 1999; 65: 155-60.

[109] Klein M, Eggers HJ, Nelsen-Salz B. Echovirus-9 protein 2C bindssingle-stranded RNA unspecifically. J Gen Virol 2000; 81: 2481-4.

[110] Pfister T, Wimmer E. Polypeptide p41 of a Norwalk-like virus is anucleic acid-independent nucleoside triphosphatase. J Virol 2001;75: 1611-9.

[111] Yao N, Hesson T, Cable M, Hong Z, Kwong AD, Le HV, et al.Structure of the hepatitis C virus RNA helicase domain. Nat StructBiol 1997; 4: 463-7.

[112] Cho HS, Ha NC, Kang LW, Chung KM, Back SH, Jang SK, et al.Crystal structure of RNA helicase from genotype 1b hepatitis C vi-rus. A feasible mechanism of unwinding duplex RNA. J Biol Chem1998; 273: 15045-52.


[113] Kim JL, Morgenstern KA, Griffith JP, Dwyer MD, Thomson JA,Murcko MA, et al. Hepatitis C virus NS3 RNA helicase domainwith a bound oligonucleotide: the crystal structure provides insightsinto the mode of unwinding. Structure 1998; 6: 89-100.

[114] Yao N, Reichert P, Taremi SS, Prosise WW, Weber PC. Molecularviews of viral polyprotein processing revealed by the crystal struc-ture of the hepatitis C virus bifunctional protease-helicase. Struc-ture Fold Des 1999; 7: 1353-63.

[115] Liu D, Wang YS, Gesell JJ, Wyss DF. Solution structure and back-bone dynamics of an engineered arginine-rich subdomain 2 of thehepatitis C virus NS3 RNA helicase. J Mol Biol 2001; 314: 543-61.

[116] Sawaya MR, Guo S, Tabor S, Richardson CC, Ellenberger T.Crystal structure of the helicase domain from the replicative heli-case- primase of bacteriophage T7. Cell 1999; 99: 167-77.

[117] Bird LE, Hakansson K, Pan H, Wigley DB. Characterization andcrystallization of the helicase domain of bacteriophage T7 gene 4protein. Nucleic Acids Res 1997; 25: 2620-6.

[118] Singleton MR, Sawaya MR, Ellenberger T, Wigley DB. Crystalstructure of T7 gene 4 ring helicase indicates a mechanism for se-quential hydrolysis of nucleotides. Cell 2000; 101: 589-600.

[119] Toth EA, Li Y, Sawaya MR, Cheng Y, Ellenberger T. The crystalstructure of the bifunctional primase-helicase of bacteriophage T7.Mol Cell 2003; 12: 1113-23.

[120] James JA, Escalante CR, Yoon-Robarts M, Edwards TA, LindenRM, Aggarwal AK. Crystal structure of the SF3 helicase fromadeno-associated virus type 2. Structure (Camb) 2003; 11: 1025-35.

[121] James JA, Aggarwal AK, Linden RM, Escalante CR. Structure ofadeno-associated virus type 2 Rep40-ADP complex: insight intonucleotide recognition and catalysis by superfamily 3 helicases.Proc Natl Acad Sci USA 2004; 101: 12455-60.

[122] Li D, Zhao R, Lilyestrom W, Gai D, Zhang R, DeCaprio JA, et al.Structure of the replicative helicase of the oncoprotein SV40 largetumour antigen. Nature 2003; 423: 512-8.

[123] Gai D, Zhao R, Li D, Finkielstein CV, Chen XS. Mechanisms ofconformational change for a replicative hexameric helicase ofSV40 large tumor antigen. Cell 2004; 119: 47-60.

[124] Story RM, Steitz TA. Structure of the recA protein-ADP complex.Nature 1992; 355: 374-6.

[125] Walker JE, Saraste M, Runswick MJ, Gay NJ. Distantly relatedsequences in the alpha- and beta-subunits of ATP synthase, my-osin, kinases and other ATP-requiring enzymes and a common nu-cleotide binding fold. Embo J 1982; 1: 945-51.

[126] Orelle C, Dalmas O, Gros P, Di Pietro A, Jault JM. The conservedglutamate residue adjacent to the Walker-B motif is the catalyticbase for ATP hydrolysis in the ATP-binding cassette transporterBmrA. J Biol Chem 2003; 278: 47002-8.

[127] Goetzinger KR, Rao VB. Defining the ATPase center of bacterio-phage T4 DNA packaging machine: requirement for a catalyticglutamate residue in the large terminase protein gp17. J Mol Biol2003; 331: 139-54.

[128] Ahmadian MR, Stege P, Scheffzek K, Wittinghofer A. Confirma-tion of the arginine-finger hypothesis for the GAP-stimulated GTP-hydrolysis reaction of Ras. Nat Struct Biol 1997; 4: 686-9.

[129] Nadanaciva S, Weber J, Wilke-Mounts S, Senior AE. Importanceof F1-ATPase residue alpha-Arg-376 for catalytic transition statestabilization. Biochemistry 1999; 38: 15493-9.

[130] Crampton DJ, Guo S, Johnson DE, Richardson CC. The argininefinger of bacteriophage T7 gene 4 helicase: role in energy coupling.Proc Natl Acad Sci USA 2004; 101: 4373-8.

[131] Lin C, Kim JL. Structure-based mutagenesis study of hepatitis Cvirus NS3 helicase. J Virol 1999; 73: 8798-807.

[132] Tai CL, Pan WC, Liaw SH, Yang UC, Hwang LH, Chen DS.Structure-based mutational analysis of the hepatitis c virus ns3helicase. J Virol 2001; 75: 8289-97.

[133] Preugschat F, Danger DP, Carter LH, 3rd, Davis RG, Porter DJ.Kinetic analysis of the effects of mutagenesis of W501 and V432of the hepatitis C virus NS3 helicase domain on ATPase andstrand-separating activity. Biochemistry 2000; 39: 5174-83.

[134] Paolini C, Lahm A, De Francesco R, Gallinari P. Mutational analy-sis of hepatitis C virus NS3-associated helicase. J Gen Virol 2000;81 Pt 7: 1649-58.

[135] Kim JW, Seo MY, Shelat A, Kim CS, Kwon TW, Lu HH, et al.Structurally Conserved Amino Acid W501 Is Required for RNAHelicase Activity but Is Not Essential for DNA Helicase Activityof Hepatitis C Virus NS3 Protein. J Virol 2003; 77: 571-582.

[136] Lam AM, Keeney D, Frick DN. Two novel conserved motifs in thehepatitis C virus NS3 protein critical for helicase action. J BiolChem 2003; 278: 44514-24.

[137] Korolev S, Hsieh J, Gauss GH, Lohman TM, Waksman G. Majordomain swiveling revealed by the crystal structures of complexesof E. coli Rep helicase bound to single-stranded DNA and ADP.Cell 1997; 90: 635-47.

[138] Velankar SS, Soultanas P, Dillingham MS, Subramanya HS, Wig-ley DB. Crystal structures of complexes of PcrA DNA helicasewith a DNA substrate indicate an inchworm mechanism. Cell 1999;97: 75-84.

[139] Subramanya HS, Bird LE, Brannigan JA, Wigley DB. Crystalstructure of a DExx box DNA helicase. Nature 1996; 384: 379-83.

[140] Soultanas P, Dillingham MS, Velankar SS, Wigley DB. DNAbinding mediates conformational changes and metal ion coordina-tion in the active site of PcrA helicase. J Mol Biol 1999; 290: 137-48.

[141] Soultanas P, Dillingham MS, Wiley P, Webb MR, Wigley DB.Uncoupling DNA translocation and helicase activity in PcrA: directevidence for an active mechanism. Embo J 2000; 19: 3799-810.

[142] Dillingham MS, Soultanas P, Wiley P, Webb MR, Wigley DB.Defining the roles of individual residues in the single-strandedDNA binding site of PcrA helicase. Proc Natl Acad Sci USA 2001;98: 8381-7.

[143] Soultanas P, Wigley DB. Unwinding the 'Gordian knot' of helicaseaction. Trends Biochem Sci 2001; 26: 47-54.

[144] Dillingham MS, Wigley DB, Webb MR. Demonstration of unidi-rectional single-stranded DNA translocation by PcrA helicase:measurement of step size and translocation speed. Biochemistry2000; 39: 205-12.

[145] Porter DJ, Short SA, Hanlon MH, Preugschat F, Wilson JE,Willard DH, Jr., et al. Product release is the major contributor tokcat for the hepatitis C virus helicase-catalyzed strand separation ofshort duplex DNA. J Biol Chem 1998; 273: 18906-14.

[146] Levin MK, Gurjar MM, Patel SS. ATP binding modulates thenucleic acid affinity of hepatitis C virus helicase. J Biol Chem2003; 278: 23311-23316.

[147] Levin MK, Wang YH, Patel SS. The functional interaction of thehepatitis C virus helicase molecules is responsible for unwindingprocessivity. J Biol Chem 2004; 279: 26005-12.

[148] Serebrov V, Pyle AM. Periodic cycles of RNA unwinding andpausing by hepatitis C virus NS3 helicase. Nature 2004; 430: 476-80.

[149] Lam AMI, Rypma RS, Frick DN. Enhanced nucleic acid binding toATP-bound hepatitis C virus NS3 helicase at low pH activatesRNA unwinding. Nucl. Acids. Res. 2004; 32: 4060-4070.

[150] Pang PS, Jankowsky E, Planet PJ, Pyle AM. The hepatitis C viralNS3 protein is a processive DNA helicase with cofactor enhancedRNA unwinding. Embo J 2002; 21: 1168-1176.

[151] Frick DN, Rypma RS, Lam AM, Gu B. The nonstructural protein 3protease/helicase requires an intact protease domain to efficientlyunwind duplex RNA. J Biol Chem 2004; 279: 1269-1280.

[152] Flajolet M, Rotondo G, Daviet L, Bergametti F, Inchauspe G,Tiollais P, et al. A genomic approach of the hepatitis C virus gen-erates a protein interaction map. Gene 2000; 242: 369-79.

[153] Khu YL, Koh E, Lim SP, Tan YH, Brenner S, Lim SG, et al. Mu-tations that affect dimer formation and helicase activity of thehepatitis C virus helicase. J Virol 2001; 75: 205-14.

[154] Tackett AJ, Chen Y, Cameron CE, Raney KD. Multiple full-lengthNS3 molecules are required for optimal unwinding of oligonucleo-tide DNA in vitro. J Biol Chem 2005.

[155] Wong I, Lohman TM. Allosteric effects of nucleotide cofactors onEscherichia coli Rep helicase-DNA binding. Science 1992; 256:350-5.

[156] Ali JA, Lohman TM. Kinetic measurement of the step size of DNAunwinding by Escherichia coli UvrD helicase. Science 1997; 275:377-80.

[157] Maluf NK, Fischer CJ, Lohman TM. A Dimer of Escherichia coliUvrD is the Active Form of the Helicase In vitro. J Mol Biol 2003;325: 913-35.

[158] Lohman TM, Bjornson KP. Mechanisms of helicase-catalyzedDNA unwinding. Annu Rev Biochem 1996; 65: 169-214.

[159] Goregaoker SP, Culver JN. Oligomerization and activity of thehelicase domain of the tobacco mosaic virus 126- and 183-kilodalton replicase proteins. J Virol 2003; 77: 3549-56.


[160] Hingorani MM, Patel SS. Cooperative interactions of nucleotideligands are linked to oligomerization and DNA binding in bacterio-phage T7 gene 4 helicases. Biochemistry 1996; 35: 2218-28.

[161] Hingorani MM, Washington MT, Moore KC, Patel SS. ThedTTPase mechanism of T7 DNA helicase resembles the bindingchange mechanism of the F1-ATPase. Proc Natl Acad Sci USA1997; 94: 5012-7.

[162] Noji H, Yasuda R, Yoshida M, Kinosita K, Jr. Direct observationof the rotation of F1-ATPase. Nature 1997; 386: 299-302.

[163] Itoh H, Takahashi A, Adachi K, Noji H, Yasuda R, Yoshida M, etal. Mechanically driven ATP synthesis by F1-ATPase. Nature2004; 427: 465-8.

[164] Egelman EH, Yu X, Wild R, Hingorani MM, Patel SS. Bacterio-phage T7 helicase/primase proteins form rings around single-stranded DNA that suggest a general structure for hexameric heli-cases. Proc Natl Acad Sci USA 1995; 92: 3869-73.

[165] VanLoock MS, Alexandrov A, Yu X, Cozzarelli NR, Egelman EH.SV40 large T antigen hexamer structure: domain organization andDNA-induced conformational changes. Curr Biol 2002; 12: 472-6.

[166] Notarnicola SM, Park K, Griffith JD, Richardson CC. A domain ofthe gene 4 helicase/primase of bacteriophage T7 required for theformation of an active hexamer. J Biol Chem 1995; 270: 20215-24.

[167] Washington MT, Rosenberg AH, Griffin K, Studier FW, Patel SS.Biochemical analysis of mutant T7 primase/helicase proteins de-fective in DNA binding, nucleotide hydrolysis, and the coupling ofhydrolysis with DNA unwinding. J Biol Chem 1996; 271: 26825-34.

[168] Monod J, Wyman J, Changeux JP. On the Nature of AllostericTransitions: A Plausible Model. J Mol Biol 1965; 12: 88-118.

[169] Kao RY, Tsui WH, Lee TS, Tanner JA, Watt RM, Huang JD, et al.Identification of novel small-molecule inhibitors of severe acuterespiratory syndrome-associated coronavirus by chemical genetics.Chem Biol 2004; 11: 1293-9.

[170] Duan J, Liuzzi M, Paris W, Liard F, Browne A, Dansereau N, et al.Oral bioavailability and in vivo efficacy of the helicase-primase in-hibitor BILS 45 BS against acyclovir-resistant herpes simplex virustype 1. Antimicrob Agents Chemother 2003; 47: 1798-804.

[171] Liuzzi M, Kibler P, Bousquet C, Harji F, Bolger G, Garneau M, etal. Isolation and characterization of herpes simplex virus type 1 re-sistant to aminothiazolylphenyl-based inhibitors of the viral heli-case-primase. Antiviral Res 2004; 64: 161-70.

[172] Spector FC, Liang L, Giordano H, Sivaraja M, Peterson MG. Inhi-bition of herpes simplex virus replication by a 2-amino thiazole viainteractions with the helicase component of the UL5-UL8-UL52complex. J Virol 1998; 72: 6979-87.

[173] Betz UA, Fischer R, Kleymann G, Hendrix M, Rubsamen-Waigmann H. Potent in vivo antiviral activity of the herpes simplexvirus primase- helicase inhibitor BAY 57-1293. Antimicrob AgentsChemother 2002; 46: 1766-72.

[174] Borowski P, Schalinski S, Schmitz H. Nucleotide triphospha-tase/helicase of hepatitis C virus as a target for antiviral therapy.Antiviral Res 2002; 55: 397-412.

[175] Borowski P, Niebuhr A, Schmitz H, Hosmane RS, Bretner M,Siwecka MA, et al. NTPase/helicase of Flaviviridae: inhibitors andinhibition of the enzyme. Acta Biochim Pol 2002; 49: 597-614.

[176] Borowski P, Lang M, Haag A, Schmitz H, Choe J, Chen HM, et al.Characterization of imidazo[4, 5-d]pyridazine nucleosides asmodulators of unwinding reaction mediated by West Nile virus nu-cleoside triphosphatase/helicase: evidence for activity on the levelof substrate and/or enzyme. Antimicrob Agents Chemother 2002;46: 1231-9.

[177] Preugschat F, Averett DR, Clarke BE, Porter DJT. A steady-stateand pre-steady-state kinetic analysis of the NTPase activity associ-ated with the hepatitis C virus NS3 helicase domain. J Biol Chem1996; 271: 24449-57.

[178] Wardell AD, Errington W, Ciaramella G, Merson J, McGarvey MJ.Characterization and mutational analysis of the helicase andNTPase activities of hepatitis C virus full-length NS3 protein. JGen Virol 1999; 80: 701-9.

[179] Locatelli GA, Gosselin G, Spadari S, Maga G. Hepatitis C virusNS3 NTPase/helicase: different stereoselectivity in nucleosidetriphosphate utilisation suggests that NTPase and helicase activitiesare coupled by a nucleotide-dependent rate limiting step. J MolBiol 2001; 313: 683-94.

[180] Kyono K, Miyashiro M, Taguchi I. Expression and purification of ahepatitis C virus NS3/4A complex, and characterization of its heli-

case activity with the Scintillation Proximity Assay system. J Bio-chem (Tokyo) 2004; 135: 245-52.

[181] Porter DJ. Inhibition of the hepatitis C virus helicase-associatedATPase activity by the combination of ADP, NaF, MgCl2, andpoly(rU). Two ADP binding sites on the enzyme-nucleic acid com-plex. J Biol Chem 1998; 273: 7390-6.

[182] Xu YW, Morera S, Janin J, Cherfils J. AlF3 mimics the transitionstate of protein phosphorylation in the crystal structure of nucleo-side diphosphate kinase and MgADP. Proc Natl Acad Sci USA1997; 94: 3579-83.

[183] Levin MK, Patel SS. Helicase from hepatitis C virus, energetics ofDNA binding. J Biol Chem 2002; 277: 29377-85.

[184] Porter DJ. A kinetic analysis of the oligonucleotide-modulatedATPase activity of the helicase domain of the NS3 protein fromhepatitis C virus. The first cycle of interaction of ATP with the en-zyme is unique. J Biol Chem 1998; 273: 14247-53.

[185] Locatelli GA, Spadari S, Maga G. Hepatitis C virus NS3 AT-Pase/helicase: an ATP switch regulates the cooperativity among thedifferent substrate binding sites. Biochemistry 2002; 41: 10332-42.

[186] Borowski P, Deinert J, Schalinski S, Bretner M, Ginalski K,Kulikowski T, et al. Halogenated benzimidazoles and benzotria-zoles as inhibitors of the NTPase/helicase activities of hepatitis Cand related viruses. Eur J Biochem 2003; 270: 1645-53.

[187] Zhang N, Chen HM, Koch V, Schmitz H, Minczuk M, Stepien P , etal. Potent inhibition of NTPase/helicase of the West Nile Virus byring-expanded ("fat") nucleoside analogues. J Med Chem 2003; 46:4776-89.

[188] Zhang N, Chen HM, Koch V, Schmitz H, Liao CL, Bretner M, etal. Ring-expanded ("fat") nucleoside and nucleotide analogues ex-hibit potent in vitro activity against flaviviridae NTPases/ helicases,including those of the West Nile virus, hepatitis C virus, and Japa-nese encephalitis virus. J Med Chem 2003; 46: 4149-64.

[189] Hosmane RS. Ring-expanded ("Fat") nucleosides as broad-spectrum anticancer and antiviral agents. Curr Top Med Chem2002; 2: 1093-109.

[190] Klug SJ, Famulok M. All you wanted to know about SELEX. MolBiol Rep 1994; 20: 97-107.

[191] Fukuda K, Vishnuvardhan D, Sekiya S, Hwang J, Kakiuchi N,Taira K, et al. Isolation and characterization of RNA aptamers spe-cific for the hepatitis C virus nonstructural protein 3 protease. Eur JBiochem 2000; 267: 3685-94.

[192] Nishikawa F, Kakiuchi N, Funaji K, Fukuda K, Sekiya S, Nishi-kawa S. Inhibition of HCV NS3 protease by RNA aptamers incells. Nucleic Acids Res 2003; 31: 1935-43.

[193] Hwang J, Fauzi H, Fukuda K, Sekiya S, Kakiuchi N, ShimotohnoK, et al. The RNA aptamer-binding site of hepatitis C virus NS3protease. Biochem Biophys Res Commun 2000; 279: 557-62.

[194] Fukuda K, Umehara T, Sekiya S, Kunio K, Hasegawa T, Nishi-kawa S. An RNA ligand inhibits hepatitis C virus NS3 protease andhelicase activities. Biochem Biophys Res Commun 2004; 325: 670-675.

[195] Hwang B, Cho JS, Yeo HJ, Kim JH, Chung KM, Han K, et al.Isolation of specific and high-affinity RNA aptamers against NS3helicase domain of hepatitis C virus. Rna 2004; 10: 1277-90.

[196] Banerjee R, Dasgupta A. Specific Interaction of Hepatitis C VirusProtease/Helicase NS3 with the 3'-Terminal Sequences of ViralPositive- and Negative-Strand RNA. J. Virol. 2001; 75: 1708-1721.

[197] Tackett AJ, Wei L, Cameron CE, Raney KD. Unwinding of nucleicacids by HCV NS3 helicase is sensitive to the structure of the du-plex. Nucleic Acids Res 2001; 29: 565-72.

[198] Bastide L, Boehmer PE, Villani G, Lebleu B. Inhibition of a DNA-helicase by peptide nucleic acids. Nucleic Acids Res 1999; 27:551-4.

[199] Joshi PJ, Fisher TS, Prasad VR. Anti-HIV inhibitors based onnucleic acids: emergence of aptamers as potent antivirals. CurrDrug Targets Infect Disord 2003; 3: 383-400.

[200] Goncalves J, Silva F, Freitas-Vieira A, Santa-Marta M, Malho R,Yang X, et al. Functional neutralization of HIV-1 Vif protein byintracellular immunization inhibits reverse transcription and viralreplication. J Biol Chem 2002; 277: 32036-45.

[201] Tessmann K, Erhardt A, Haussinger D, Heintges T. Cloning andmolecular characterization of human high affinity antibody frag-ments against Hepatitis C virus NS3 helicase. J Virol Methods2002; 103: 75-88.


[202] Artsaenko O, Tessmann K, Sack M, Haussinger D, Heintges T.Abrogation of hepatitis C virus NS3 helicase enzymatic activity byrecombinant human antibodies. J Gen Virol 2003; 84: 2323-32.

[203] Prabhu R, Khalap N, Burioni R, Clementi M, Garry RF, Dash S.Inhibition of hepatitis C virus nonstructural protein, helicase activ-ity, and viral replication by a recombinant human antibody clone.Am J Pathol 2004; 165: 1163-73.

[204] Sullivan DE, Mondelli MU, Curiel DT, Krasnykh V, Mikheeva G,Gaglio P, et al. Construction and characterization of an intracellularsingle-chain human antibody to hepatitis C virus non-structural 3protein. J Hepatol 2002; 37: 660-8.

[205] Mondelli MU, Cerino A, Boender P, Oudshoorn P, Middeldorp J,Fipaldini C, et al. Significance of the immune response to a major,conformational B-cell epitope on the hepatitis C virus NS3 regiondefined by a human monoclonal antibody. J Virol 1994; 68: 4829-36.

[206] White PW, Titolo S, Brault K, Thauvette L, Pelletier A, WelchnerE, et al. Inhibition of human papillomavirus DNA replication bysmall molecule antagonists of the E1-E2 protein interaction. J BiolChem 2003; 278: 26765-72.

[207] Faucher AM, White PW, Brochu C, Grand-Maitre C, Rancourt J,Fazal G. Discovery of small-molecule inhibitors of the ATPase ac-tivity of human papillomavirus E1 helicase. J Med Chem 2004; 47:18-21.

[208] Venkatesan M, Silver LL, Nossal NG. Bacteriophage T4 gene 41protein, required for the synthesis of RNA primers, is also a DNAhelicase. J Biol Chem 1982; 257: 12426-34.

[209] Raney KD, Benkovic SJ. Bacteriophage T4 Dda helicase translo-cates in a unidirectional fashion on single-stranded DNA. J BiolChem 1995; 270: 22236-42.

[210] Dudas KC, Kreuzer KN. UvsW protein regulates bacteriophage T4origin-dependent replication by unwinding R-loops. Mol Cell Biol2001; 21: 2706-15.

[211] Ziegelin G, Scherzinger E, Lurz R, Lanka E. Phage P4 alpha pro-tein is multifunctional with origin recognition, helicase and primaseactivities. Embo J 1993; 12: 3703-8.

[212] Lemonnier M, Ziegelin G, Reick T, Munoz Gomez A, Diaz-OrejasR, Lanka E. Bacteriophage P1 Ban protein is a hexameric DNAhelicase that interacts with and substitutes for Escherichia coliDnaB. Nucleic Acids Res 2003; 31: 3918-28.

[213] Tabor S, Richardson CC. Template recognition sequence for RNAprimer synthesis by gene 4 protein of bacteriophage T7. Proc NatlAcad Sci USA 1981; 78: 205-9.

[214] Ayora S, Weise F, Mesa P, Stasiak A, Alonso JC. Bacillus subtilisbacteriophage SPP1 hexameric DNA helicase, G40P, interacts withforked DNA. Nucleic Acids Res 2002; 30: 2280-9.

[215] Laufs S, Lu A, Arrell K, Carstens EB. Autographa californicanuclear polyhedrosis virus p143 gene product is a DNA-bindingprotein. Virology 1997; 228: 98-106.

[216] Scheffner M, Knippers R, Stahl H. Simian-virus-40 large-T-antigen-catalyzed DNA and RNA unwinding reactions. Eur J Bio-chem 1991; 195: 49-54.

[217] Yang L, Mohr I, Fouts E, Lim DA, Nohaile M, Botchan M. The E1protein of bovine papilloma virus 1 is an ATP-dependent DNAhelicase. Proc Natl Acad Sci USA 1993; 90: 5086-90.

[218] Shuman S. Vaccinia virus RNA helicase. Directionality and sub-strate specificity. J Biol Chem 1993; 268: 11798-802.

[219] Merits A, Fedorkin ON, Guo D, Kalinina NO, Morozov SY. Ac-tivities associated with the putative replication initiation protein ofcoconut foliar decay virus, a tentative member of the genus Nano-virus. J Gen Virol 2000; 81: 3099-106.

[220] Jindal HK, Yong CB, Wilson GM, Tam P, Astell CR. Mutations inthe NTP-binding motif of minute virus of mice (MVM) NS-1 pro-tein uncouple ATPase and DNA helicase functions. J Biol Chem1994; 269: 3283-9.

[221] Kainov DE, Pirttimaa M, Tuma R, Butcher SJ, Thomas GJ, Jr.,Bamford DH, et al. RNA packaging device of double-strandedRNA bacteriophages, possibly as simple as hexamer of P4 protein.J Biol Chem 2003; 278: 48084-91.

[222] Stauber N, Martinez-Costas J, Sutton G, Monastyrskaya K, Roy P.Bluetongue virus VP6 protein binds ATP and exhibits an RNA-dependent ATPase function and a helicase activity that catalyze theunwinding of double-stranded RNA substrates. J Virol 1997; 71:7220-6.

[223] Ahola T, den Boon JA, Ahlquist P. Helicase and capping enzymeactive site mutations in brome mosaic virus protein 1a cause de-fects in template recruitment, negative-strand RNA synthesis, andviral RNA capping. J Virol 2000; 74: 8803-11.

[224] Til'kunova T, Zinovkin RA, Agranovskii AA. RNA-binding prop-erties of proteins of the beet yellows closterovirus. Mol Biol(Mosk) 2004; 38: 553-8.

[225] Kalinina NO, Rakitina DV, Solovyev AG, Schiemann J, MorozovSY. RNA helicase activity of the plant virus movement proteinsencoded by the first gene of the triple gene block. Virology 2002;296: 321-9.

[226] Eagles RM, Balmori-Melian E, Beck DL, Gardner RC, Forster RL.Characterization of NTPase, RNA-binding and RNA-helicase ac-tivities of the cytoplasmic inclusion protein of tamarillo mosaicpotyvirus. Eur J Biochem 1994; 224: 677-84.

[227] Kadare G, David C, Haenni AL. ATPase, GTPase, and RNA bind-ing activities associated with the 206-kilodalton protein of turnipyellow mosaic virus. J Virol 1996; 70: 8169-74.

[228] Warrener P, Collett MS. Pestivirus NS3 (p80) protein possessesRNA helicase activity. J Virol 1995; 69: 1720-6.

[229] Gu B, Liu C, Lin-Goerke J, Maley DR, Gutshall LL, FeltenbergerCA, et al. The RNA helicase and nucleotide triphosphatase activi-ties of the bovine viral diarrhea virus NS3 protein are essential forviral replication. J Virol 2000; 74: 1794-800.

[230] Zhong W, Ingravallo P, Wright-Minogue J, Skelton A, Uss AS,Chase R, et al. Nucleoside triphosphatase and RNA helicase ac-tivities associated with GB virus B nonstructural protein 3. Virol-ogy 1999; 261: 216-26.

[231] Gwack Y, Yoo H, Song I, Choe J, Han JH. RNA-Stimulated AT-Pase and RNA helicase activities and RNA binding domain ofhepatitis G virus nonstructural protein 3. J Virol 1999; 73: 2909-15.

[232] Gomez de Cedron M, Ehsani N, Mikkola ML, Garcia JA, Kaari-ainen L. RNA helicase activity of Semliki Forest virus replicaseprotein NSP2. FEBS Lett 1999; 448: 19-22.

[233] Gros C, Wengler G. Identification of an RNA-stimulated NTPasein the predicted helicase sequence of the Rubella virus nonstruc-tural polyprotein. Virology 1996; 217: 367-72.

[234] Iyer LM, Leipe DD, Koonin EV, Aravind L. Evolutionary historyand higher order classification of AAA+ ATPases. J Struct Biol2004; 146: 11-31.

[235] Leipe DD, Wolf YI, Koonin EV, Aravind L. Classification andevolution of P-loop GTPases and related ATPases. J Mol Biol2002; 317: 41-72.

[236] Frick DN, Rypma RS, Lam AM, Frenz CM. Electrostatic analysisof the hepatitis C virus NS3 helicase reveals both active and allos-teric site locations. Nucleic Acids Res 2004; 32: 5519-28.

[237] Brunetto MR, Bonino F. Treatment of chronic hepatitis B: fromresearch to clinical practice via the consensus conferences. CurrPharm Des 2004; 10(17): 2063-75.

[238] Camma C, Di Bona D, Craxi A. The impact of antiviral treatmentson the course of chronic hepatitis C: an evidence-based approach.Curr Pharm Des 2004; 10(17): 2123-30.

[239] D'Cruz OJ, Uckun FM. Clinical development of microbicides forthe prevention of HIV infection. Curr Pharm Des 2004; 10(3): 315-36.

Documents

Understanding Helicases as a Means of Virus Control