1

DELIVERY OF ANIMAL VIRUS DNA INTO THE NUCLEUS

Urs F. GreberInstitute of ZoologyUniversity of Zürich

Winterthurerstrasse 190CH-8057 Zürich

SwitzerlandPhone: 41 1 635 4841, Fax: 41 1 635 6817, email: ufgreber@zool.unizh.ch

1. INTRODUCTION

2. CELL SURFACE BINDING AND UPTAKE

Non-enveloped viruses2.1. Adenoviridae: Adenovirus type 2 and type 5 (Ad-2 and Ad-5)2.2. Papovaviridae: Polyoma virus and Simian virus 40 (SV40)2.3. Parvoviridae: Parvovirus and Adeno-associated virus (AAV)

Enveloped viruses2.4. Herpesviridae: Herpes simplex virus type 1 (HSV-1)2.5. Hepadnaviridae: Hepatitis B virus (HBV)2.6. Baculoviridae: Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV)

3. NUCLEAR ENVELOPE TARGETING

3.1. Herpes simplex virus type 1 (HSV-1)3.2. Nuclear polyhedrosis virus (NPV)3.3. Other viruses

4. NUCLEAR IMPORT: UNCOATING, DOCKING TRANSLOCATION

4.1. Adenovirus type 2 (Ad-2)4.2. Simian virus 40 (SV40)4.3. Herpes simplex virus type 1 (HSV-1)4.4. Hepatitis B virus (HBV)4.5. Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV)

5. CONCLUSIONS

6. TABLES

7. REFERENCES

Abbreviations:AAV: Adeno-associated virusAcNPV: Autographa californic Nuclear polyhedrosis virusAd: AdenovirusGV: Granulosis virusdHBV: Duck Hepatitis B virushHBV: Human Hepatitis B virusHSV: Herpes simplex virusNPV: Nuclear polyhedrosis virus

MMV: Mouse minute virusNLS: Nuclear localization sequenceNPC: Nuclear pore complexNE: Nuclear envelopeSV40: Simian virus 40

c: circular, ds: double stranded, kb: kilo bases, l: linear, pds: partly double-stranded, ss: single stranded

2

1. INTRODUCTION

Viruses are natural carriers of genetic information between cells. They are made up of proteins, nucleic acidsand often lipids. Viruses are usually smaller than ribosomes, but can be as big as a bacterial cell. Their DNAor RNA genome is tightly packed and well protected with proteins and often enwrapped with a lipid envelope.Within a virus particle, which is outside a host organism, the genome is inactive. The genome only becomesactivated when the virus interacts with a host cell in a very specific manner. By utilizing the host cell’smechanisms, a virus particle enters, unpacks the genome and initiates its replication and transcriptionprograms. It finally directs the cell to assemble and release progeny virions into the extracellular milieu for anew round of infection.

Viruses that infect eukaryotic cells replicate either in the cytoplasm or in the nucleus. Viruses replicating inthe nucleus usually contain a DNA or, seldomly, an RNA genome. These viruses take advantage of thenuclear compartment for their DNA synthesis, for generation and processing of their mRNA and in mostcases, they also assemble progeny virions within the nucleus (for review, see Fields, et al., 1996). RNAviruses replicating in the nucleus, such as the orthomyxovirus influenza virus, or the RNA lentiviruses,examplified by the human immunodeficiency virus (HIV), follow a similar strategy as the DNA viruses, butform their progeny outside the nucleus (for review, see Freed, et al., 1995, Stevenson, 1996, Trono, 1995,Whittaker, et al., 1996). For HIV, the incoming viral RNA is reverse transcribed in the cytoplasm to formlinear double stranded DNA, the proviral DNA, which is then imported into the nucleus as a high molecularweight nucleo-protein complex. The DNA viruses, which do not replicate in the nucleus, such as poxvirusesand iridoviruses, do not need nuclear targeting information. They must, however, contain additional geneticinformation to build up their own DNA and mRNA synthesis factories within the cytoplasm (for review, seeMoss, 1996).

The genome of DNA viruses replicating in the nucleus must cross three barriers, the plasma membrane, thecytosol and the nuclear envelope. A virus particle gets across the plasma membrane by attachment with highaffinity to a suitable cell surface receptor. The cytosol is traversed either by passive diffusion in the case ofsmall viruses or by exploitation of the cell’s motile functions in the case of large viruses. To overcome thenuclear envelope barrier nuclear DNA or RNA viruses have developed two principal strategies. The onco-retroviruses wait for the cell to break down its nuclear envelope during cell division. They then integrate theirreverse transcribed DNA genome into the host chromatin (for review, see Coffin, 1996). The nuclear DNA-containing viruses, typified by the adenoviridae, parvoviridae, papovaviridae, baculoviridae, hepadnaviridae andherpesviridae have chosen to import their genome into the nucleus across an intact nuclear envelope (seeTable 1). Emerging evidence suggests that DNA viruses are using elements of the same pathways, whichcontrol transport of cellular proteins and nucleic acids into the nucleus.

In this review, I will survey evidence on different mechanisms of DNA virus entry. I will briefly describe theviruses and their portals of entry into cells, and then discuss various nuclear envelope targeting and DNAimport mechanisms.

3

2. CELL SURFACE BINDING AND UPTAKE

The first step in virus infection of a host cell is attachment to the cell surface. Attachment can be mediated bya large variety of cell surface molecules (for review of different virus receptors, see Wimmer, 1994, see alsoTable 2). Attachment is necessary but not always sufficient for a successful infection, since subsequent steps,such as membrane penetration and disassembly often require virus binding to specialized surface receptors.The presence of specific receptors is often an important determinant of host range and cell and tissue tropismof a virus, but other factors acting in different phases of the replication cycle can be important for productiveinfection as well. A variety of experimental strategies has been employed over the years to identify specificviral receptors. Most of these studies, however, have not conclusively demonstrated that a putative receptoractually serves to initiate productive virus entry. The most stringent criteria of biological significance of aputative receptor are the transformation of receptor-negative nonpermissive cells to a permissive phenotype bymeans of recombinant DNA technology and the concomitant block of permissiveness of the engineered cellswith a specific antibody applied to the cell surface. This technology is readily applicable if the receptor isencoded by a single gene. If the receptor is a carbohydrate, one can restore the permissiveness by addingexogenous glycolipids of exactly defined composition to nonpermissive cells. In such reconstitutionexperiments, glycolipids have an advantage over glycoproteins, since they can contain a single carbohydratechain and can insert themselves into the cell membranes.

Emerging evidence indicates that virus binding to the cell surface is a multistep process for many viruses.Multiple receptors can either act together or sequentially. Binding to a first receptor can cause changes in thevirus or the host, which then favor interactions with a second receptor. For blood- or respiratory secretions-born viruses, the initial binding must effect rapid virus docking. In this case, the rate of binding can be moreimportant than the affinity, which is an equilibrium measurement (for review, see Williams, 1991). If the firstreceptor allows fast binding, the second receptor could be responsible for any slower processes, such asstable binding, endocytosis or penetration. To understand dynamic interactions between viruses and cells weneed to understand questions like, how many molecules are engaged in the interactions and how long do theyneed to be associated to confer a biological effect. For virus-cell interactions we are just at the beginning ofthis new phase of understanding.

NON-ENVELOPED VIRUSES

2.1. Adenovirus 2 and 5 (Ad-2 and Ad-5)

Ad-2 and Ad-5 belong to the subgroup C adenoviruses. These viruses are non-oncogenic and widely used asgene transfer vehicles into somatic cells, when rendered replication defective (for review, see Yeh andPerricaudet, 1997). Adenoviruses naturally enter human airway cells and produce progeny virions within thenucleus of the infected cell (for review, see Horwitz, 1990). The Ad-2 particle is icosahedral and has adiameter of approximately 90 nm. It contains at least 11 different structural polypeptides and a linear doublestranded DNA molecule, which is condensed with proteins V, VII, µ. The DNA also contains about 10copies of the cysteine protease p23 and is covalently associated with a terminal protein at its 5’ ends (Mangel,

4

et al., 1993, Stewart, et al., 1993). The chromosome is connected to the inside wall of the capsid via proteinVI. The capsid consists of 6 to 7 different proteins. More than 75% of its mass is contributed by the hexonprotein. This major protein is held together by homophilic interactions and by the minor protein IX. ProteinIIIa is another capsid stabilizing component linking adjacent facets of the icosahedron. The vertices of thecapsid are made up of pentameric penton base and protruding trimeric fibers. The fibers are built by a linearshaft and a carboxy-terminal globular head domain of 200 amino acids containing the receptor bindingdeterminant (Louis, et al., 1994, Xia, et al., 1995).

For entry into cells, Ad-2 and Ad-5 use an immunoglobulin gene family protein, termed CAR (Coxsackie Bvirus, an unrelated RNA virus, and Adenovirus Receptor), as an initial attachment site (Bergelson, et al., 1997,Tomko, et al., 1997). This receptor binds with high affinity to the fiber head domain. The precise nature ofthe interaction, i.e., the on and off rates or amino acid residues making up the interface, are not yet known.The Ig domain is most likely directly involved in fiber binding, since recently, the Ig domain containing MHCclass I protein has also been identified as a functional primary receptor for Ad-5 (Hong, et al., 1997). Besidesthe fiber receptor, the alpha m-beta 2 integrin of hematopoietic cells has also been proposed to function as ahigh affinity attachment site in the absence of fiber proteins (Huang, et al., 1996). Purified trimeric fiberbinds with high affinity to epitheloid cells, but is not endocytozed (Wickham, et al., 1993). For endocytosis,the virus utilizes a secondary receptor, the fibronectin binding alpha v-beta 3/5 integrin, as demonstrated bycDNA transfection experiments in integrin deficient cell lines (Wickham, et al., 1993). DNA transfectionexperiments in human airway cells or lymphocytes have confirmed that expression of the alpha v-beta 3/5integrins is essential for efficient virus infection (Goldman and Wilson, 1995, Huang, et al., 1995). It is,however, not established if integrins are true mediators of virus endocytosis or whether they exert an assistingfunction. Recently, other integrins, such as alpha 5-beta 1 have also been suggested to assist Ad5internalization thus raising the possibility that multiple related entry pathways could be used by differentadenoviruses (Davison, et al., 1997).

For virus uptake, the penton base plays a critical role. Its arginine-glycine-aspartate (RGD)-domain isexposed in five small protrusions at each of the 12 vertices of the icosahedron as indicated by cryo electronmicroscopy and cristallographic modeling (Stewart, et al., 1997). It engages with alpha v and thus somehowtriggers clathrin-dependent endocytosis (Bai, et al., 1993, Greber, et al., 1996, Svensson, 1985, Varga, et al.,1991, Wickham, et al., 1994, Wickham, et al., 1993). The RGD motive is present in penton base proteins offour different subgroups (A, B, C, E). Synthetic RGD peptides, which prevent fibronectin binding tointegrins, block Ad-2 endocytosis, but not attachment to cells suggesting that the primary receptor binds tofiber independently of integrins. Endocytosis of Ad-2 is highly efficient and accompanied by quantitativeshedding of the fibers from the capsid (Greber, et al., 1993). It is possible that fiber detachment is aprerequisite for the virus endocytosis. However, virus uptake via integrins can also occur, if fibers are notquantitatively released from the capsid as demonstrated with the ts1 Ad-2 mutant grown at the nonpermissivetemperature (Greber, et al., 1996). This virus lacks functional protease and contains a capsid which is notproteolytically processed (Anderson, 1990, Rancourt, et al., 1995). It presumably binds to the same primaryreceptor as the wild type virus, is endocytozed into the cells with indistinguishable efficiency and kinetics aswild type virus, but unable to penetrate the endosomal membrane and instead is recycled back to the plasma

5

membrane (Greber, et al., 1996). It is possible that ts1 fibers are shed at only those vertices which mediatecell contact, but left attached on opposite pentons.

With wild type virus, integrin binding to penton base somehow assists capsid penetration of the endosomalmembrane, which takes place approximately 15 min after internalization (Greber, et al., 1993, Wickham, et al.,1994). Penetration is enhanced by acidic endosomal pH (Pastan, et al., 1986, Prchla, et al., 1995), but theprecise mechanism is unknown. In particular, at high multiplicity of infection, penetration can occur in theabsence of acidic endosomal pH (Greber, unpublished results, and Rodriguez and Everitt, 1996). Pentonbase-integrin interactions together with reducing agents in endosomes or the cytosol then reactivate the viralcysteine protease p23 inside the capsid. p23 degrades the internal protein VI (Cotten and Weber, 1995,Greber, et al., 1996). This step is essential to weaken the capsid and to prepare it for final dissociation andDNA import into the nucleus.

2.2. Papovaviridae: Polyoma virus and Simian virus 40 (SV40)

Papovaviridae are a family of small non-enveloped viruses with icosahedral capsids. The name is derivedfrom three prototypic members, rabbit papilloma virus, mouse polyoma virus and simian virus 40 (SV40)(earlier called vacuolating virus). The genomes of these tumorigenic viruses are made up of a single circulardouble stranded DNA molecule, which is replicated in the nucleus. Particularly, polyoma virus and SV40have been intensely studied by virologists and molecular biologists during the past 30 years (for review, seeCole, 1996). A considerable amount of information is also available about their modes of cell entry. Incontrast, much less is known about entry mechanisms of the related papilloma viruses, largely because ofdifficulties propagating these agents in the laboratory (see zur Hausen and de Villiers, 1994).

Polyoma virus and SV40 capsids have a diameter of approximately 50 nm and consist of three structuralproteins Vp1, Vp2, and Vp3 (Stehle, et al., 1996). The capsid enwraps a double stranded closed circular DNAof about 5 kb, which is condensed into a minichromosome by four core histones, H2A, H2B, H3 and H4(Hendrix and Garcea, 1994). The major coat protein Vp1 forms a total of 72 pentameric capsomers. Eitherof the two interiorly located minor capsid proteins, Vp2 and Vp3, is thought to contact both, theminichromosome and the Vp1 pentameric capsomer (Clever, et al., 1993).

Polyoma virus and SV40 infect a large variety of mammalian cells and tissue. The outcome of the infectioncan be productive or non-productive, largely depending on how the newly synthesized viral large T antigeninteracts with the host replication machinery. Infection starts with virus attachment to a surface receptor.Competiton experiments indicated that polyoma virus and SV40 use different receptors for attachment(Basak, et al., 1992). Polyoma virus Vp1 binds to a cell surface sialic acid determinant (Stehle and Harrison,1996, Stehle, et al., 1994). This step enhances the tumorigenicity of the virus and is thus thought to beinvolved in the productive entry pathway. SV40 appears to enter rhesus monkey kidney cells by endocytosisinvolving attachment to MHC class I molecules at the cell surface as suggested by antibody inhibitionexperiments (Breau, et al., 1992). SV40 adsorption to lymphoblastoid cells lacking MHC class I expressionon the surface due to failure of beta 2 microglobulin or HLA class I expression has not been observed.

6

Different results were, however, obtained with different cells. Polarized monkey kidney cells express HLAclass I on both sides, apically and basolaterally, yet SV40 only attaches to the apical membrane (Basak, et al.,1992). These results suggest that MHC class I may be a possible binding determinant for SV40, but theremay be additional binding factors present on cellular membranes.

After surface binding, SV40 is internalized via smooth, clathrin-free vesicles, which were identified as caveolae(Anderson, et al., 1996, Stang, et al., 1997). Caveolae are cholesterol-rich, pot-shaped, smooth plasmamembrane invaginations of approximately 70 to 90 nm width. Their precise function is unknown, but theyhave been implicated in endocytosis, transcytosis and intracellular signalling (for reviews, see Dangoria, et al.,1996, Ikonen, 1997, Parton, 1996, Stralfors, 1997). It is possible that caveolae are internalized and targeted tointernal membranes, such as the ER. It is interesting that the majority of the incoming SV40 particles arefound within vesicular compartments including endosomes and the ER and rarely also the nuclear envelopelumen (Kartenbeck, et al., 1989, Maul, et al., 1978, Yamada and Kasamatsu, 1993). There is, however, noconcrete evidence that entry of SV40 into the ER is a productive route of delivering DNA into thenucleoplasm (Greber and Kasamatsu, 1996, Norkin and Anderson, 1996). It is possible that the entrypathway via caveolae and endosomes is far more complex than anticipated. Conceivably, productive SV40entry could occur by attachment to MHC class I or other receptors in the vicinity of caveolae. Internalizationand membrane penetration could then be mediated by some secondary receptor(s) or other trigger factors,perhaps similar to entry of adenovirus.

2.3. Parvoviridae: Parvovirus and adeno-associated virus (AAV)

Parvoviruses are among the smallest of the DNA viruses. Within an icosahedral capsid of about 18 to 26 nm,they contain a linear single stranded DNA molecule of about 5’000 bases (for review, see Berns, 1996).Autonomous parvoviruses, examplified by human parvovirus B19, replicate without a helper virus, whiledependoviruses, such as adeno-associated virus AAV, require functions of helper viruses, like adenovirus orherpes virus, for their replication. Usually, the capsids are made up of three viral proteins, VP1, VP2 and themajor capsid protein VP3. Newly synthesized structural proteins and a nonstructural protein NS1 aredirected into the nucleus for progeny virus assembly (Nuesch and Tattersall, 1993, Wistuba, et al., 1997).Based on tryptic digests, VP1, VP2 and VP3 seem to be related to each other, perhaps even derived from acommon DNA sequence.

Although AAV and parvovirus vectors, together with adenoviruses, are among the most popular vehicles for invivo gene transfer into non-dividing cells, very little is known about their entry mode. Hemagglutination andantibody inhibition studies together with cryoelectron microscopy suggested that human parvovirus B19attached to the blood group P antigen, a glycolipid called globoside (Brown, et al., 1993, Chipman, et al.,1996). VP1 of the autonomous mouse minute virus is probably required to initiate a productive infection, butit may not be needed for virus attachment to cells or DNA replication (Tullis, et al., 1993). Perhaps, VP1 isrequired during virus entry into cells. For entry of AAV into permissive human cell lines, N-linked glycansof a 150 kD glycoprotein have been suggested as a cell surface attachment factor (Mizukami, et al., 1996). Towhich viral capsid protein this determinant binds and whether it is both, a necessary and sufficient factor for

7

AAV infection is presently unknown. Furthermore, virtually nothing is known about the subsequent steps ofnuclear targeting and import of viral DNA.

ENVELOPED VIRUSES

2.4. Herpesviridae: Herpes simplex virus type 1 (HSV-1)

Herpes simplex virus types 1 and 2 are the prototypic human herpes viruses. They are among the mostcomplex but also most intensely studied viruses (for review, see Roizman and Sears, 1996). The HSVparticle is made up of four structural elements, an electron dense inner core containing a linear doublestranded DNA molecule of about 150 kb, an icosahedral capsid around the core, an amorphous mass calledtegument around the capsid, and an outer lipid envelope containing viral membrane proteins. It is estimatedthat the complete virion contains about 40 or so different proteins, many of which are surface glycoproteins.From numerous studies over the past years it became apparent that incoming virus can use multiple pathwaysfor attachment. There is no single viral surface protein responsible for productive engagement with a surfacereceptor of nonpolarized epithelial cells.

The most prominent among the cellular receptor molecules described for HSV is heparan sulfate, a heparinbinding component of the extracellular matrix (Seck, et al., 1994, Spear, 1993). It occurs in limiting amountsand accounts for at least 85% of the virus attachment sites in nonpolarized epithelial cells. Other receptorswere proposed for various viral surface glycoproteins and include basic fibroblast growth factor (Baird, et al.,1990, Kaner, et al., 1990), mannose 6-phosphate receptor (Brunetti, et al., 1995), Fc receptors (Johnson, et al.,1988) and HVEM, a herpes virus entry mediator protein of the tumor necrosis factor gene family(Montgomery, et al., 1996, Whitbeck, et al., 1997). Whether all of these surface molecules can separatelymediate virus entry or whether they act as co-receptors is under investigation.

After HSV attachment to the cell surface, fusion between viral and cellular membranes is mediated by viralsurface glycoproteins. Fusion can occur efficiently at neutral pH suggesting that it may take place at the cellsurface rather than in acidic endosomes (for review, see Roizman and Sears, 1996). It is, however, possiblethat under certain conditions, the virus utilizes the endocytic uptake machinery for passing through the corticalactin underneath the cell membrane and then fuses in a pH-independent manner out of endosomes (Marshand Pelchen-Matthews, 1994). In cases where virus does not enter by endocytosis, it is conceivable that thecapsid passes through the cortical actin with the help of an actin-dependent motor protein, such as a memberof the myosin family implicated in organellar trafficking (Lin, et al., 1997, Pollard and Ostap, 1996, Welch, etal., 1997). Interestingly, cytochalasin B, which interferes with filamentous actin, has been reported to inhibitHSV-1 entry into neurites of rat dorsal root ganglia (Lycke, et al., 1984).

2.5. Hepadnaviridae: Hepatitis B virus (HBV)

Hepatitis B virus (HBV) is the major cause of human hepatitis (for reviews, see Ganem, 1996, Schaefer andGerlich, 1995). Among the three virus related particles existing in an infected individual, only the enveloped

8

DNA containing particle, the so called “Dane” particle named after its discoverer, confers infectivity. Thegenome of “Dane” particles is a relaxed circular, partially duplexed DNA molecule of about 3.2 kb. Itencodes surface proteins, the nucleocapsid forming C protein, the DNA-associated polymerase andpotentially other polypetides of unknown functions.

Despite a great deal of efforts, little is known about the early steps of infection. This is partly due todifficulties of efficiently reproducing early steps in cultured cells. Most information about the early steps ofinfection comes from studies of duck hepatitis B virus (dHBV). The cell biology of this virus is in manyrespects similar to that of human hepatitis B virus (hHBV). dHBV efficiently enters freshly isolated primaryduck hepatocytes. It is thought that the amino-terminal domain, pre-S1, of the large lipid-anchored surfaceglycoprotein L contains a receptor binding determinant (Klingmuller and Schaller, 1993, Qiao, et al., 1994).The subsequent fusion between the viral and cellular membrane apparently occurs at neutral pH (Rigg andSchaller, 1992), although the underlying mechanism is unknown (Offensperger, et al., 1991). Thenucleocapsid is then released into the cytoplasm and transported to the nucleus, where the genomic DNA isrepaired into a closed circular DNA molecule for ensuing RNA polymerase II transcription and packaging ofcomplete RNA into newly assembled nucleocapsids in the cytoplasm. By reverse transcription, RNA withinthe cytoplasmic capsids is then copied into a double-stranded DNA molecule. Virus particles bud throughinternal membranes and are finally released by vesicular transport into the extracellular medium.

2.6. Baculoviridae: Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV)

Baculoviruses are a diverse group of large DNA viruses characterized by a circular double stranded DNAgenome, which is packaged into a rod-shaped (latin “baculum”) capsid and enveloped with a lipid membrane(for recent review, see Miller, 1996). They occur in two genera, nuclear polyhedrosis viruses (NPVs) andgranulosis viruses (GVs). They replicate and produce progeny capsids in the nucleus of insect cells. Theyare often named according to their host. For example, the commonly used baculovirus vector for proteinexpression in insect cells is the alfalfa looper Autographa californica nuclear polyhedrosis virus (AcNPV).During their life cycle, baculoviruses occur in two forms, an occluded form and an open form. Occludedcomplete virions are embedded in a characteristic proteinacious matrix in the nucleus of the infected cell.After cell lysis in the infected insect, these inclusions are dissolved in the acidic environment of the midgutlumen and released viruses can be taken up by another epithelial cell. Alternatively, nucleocapsids cansomehow exit the nucleus, bud across the plasma membrane and thus aquire a lipid membrane in a nonlyticprocess. This process may be mechanistically similar to virus budding in mammalian cells, such as HSV-1budding from human epitheloid cells (see above). Extracellular virus then enters non-infected cells byreceptor-mediated endocytosis involving the viral surface protein gp64, passes through acidic endosomes andreleases the nucleocapsid after membrane fusion into the cytoplasm (Volkman and Goldsmith, 1985,Volkman and Goldsmith, 1988). Whether a less efficient pH-independent fusion at the plasma membranecan be an alternative infectious pathway is currently uncertain. Likewise, a cell surface receptor and additionalstimuli for membrane penetration are not known.

3. NUCLEAR ENVELOPE TARGETING

9

While penetration of cellular membranes can be accomplished by viral capsid proteins without the activeassistance of host proteins (for review of enveloped virus fusion mechanisms see White, 1990), the movementof large virus particles through the cytoplasm requires cellular activities. The 90 nm large adenovirus particle,for example, is not transported to the nuclear envelope when cell metabolism is slowed down (Greber andKasamatsu, 1996). Likewise, passive diffusion of synthetic beads larger than 40 to 50 nm in the cytoplasmdoes not occur due to steric hindrance and high viscosity, even under physiological conditions (Luby-Phelps,1994).

Currently, there is evidence for two different modes of virus transport through the cytoplasm, microtubule-dependent movement and actin-driven motility. In principle, both mechanisms could be operating on eithernaked cytoplasmic capsids or lipid-enveloped capsids, such as incoming endosomal or newly synthesizedviruses (Cudmore, et al., 1997, Greber and Kasamatsu, 1996). Cell-assisted motility thus allows viruses torapidly proceed to the intracellular site of replication or find a selective path for egress.

3.1. Herpes simplex virus (HSV-1)

Targeting of HSV-1 to the nucleus is one of the best studied intracellular movements of an incoming virusparticle, since this process is important for cell to cell spread of infectivity within the neuronal system of both,animals and humans. HSV infects epithelial cells of the mucosa and can be latent or lytic in a variety ofneurons in the periphery and the central nervous system (Roizman and Sears, 1996). It is thought to enterneuronal cells at the presynatic membrane. Transport to the cell nucleus then occurs via retrograde movementof the capsid along microtubules (Bosem, et al., 1990, Kristensson, et al., 1986, Lycke, et al., 1984, Topp, etal., 1994). Electron microscopy and indirect immunofluorescence studies in fibroblastic Vero cells haverecently demonstrated that incoming HSV-1 capsids are closley associated with microtubules (Sodeik, et al.,1997). In these studies, cytoplasmic dynein has been found in the vicinty of HSV-1 capsids in closeproximity to microtubules. In many cells, dynein occurs in the dynactin complex as a large protein assemblyof about 1.2 MDa implicated in transporting cellular vesicles along microtubules (Rickard and Kreis, 1996,Schroer, 1994, Vallee and Sheetz, 1996). When most of the microtubules in Vero cells were depolymerizedwith nocodazole or cholchicine, capsid targeting to the nucleus was slowed down, but not completely blocked.It is possible that those viruses, which attached to the plasma membrane proximal to the nucleus, arrived at thenuclear envelope independently of microtubules. Alternatively, it is conceivable that nocodazole-resistantmicrotubules still served as tracks for nuclear targeting. When microtubules were collapsed intoparacristalline arrays around the nucleus by treating cells with vincristine, nuclear targeting of HSV wasinhibited similar to the nocodazole results. The data would thus suggest that the retrograde-directed motorprotein dynein could carry HSV-1 along microtubules. Which of the capsid or tegument proteins iscontacting the dynein/dynactin complex and if dynein is sufficient to generate retrograde HSV movementremains to be determined.

3.2. Nuclear polyhedrosis virus (NPV)

10

Morphological studies on Autographa californica NPV entry into insect cells have suggested thatcytoplasmic viral capsids induced the formation of actin cables (Charlton and Volkman, 1993). Actinpolymerization did not require protein synthesis and was only observed after the capsid penetrated theendosomal membrane. Often times, virion capsids appeared to be located at the tips of actin filaments. Twovirion derived proteins have been implicated so far in the regulation of actin polymerization, an actin-bindingand perhaps actin-polymerizing protein and an actin degrading viral protease (Lanier, et al., 1996). Whethercapsid motility within the cytoplasm is entirely derived from polymerizing actin similar to the mobile bacteriaShigella and Listeria (Pollard, 1995), or whether microtubules are also involved in driving NPV through thecytoplasm will be important to determine. Interestingly, treatment of insect cells with the microtubule-stabilizing agents taxol was reported to delay virus replication, but colchicine had no effect (Volkman andZaal, 1990).

3.3. Other viruses

Much less is known about nuclear targeting of other DNA viruses. Purified Ad-5 has been shown toassociate in vitro with polymerized microtubules, which contained high molecular weight microtubule-associated proteins (Luftig and Weihing, 1975, Weatherbee, et al., 1977). Electron microscopy in humanepithelial cells has visualized incoming Ad-2 and Ad-5 particles in association with microtubules (Dales andChardonnet, 1973, Miles, et al., 1980). Often times, distinctive globular projections of 10 to 20 nm were seenat the vertices of the capsids in these images. Whether these projections represent a physiological motor or acytoplasmic linker protein is unknown.

The mechanisms, by which SV40 is transported through the cytoplasm are controversial. Electronmicroscopy and measurement of early gene expression in CV-1 cells suggested that disruption ofmicrotubules with a variety of inhibitors blocked virus transport to the nucleus (Shimura, et al., 1987).However, when purified SV40 was microinjected into fibroblastic cells, disruption of microtubules bynocodazole treatment had little effect on capsid protein targeting to the nucleus (for review, see Greber andKasamatsu, 1996). It is possible that SV40 naturally enters cells by passing through endosomes and thattransport of virions within endosomes towards the nucleus requires microtubules or, alternatively, thatnocodazole resistant microtubules were mediating nuclear targeting. Interestingly, receptor-mediated uptakeof the SV40-related bovine papilloma virus into primary hepatocytes required functional dynamicmicrotubules and actin microfilaments as suggested by taxol and cytochalasin B inhibition experiments(Muller, et al., 1995, Zhou, et al., 1995).

4. NUCLEAR IMPORT: UNCOATING, DOCKING, TRANSLOCATION

Nuclear targeting of a virus capsid is generally not sufficient for import of the genomic DNA into thenucleoplasm. The capsid around the DNA must be broken up, the DNA released and translocated across thenuclear envelope. All nuclear DNA viruses studied so far, appear to be using nuclear import pathways thatconverge at nuclear pore complexes (NPCs).

11

NPCs are large molecular assemblies of about 124 MDa embedded in the double lipid bilayer of the nuclearenvelope (Reichelt, et al., 1990). In an average HeLa cell nucleus, about 3000 NPCs are evenly distributedover approximately 300 µm2 (Maul, 1977). The NPC is an eight fold symmetrical structure with peripheralnuclear and cytoplasmic ring elements and a central plaque-spokes complex (for review, see Davis, 1995).The plaque-spokes complex harbours two types of channels, eight peripherally located aqueous channels forpassive diffusion of small proteins and solutes (Hinshaw, et al., 1992), and a central channel harboring a socalled transporter complex. The peripheral diffusional channels are thought to allow passage of smallmolecules up to about 50 kDa. The maximal NPC diameter is thought to be located in the center of the NPC.It functions in an energy-dependent manner and can - in exponentially growing cells - accommodate colloidalgold particles as large as 27 nm (Feldherr and Akin, 1991). Filaments radiating from the nuclear rings inwardare forming a basket-like structure with a terminal ring element. Cytoplasmic filaments etending from thecytoplasmic rings outward are somewhat thicker in appearance than the nuclear filaments and are thought toprovide a binding site for incoming cargo. NPCs are anchored in the membrane by the pore membranedomain, a specialized subdomain of the nuclear envelope (NE), which lines the pore and links the outer andinner nuclear membranes (for reviews see Davis, 1995, Panté and Aebi, 1996). The pore membrane domain isan integral part of the scaffolding framework and contains transmembrane proteins, such as the major NPCglycoprotein gp210 or Pom121 (for review see Gerace and Foisner, 1994).

Nuclear import of most cellular proteins depends on nuclear localization signals (NLSs) and signal decodingmachineries (for different models, see Duverger, et al., 1995, Görlich and Mattaj, 1996, Melchior and Gerace,1995, Panté and Aebi, 1996, Pollard, et al., 1996, Rexach and Blobel, 1995). Classical NLSs have beendefined by the SV40 large T antigen sequence PKKKRKV and the bipartite nucleoplasmin motifKRPAATKKAGQAKKK (capital letters representing single amino acid code: P=Proline, K=Lysine;R=Arginine; V=Valine; A=Alanine; T=Threonine; G=Glycine; Q=Glutamine) (Dingwall and Laskey, 1991,Kalderon, et al., 1984). These sequences are recognized in the cytosol by karyopherin/importin alpha. Thealpha receptor then docks with the cargo to the karyopherin/importin beta at the cytoplasmic filaments of theNPC. O-linked glycoproteins of the NPC are directly or indirectly involved in this docking process (Adamand Adam, 1994). Recently, additional NLSs have emerged, such as the glycine rich M9 sequence firstidentified in the heterogeneous nuclear ribonucleoprotein (hnRNP) A1 (Siomi and Dreyfuss, 1995). Acytosolic M9 receptor, termed transportin, has been identified in mammalian cells, and also in yeast cells,termed Kar104p (Aitchison, et al., 1996, Pollard, et al., 1996). Translocation of the signal bearing moleculethen occurs via the central transporter region of the NPC. This step is GTP- and temperature-dependent andrequires the small GTP-binding protein Ran/TC4, and other cytosolic factors.

Lumenal factors of the NE also have a role in maintaining the functionality of the NPC. The NE cisternae aremajor intracellular calcium stores (for review see Pozzan, et al., 1994). Depletion of calcium from internalstores by calcium ionophores or the calcium ATPase inhibitor thapsigargin blocked passive diffusion andsignal-mediated transport across the nuclear envelope in somatic mammalian cells and frog oocytes (Greberand Gerace, 1995, Stehno-Bittel, et al., 1995, Sweitzer and Hanover, 1996). The inhibition is most likely dueto a sterical block of the central and peripheral NPC transport channels, as suggested by atomic forcemicroscopy in Xenopus nuclear envelopes (Perez-Terzic, et al., 1996). Such a transport block can be

12

bypassed by elevating the cytosolic calcium concentrations. Under these conditions, nuclear import ofclassical NLS-bearing proteins became calmodulin-dependent and no longer required Ran-TC4, but stilloperated through NPCs in an ATP dependent manner (Sweitzer and Hanover, 1996).

4.1. Adenovirus type 2 (Ad-2)

A number of electron microscopy studies indicated that numerous incoming adenovirus capsids attach to thecytoplasmic side of NPCs, but never was a capsid found within the nucleus (for reviews, see Dales, 1973,Greber and Kasamatsu, 1996, Horwitz, 1990). Quantitative electron microscopy of thin sectioned epitheloidHeLa cells indicated that about 29% of the capsids, which were located within 2 capsid diameters at the NE,were attached to NPCs (Greber, et al., 1996). This number is probably even underestimating the real extent ofcapsid association with NPCs, since it is possible that thin sections crossed through a capsid at an NPC, butspared the underlying NPC. Interestingly, only partially disassembled capsids, but not so called “empty”capsids were found in association with NPCs,. It is possible that the weakly stained “empty” particlesobserved at the NE in earlier studies represented partially disassembled capsids (Dales, 1973).

The first adenovirus capsids arrive at the nuclear envelope of HeLa cells about 20 min post infection,depending on the distance between the portal of entry at the plasma membrane and the nuclear envelope (NE).At 60 min post infection, the majority of incoming capsids were found at the NE as indicated by fluorescentlylabeled virions (Greber, et al., 1997). Cytoplasmic capsids contained fewer proteins than the extracellularparticles and mainly consisted of the major hexon protein (present in 720 copies per intact virion) and 15 to40% of the penton base (about 10 to 30 copies) (Greber, et al., 1993). During entry capsid proteins wereshed in a stepwise process. The first protein released was the primary cell surface attachment factor, fiber,followed by the capsid-stabilizing proteins IIIa and VIII. Protein IX, a cementing factor keeping the hexonstogether was removed between 30 and 60 min after entry. Removal of protein IX coincided with the onset ofcapsid disassembly, but was not sufficient for disassembly. Capsid disassembly also required the activity ofa virus-resident cysteine protease, p23 (Cotten and Weber, 1995, Greber, et al., 1996, Weber, 1995). If p23was inactivated either chemically or by mutagensis, capsid disassembly was inhibited and cell infectionimpaired. During virus entry, p23 gets reactivated by at least two different triggers, virus attachment to cellsurface integrins and reducing intracellular milieu. Activated p23 degraded an internal capsid protein, VI, andthus enabled the detachment of the viral DNA from the shell. The loss of stabilizing proteins includingprotein VI degradation, was still not sufficient for capsid disassembly and DNA import into the nucleus. Theincoming capsid must also associate with the NPC thus triggering DNA release (Greber, et al., 1997). Thiswas demonstrated by specific inhibitors of O-linked glycoproteins of the NPC, the RL1 antibody and wheatgerm agglutinin. In living cells, these inhibitors blocked virus attachment to the NE and also capsiddisassembly. It can be speculated that some surface-exposed determinants of either hexon or penton base areinvolved in attaching the capsid to the NPC. Protein IX is less likely to be involved in attachment andtriggering disassembly, since it does not appear to be located at the capsid surface (Stewart, et al., 1993).Whether capsid disassembly is triggered by an integral NPC component directly or together with aperipherally attached cytosolic component is not known yet.

13

After capsid disassembly, DNA and associated protein VII were imported into the nucleus through NPCs.This was demonstrated by immunocytochemistry and sensitive fluorescent in situ hybridization protocolscombined with confocal laser scanning microscopy (Greber, et al., 1997). NPC function was blocked bydepletion of calcium from internal stores using ionophores or a calcium ATPase inhibitor, thapsigargin.Calcium store depletion did not affect virus targeting to NPCs or capsid disassembly, but excluded incomingviral DNA from the nuclear interior. This data directly implies that functional NPCs are required for nuclearimport of incoming viral genome through NPCs. A classical NLS of the terminal protein p55, which iscovalently attached to the incoming viral DNA, has been identified (Zhao and Padmanabhan, 1988). Whetherthis signal is involved in threading the linear DNA through the NPC is not known. p55 is, however, requiredfor directing the viral DNA to specific sites of the nuclear matrix and for initiation of viral transcription (forreview, see Shenk, 1996).

4.2. Simian virus 40 (SV40)

In contrast to adenoviruses, SV40 executes a much less efficient entry program in cultured fibroblastic celllines. Only one out of 200 particles is able to produce progeny in these in vitro systems (Cole, 1996). It isnot entirely clear what the limiting step(s) is. Possibly, many of the isolated particles are damaged and thusnot infectious. When SV40 particles were microinjected into the cytoplasm or the nucleus, every 10th or evenevery single particle, respectively, lead to the expression of large T antigen (Diacumakos and Gershey, 1977,Gersey and Diacumakos, 1978). This data would suggest that damaged particles were retained at the earliestbarriers, the plasma membrane and the cytoplasm. Indeed, cytoplasmically injected chromosal SV40 DNAdevoid of capsid proteins was not imported into the nucleus, although it was complexed with NLS-bearinghistones (Nakanishi, et al., 1996). Conversely, microinjected particles lacking DNA readily entered thenucleus suggesting that the capsid contained some nuclear targeting information.

Mutagenesis experiments on individual capsid proteins have identified classical NLSs in the structuralproteins Vp1, Vp2 and Vp3 (Ishii, et al., 1996). The NLS of Vp1 is a bipartite signal comprising the amino-terminal 19 residues, in which two clusters of basic residues are independently important for nuclearlocalization activity. In the intact virion, there are 360 Vp1 NLSs hidden in the capsid near theminichromosome (Liddington, et al., 1991). The classical NLS of Vp2 and Vp3 is located close to thecarboxyl end of the both proteins, but the precise location of the Vp2 and Vp3-NLS in the interior of thevirion is not known (Ishii, et al., 1996).

SV40 particles injected into the cytoplasm accessed the nucleus via NPCs (Clever, et al., 1991, Yamada andKasamatsu, 1993). Most likely, this route of entry represents the natural infectious pathway, sincecytoplasmically administered antibodies against capsid proteins or NPC inhibitors blocked the expression ofT antigen (Nakanishi, et al., 1996). In electron microscopy studies, the presence of electron dense virion-likeparticles in the nucleus has been noted (Yamada and Kasamatsu, 1993). Whether these particles truelyrepresent incoming capsids and contain the viral genome is unknown. In addition, it is not clear how the 50nm large SV40 capsid is transported through the NPC channel, which can maximally accommodate goldparticles of 27 nm diameter (Davis, 1995). One possibility is that conformational changes alter the shape of

14

the cytoplasmic capsids, as suggested by in vitro uncoating experiments (Colomar, et al., 1993, Frost andBourgaux, 1975). It is possible that such changes expose a latent NLS on some SV40 capsid proteinmediating capsid association with the NPC and finally nuclear import. In what kind of a configuration theviral DNA enters the nucleus is, however, not known. It is possible that subsequent to nuclear import someadditional disassembly steps are necessary for viral transcription, as suggested by nuclear microinjections ofantibodies against the Vp3 capsid protein (Nakanishi, et al., 1996). It is, however, uncertain if all papovaviruses are taken into the nucleus for disassembly or if disassembly could mainly occur in the cytoplasm.Bovine papilloma virus capsid proteins, for example, are imported into the nucleus via classical NLSs, butincoming capsids have not been observed in the nucleoplasm supporting the notion that the virus capsiddissociated before reaching the nucleoplasm (Zhou, et al., 1995).

4.3. Herpes simplex virus type 1 (HSV-1)

As with adenoviruses, incoming HSV-1 capsids have been seen in large numbers at the nuclear envelope inassociation with NPCs (for review, see Roizman and Sears, 1996). Interestingly, also so called “empty”,electron luscent capsids were frequently seen at NPCs. It is, however, not known if these particles are a trueintermediates of the DNA release process or if they still contain DNA, perhaps in a different configurationthan the native capsids. Studies with the HSV-1 mutants tsB7 and 50B indicated that one or several viralfunctions are involved in DNA release from the capsids (Batterson, et al., 1983, Tognon, et al., 1981),analogous to the adenovirus protease p23, required for capsid dissociation and DNA release (Greber, et al.,1996). At the restrictive temperature, the tsB7 capsids at the nuclear envelope have an electron denseappearance, but become electron luscent after lowering the temperature suggesting that some changes in thecapsids have occurred (Batterson, et al., 1983). Unfortunately, we do not know exactly which capsidcomponents are affected in the tsB7 or B50 mutants. Whether the DNA is released and then transportedthrough the NPC also remains to be determined.

4.4. Hepatitis B virus (HBV)

The viral capsid, which comprises DNA, complexed core protein, and polymerase, is released into thecytoplasm, and somehow transported to the nuclear envelope. Within the nucleus, genome replication occursvia an RNA polymerase II-generated RNA intermediate. (Ganem, 1996). Thus, DNA must be imported intothe nucleus to initiate the infectious cycle.

Mutagenesis studies on the core protein have identified a classical NLS (Eckhardt, et al., 1991). Since it isthought that this NLS could overlap with the nucleic acid binding domain, it may not be functional on anucleocapsid (for references, see Kann, et al., 1997). In vitro binding studies have shown thatphosphorylation of the core protein by a protein kinase C (PKC) activity supresses RNA binding (Kann andGerlich, 1994). Since a PKC-like activity was found in isolated human HBV (Gerlich, et al., 1982, Kann andGerlich, 1994), it is possible that core phosphorylation exposes an NLS on the surface of the capsids. Suchan NLS then might mediate capsid association with the nuclear envelope. A nuclear membrane association ofincoming capsid-containing DNA was suggested by biochemical fractionation experiments using the hepatic

15

HepG2 cells (Qiao, et al., 1994). It is, however, not known if capsid association with the nuclear membranewas entirely due to attachment at NPCs or if the nuclear membrane interaction was of indirect nature in theseexperiments. In addition, nothing is known about the configuration of the capsid associated with the nuclearenvelope.

How nuclear import of the DNA actually occurs is still under investigation. HBV DNA-polymerase complexisolated from woodchock liver was apparently sufficient for nuclear import in permeabilized cultured cells asassayed by semi-quantitative polymerase chain reaction (Kann, et al., 1997). Nuclear import of DNA-polymerase complex was ATP- and cytosol-dependent and in this respect, similar to signal-mediated nuclearprotein import. These data would suggest that capsid proteins were not essential for DNA translocation.Redundancy in the involved signals is, however, still possible, since small amounts of capsid protein couldhave been present in the isolated polymerase-DNA complexes. It will be interesting to determine the signalsand mechanisms of DNA translocation through the NPC.

4.5. Nuclear polyhedrosis virus (NPV) and Granulosis virus (GV)

As with the other viruses described here, association of the nucleocapsid of baculoviruses with the NPC hasbeen described by electron microscopy without many functional biochemical or genetic studies (Miller, 1996,Summers, 1971). Even though so called “empty” capsids were found attached to NPCs, it is not clear ifthese capsids represented true intermediates in the release process of the DNA or whether they were notrelated to the actual import process at all. Based on the dimensions of the capsids (approximately 50 nm), itcan be expected that the rod-like nucleocapsids are dissociated at least to some extent before the DNA transitsacross the NE.

5. CONCLUSIONS

Most DNA viruses replicate their genomes in the cell nucleus. The mechanisms of DNA delivery to thenucleus are different for different virus families and may differ even among members within a family.Common steps for nuclear delivery include virus attachment to a suitable cell surface receptor, penetration ofthe plasma or endosomal membrane, targeting to the nuclear envelope and finally translocation across thenuclear pore complex (see Tables 1 and 2). As the virus passes from one step to the next, its structure isaltered. Completion of one entry step is often necessary to facilitate a correct excecution of a subsequentstep.

If we want to figure out, how DNA viruses manage to overcome the barriers separating the nucleus from theextracellular milieu we will have to understand the interdependent virus entry steps in correlation with adetailed structural map of the virus particle. We have to extend the large body of existing literature onmorphological aspects of virus entry by functional cell biological and genetic assays, and directly measure theefficiency of a particular entry step. With the advent of sensitive fluorescent DNA in situ hybridizationprotocols detecting single copies of incoming DNA genomes, as described for adenovirus, some of the toolsare now readily available for addressing these burning questions. If we understand the mechanisms

16

regulating intracellular trafficking of viral genomes, we will not only expand our knowledge of normal andpathological cellular function, but will also know better how to use viruses for gene delivery protocols inmolecular medicine.

ACKNOWLEDGEMENT

Work from the author’s laboratory was supported by the Kanton of Zurich and a grant from the SwissNational Science Foundation.

17

6. TABLES

Table 1: Animal DNA viruses replicating in the nucleus

Virus Familiy Genome(type)

Genome(kb)

Nucleocapsid(nm)

Prototypic Virus Host

Non-enveloped

Adenoviridae l-ds 34 90 Adenovirus type 2 or 5 (Ad-2, Ad-5)

Human

Papovaviridae c-ds 5-8 40-50 Papilloma virusPolyoma virusSimian virus 40 (SV40)

RabbitMouseMonkey

Parvoviridae l-ss 5 18-26 Adeno-associated virus(AAV)Parvovirus B19

Human

HumanDog

Enveloped

Baculoviridae c-ds 90-200 ca 40 x 400 Nuclear polyhedrosis virus(NPV)

Insects (e.g.alfalfalooper)

Hepadnaviridae c-pds 3.2 25 Hepatitis B virus (HBV) HumanDuck

Herpesviridae l-ds 150 100 Herpes simplex virus type 1(HSV-1)

Human

18

Table 2: Virus-cell interactions in the delivery of DNA to the nucleus

Event Cellular mediator Virus References

Surface attachment Ig domain (CAR, MHC I)

Sialic acidMHC class IGlycolipidN-linked glycansHeparan sulfate and additional factors

Factor X binding to gp64

Ad-2, Ad-5

Polyoma virusSV40Parvovirus B19MMVHSV-1

AcNPV

(Bergelson, et al., 1997, Hong, et al., 1997,Tomko, et al., 1997)(Stehle, et al., 1994)(Breau, et al., 1992)(Brown, et al., 1993, Chipman, et al., 1996)(Mizukami, et al., 1996)(Brunetti, et al., 1995, Montgomery,et al., 1996, Roizman and Sears, 1996,Spear, 1993, Whitbeck, et al., 1997)(Volkman and Goldsmith, 1988)

Plasma membrane fusion(pH independent)

???

HSV-1dHBVAcNPV

(Roizman and Sears, 1996)(Ganem, 1996)(Miller, 1996)

Endocytosis pH dependent penetration

pH independent penetration

Integrin αv β5 and ?

?Caveolae

Ad-2

AcNPVSV40

(Greber, et al., 1993, Nemerow, et al., 1994,Pastan, et al., 1986, Varga, et al., 1991)(Miller, 1996)(Anderson, et al., 1996, Stang, et al., 1997)

Nuclear targeting Microtubules

Microtubules?

Microtubules?

Microtubules/ActinActin polymerization?

HSV-1

Ad-2

SV40

Papilloma virusAcNPV

(Lycke, et al., 1984, Penfold, et al., 1994,Sodeik, et al., 1997, Topp, et al., 1994)

(Dales and Chardonnet, 1973, Luftig andWeihing, 1975, Miles, et al., 1980,Weatherbee, et al., 1977)(Greber and Kasamatsu, 1996,Shimura, et al., 1987)(Muller, et al., 1995, Zhou, et al., 1995)(Charlton and Volkman, 1993)

Capsid disassembly NPCNucleoplasmic factor(s)?

Ad-2SV40

(Greber, et al., 1997)(Cole, 1996)

Nuclear import NPCNPC?NPC

NPC?NPC?NPC?

Ad-2Polyoma virusSV40

HSV-1dHBVAcNPV

(Greber and Kasamatsu, 1996)(Zhou, et al., 1995)(Clever, et al., 1991,Yamada and Kasamatsu, 1993)(Batterson, et al., 1983, Tognon, et al.,1981)(Kann, et al., 1997)(Miller, 1996, Summers, 1971)

19

7. REFERENCES

Adam EJH and Adam SA (1994) Identification of cytosolic factors required for nuclear location sequence-mediated binding to the nuclear envelope J. Cell Biol. 125, 547-555

Aitchison JD, Blobel G and Rout MP (1996) Kap104p - a Karyopherin Involved In the Nuclear Transport OfMessenger Rna Binding Proteins Science 274, 624-627

Anderson CW (1990) The proteinase polypeptide of adenovirus serotype 2 virions Virol. 177, 259-272

Anderson HA, Chen YZ and Norkin LC (1996) Bound Simian Virus 40 Translocates to Caveolin-EnrichedMembrane Domains, and Its Entry Is Inhibited By Drugs That Selectively Disrupt Caveolae MolecularBiology of the Cell 7, 1825-1834

Bai M, Harfe B and Freimuth P (1993) Mutations that alter an Arg-Gly-Asp (RGD) sequence in theadenovirus type 2 penton base protein abolish its cell-rounding activity and delay virus reproduction in flatcells J. Virol. 67, 5198-5205

Baird A, Florkiewicz RZ, Maher PA, Kaner RJ and Hajjar DP (1990) Mediation of virion penetration intovascular cells by association of basic fibroblast growth factor with herpes simplex virus type 1 Nature 348,344-346

Basak S, Turner H and Compans RW (1992) Expression of SV40 receptors on apical surfaces of polarizedepithelial cells Virol. 190, 392-402

Batterson W, Furlong D and Roizman B (1983) Molecular genetics of Herpes simplex virus. VIII. Furthercharacterization of a temperature-sensitive mutant defective in release of viral DNA and in other stages of theviral reproductive cycle J. Virol. 45, 397-407

Bergelson JM, Cunningham JA, Droguett G, Kurt-Jones EA, Krithivas A, Hong JS, Horwitz MS, Crowell RLand Finberg RW (1997) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5Science 275, 1320-1323

Berns KI (1996) Parvoviridae: the viruses and their replication. In Fundamental virology, Fields BN, KnipeDM and Howley PM eds.), Raven Press, Philadelphia, pp. 1017-1041.

Bosem ME, Harris R and Atherton SS (1990) Optic nerve involvement in viral spread in herpes simplex virustype 1 retinitis Invest Ophthalmol Vis Sci 31, 1683-1689

Breau WC, Atwood WJ and Norkin LC (1992) Class I major histocompatibility proteins are an essentialcomponent of the Simian Virus 40 receptor J. Virol. 66, 2037-2045

20

Brown KE, Anderson SM and Young NS (1993) Erythrocyte P antigen: cellular receptor for B19 parvovirusScience 262, 114-117

Brunetti CR, Burke RL, Hoflack B, Ludwig T, Dingwell KS and Johnson DC (1995) Role of mannose-6-phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission J Virol 69, 3517-3528

Charlton CA and Volkman LE (1993) Penetration of Autographa californica nuclear polyhedrosis virusnucleocapsids into IPLB Sf 21 cells induces actin cable formation Virology 197, 245-254

Chipman PR, Agbandje-McKenna M, Kajigaya S, Brown KE, Young NS, Baker TS and Rossmann MG(1996) Cryo-electron microscopy studies of empty capsids of human parvovirus B19 complexed with itscellular receptor Proc Natl Acad Sci U S A 93, 7502-7506

Clever J, Dean DA and Kasamatsu H (1993) Identification of a DNA binding domain in simian virus 40capsid proteins Vp2 and Vp3 J. Biol. Chem. 268, 20877-20883

Clever J, Yamada M and Kasamatsu H (1991) Import of simian virus 40 virions through nuclear porecomplexes Proc. Natl. Acad. Sci. U.S.A. 88, 7333-7337

Coffin JM (1996) Retroviridae: the viruses and their replication. In Fundamental Virology, Fields BN, KnipeDM and Howley PM eds.), Lippincott-Raven, New York.

Cole CN (1996) Polyomavirinae: The viruses and their replication. In Fundamental Virology, Fields BN,Knipe DM and Howley PM eds.), Lippincott-Raven, New York, pp. 917-945.

Colomar MC, Degoumois-Sahli C and Beard P (1993) Opening and refolding of simian virus 40 and in vitropackaging of foreign DNA J. Virol. 67, 2779-2786

Cotten M and Weber JM (1995) The adenovirus protease is required for virus entry into host cells Virol.213, 494-502

Cudmore S, Reckmann I and Way M (1997) Viral manipulations of the actin cytoskeleton Trends Microbiol5, 142-148

Dales S (1973) Early events in cell-animal virus interactions Bacteriol. Rev. 37, 103-135

Dales S and Chardonnet Y (1973) Early events in the interaction of Adenoviruses with HeLa cells. IV.Association with microtubules and the nuclear pore complex during vectorial movement of the inoculumVirol. 56, 465-483

21

Dangoria NS, Breau WC, Anderson HA, Cishek DM and Norkin LC (1996) Extracellular Simian Virus 40Induces an Erk/Map Kinase-Independent Signalling Pathway That Activates Primary Response Genes andPromotes Virus Entry Journal of General Virology 77, 2173-2182

Davis LI (1995) The nuclear pore complex Annu. Rev. Biochem. 64, 865-896

Davison E, Diaz RM, Hart IR, Santis G and Marshall JF (1997) Integrin Alpha-5-Beta-1-MediatedAdenovirus Infection Is Enhanced By the Integrin-Activating Antibody Ts2/16 Journal of Virology 71, 6204-6207

Diacumakos EG and Gershey EL (1977) J. Virol. 24, 903-906

Dingwall C and Laskey RA (1991) Nuclear targeting sequences--a consensus. [Review] Trends inBiochemical Sciences 16, 478-481

Duverger E, Pellerinmendes C, Mayer R, Roche AC and Monsigny M (1995) Nuclear Import OfGlycoconjugates Is Distinct From the Classical Nls Pathway Journal of Cell Science 108, 1325-1332

Eckhardt SG, Milich DR and McLachlan A (1991) Hepatitis B virus core antigen has two nuclear localizationsequences in the arginine-rich carboxyl terminus J Virol 65, 575-582

Feldherr CM and Akin D (1991) Signal-mediated nuclear transport in proliferating and growth-arrestedBALB/c 3T3 cells Journal of Cell Biology 115, 933-939

Fields BN, Knipe DM and Howley PM (1996) Fundamental Virology: 3 Edition, Lippincott-Raven, NewYork, pp. Pages.

Freed EO, Englund G and Martin MA (1995) Role of the basic domain of human immunodeficiency virustype 1 matrix in macrophage infection J Virol 69, 3949-3954

Frost E and Bourgaux P (1975) Decapsidation of polyoma virus: identification of subviral species Virology68, 245-255

Ganem D (1996) Hepadnaviridae and their replication. In Fundamental Virology, Fields BN, Knipe DM andHowley PM eds.), Lippincott-Raven, New York, pp. 1199-1233.

Gerace L and Foisner R (1994) Integral membrane proteins and dynamic organization of the nuclear envelopeTrends Cell Biol. 4, 127-131

Gerlich WH, Goldmann U, Muller R, Stibbe W and Wolff W (1982) Specificity and localization of thehepatitis B virus-associated protein kinase J Virol 42, 761-766

22

Gersey EL and Diacumakos EG (1978) J. Virol. 28, 415-416

Goldman MJ and Wilson JM (1995) Expression of alpha-v-beta-5 integrin is necessary for efficientadenovirus-mediated gene transfer in the human airway J. Virol. 69, 5951-5958

Görlich D and Mattaj IW (1996) Protein Kinesis - Nucleocytoplasmic Transport Science 271, 1513-1518

Greber UF and Gerace L (1995) Depletion of calcium from the lumen of the endoplasmic reticulumreversibly inhibits passive diffusion and signal-mediated transport into the nucleus J. Cell Biol. 128, 5-14

Greber UF and Kasamatsu H (1996) Nuclear targeting of adenovirus and simian virus SV40 Trends CellBiol. 6, 189-195

Greber UF, Nakano MY and Suomalainen M (1997) Adenovirus entry into cells by quantitative fluorescencemicroscopy Meth. Mol. Biol. in press

Greber UF, Suomalainen M, Stidwill RP, Boucke K, Ebersold M and Helenius A (1997) The role of thenuclear pore complex in adenovirus entry EMBO J. in press

Greber UF, Webster P, Weber J and Helenius A (1996) The role of the adenovirus protease in virus entryinto cells EMBO J. 15, 1766-1777

Greber UF, Willetts M, Webster P and Helenius A (1993) Stepwise dismantling of adenovirus 2 during entryinto cells Cell 75, 477-486

Hendrix RW and Garcea RL (1994) Capsid assembly of dsDNA viruses Sem. Virol. 5, 15-26

Hinshaw JE, Carragher BO and Milligan RA (1992) Architecture and design of the nuclear pore complexCell 69, 1133-1141

Hong SS, Karayan L, Tournier J, Curiel DT and Boulanger PA (1997) Adenovirus type 5 fiber knob binds toMHC class I alpha2 domain at the surface of human epithelial and B lymphoblastoid cells EMBO J. 16,2294-2306

Horwitz MS (1990) Adenoviridae and their replication. In Virology, Fields BN and Knipe DM eds.), RavenPress, New York, pp. 1679-1721.

Horwitz MS (1990) Adenoviruses. In Virology, Fields BN and Knipe DM eds.), Raven Press, New York, pp.1723-1740.

23

Huang S, Endo RI and Nemerow GR (1995) Upregulation of integrins alpha v beta 3 and alpha v beta 5 onhuman monocytes and T lymphocytes facilitates adenovirus-mediated gene delivery J Virol 69, 2257-2263

Huang S, Kamata T, Takada Y, Ruggeri ZM and Nemerow GR (1996) Adenovirus Interaction With DistinctIntegrins Mediates Separate Events In Cell Entry and Gene Delivery to Hematopoietic Cells Journal ofVirology 70, 4502-4508

Ikonen E (1997) Molecular mechanisms of intracellular cholesterol transport Curr Opin Lipidol 8, 60-64

Ishii N, Minami N, Chen EY, Medina AL, Chico MM and Kasamatsu H (1996) Analysis of a nuclearlocalization signal of simian virus 40 major capsid protein Vp1 J. Virol. 70, 1317-1322

Johnson DC, Frame MC, Ligas MW, Cross AM and Stow ND (1988) Herpes simplex virusimmunoglobulin G Fc receptor activity depends on a complex of two viral glycoproteins, gE and gI J Virol62, 1347-1354

Kalderon D, Richardson WD, Markham AF and Smith AE (1984) Sequence requirements for nuclearlocation of simian virus 40 large-T antigen Nature (London) 311, 33-38

Kaner RJ, Baird A, Mansukhani A, Basilico C, Summers BD, Florkiewicz RZ and Hajjar DP (1990)Fibroblast growth factor receptor is a portal of cellular entry for herpes simplex virus type 1 [see comments]Science 248, 1410-1413

Kann M, Bischof A and Gerlich WH (1997) In Vitro Model For the Nuclear Transport Of the HepadnavirusGenome J. Virol. 71, 1310-1316

Kann M and Gerlich WH (1994) Effect of core protein phosphorylation by protein kinase C onencapsidation of RNA within core particles of hepatitis B virus J. Virol. 68, 7993-8000

Kartenbeck J, Stukenbrok H and Helenius A (1989) Endocytosis of Simian Virus 40 into the endoplasmicreticulum J. Cell Biol. 109, 2721-2729

Klingmuller U and Schaller H (1993) Hepadnavirus infection requires interaction between the viral pre-Sdomain and a specific hepatocellular receptor J Virol 67, 7414-7422

Kristensson K, Lycke E, Roytta M, Svennerholm B and Vahlne A (1986) Neuritic transport of herpessimplex virus in rat sensory neurons in vitro. Effects of substances interacting with microtubular function andaxonal flow [nocodazole, taxol and erythro-9-3-(2-hydroxynonyl)adenine] J Gen Virol 67, 2023-2028

Lanier LM, Slack JM and Volkman LE (1996) Actin binding and proteolysis by the baculovirus AcMNPV:the role of virion-associated V-CATH Virology 216, 380-388

24

Liddington RC, Yan Y, Moulai J, Sahli R, Benjamin TL and Harrison SC (1991) Structure of simian virus 40at 3.8-A resolution Nature 354, 278-284

Lin CH, Espreafico EM, Mooseker MS and Forscher P (1997) Myosin drives retrograde F-actin flow inneuronal growth cones Biol Bull 192, 183-185

Louis N, Fender P, Barge A, Kitts P and Chroboczek J (1994) Cell-binding domain of adenovirus serotype 2fiber J Virol 68, 4104-4106

Luby-Phelps K (1994) Physical properties of the cytoplasm Curr. Op. Cell Biol. 6, 3-9

Luftig RB and Weihing RR (1975) Adenovirus binds to rat brain microtubules in vitro J. Virol. 16, 696-706

Lycke E, Kristensson K, Svennerholm B, Vahlne A and Ziegler R (1984) Uptake and transport of herpessimplex virus in neurites of rat dorsal root ganglia cells in culture J Gen Virol 65, 55-64

Mangel WF, McGrath WJ, Toledo DL and Anderson CW (1993) Viral DNA and a viral peptide can act ascofactors of adenovirus virion proteinase activity Nature (London) 361, 274-275

Marsh M and Pelchen-Matthews A (1994) The endocytic pathway of virus entry. In Cellular receptors foranimal viruses, Wimmer E (ed.), Cold Spring Harbor Laboratory Press, New York, pp. 215-240.

Maul GG (1977) The nuclear and cytoplasmic pore complex. Structure, dynamics, distribution and evolutionInt. Rev. Cytol. Suppl. 6, 75-186

Maul GG, Rovera G, Vorbrodt A and Abramczuk J (1978) Membrane fusion as a mechanism of simian virus40 entry into different cellular compartments J Virol 28, 936-944

Melchior F and Gerace L (1995) Mechanisms of nuclear protein import Curr. Op. Cell Biol. 7, 310-318

Miles BD, Luftig RB, Weatherbee JA, Weihing RR and Weber J (1980) Quantitation of the interactionbetween adenovirus types 2 and 5 and microtubules inside infected cells Virol. 105, 265-269

Miller L (1996) Insect viruses. In Fundamental Virology, Fields BN, Knipe DM and Howley PM eds.),Lippincott-Raven, New York, pp. 401-424.

Mizukami H, Young NS and Brown KE (1996) Adeno-associated virus type 2 binds to a 150-kilodalton cellmembrane glycoprotein Virology 217, 124-130

Montgomery RI, Warner MS, Lum BJ and Spear PG (1996) Herpes simplex virus-1 entry into cellsmediated by a novel member of the TNF/NGF receptor family Cell 87, 427-436

25

Moss B (1996) Poxviridae: the viruses and their replication. In Fundamental Virology, Fields BN, Knipe DMand Howley PM eds.), Raven Press, Philadelphia, pp. 1163-1197.

Muller M, Gissmann L, Cristiano RJ, Sun XY, Frazer IH, Jenson AB, Alonso A, Zentgraf H and Zhou JA(1995) Papillomavirus Capsid Binding and Uptake By Cells From Different Tissues and Species Journal ofVirology 69, 948-954

Nakanishi A, Clever J, Yamada M, Li PP and Kasamatsu H (1996) Association With Capsid ProteinsPromotes Nuclear Targeting Of Simian Virus 40 Dna Proceedings of the National Academy of Sciences ofthe United States of America 93, 96-100

Nemerow GR, Cherish DA and Wickham TJ (1994) Adenovirus entry into host cells: a role for αv integrins

Trends Cell Biol. 4, 52-55

Norkin LC and Anderson HA (1996) Multiple stages of virus-receptor interactions as shown by simian virus40 Adv Exp Med Biol 408, 159-167

Nuesch JP and Tattersall P (1993) Nuclear targeting of the parvoviral replicator molecule NS1: evidence forself-association prior to nuclear transport Virology 196, 637-651

Offensperger WB, Offensperger S, Walter E, Blum HE and Gerok W (1991) Inhibition of duck hepatitis Bvirus infection by lysosomotropic agents Virology 183, 415-418

Panté N and Aebi U (1996) Sequential binding of import ligands to distinct nucleopore regions during theirnuclear import Science 273, 1729-1732

Panté N and Aebi U (1996) Toward the molecular dissection of protein import into nuclei Curr. Op. CellBiol. 8, 397-406

Parton RG (1996) Caveolae and caveolins Curr Opin Cell Biol 8, 542-548

Pastan I, Seth P, FitzGerald D and M. W (1986) Adenovirus entry into cells: some new observations on anold problem, Springer Verlag, New York, pp. Pages.

Penfold ME, Armati P and Cunningham AL (1994) Axonal transport of herpes simplex virions to epidermalcells: evidence for a specialized mode of virus transport and assembly Proc. Natl. Acad. Sci. U S A 91, 6529-6533

Perez-Terzic C, Pyle J, Jaconi M, Stehno-Bittel L and Clapham DE (1996) Conformational states of thenuclear pore complex induced by depletion of nuclear calcium stores Science 273, 1875-1877

26

Pollard TD (1995) Actin Cytoskeleton - Missing Link For Intracellular Bacterial Motility Current Biology 5,837-840

Pollard TD and Ostap EM (1996) The chemical mechanism of myosin-I: implications for actin-based motilityand the evolution of the myosin family of motor proteins Cell Struct Funct 21, 351-356

Pollard VW, Michael WM, Nakielny S, Siomi MC, Wang F and Dreyfuss G (1996) A Novel Receptor-Mediated Nuclear Protein Import Pathway Cell 86, 985-994

Pozzan T, Rizzuto R, Volpe P and Meldolesi J (1994) Molecular and cellular physiology of intracellularcalcium stores Physiol. Rev. 74, 595-636

Prchla E, Plank C, Wagner E, Blaas D and Fuchs R (1995) Virus-mediated release of endosomal content invitro: different behavior of adenovirus and rhinovirus serotype 2 J. Cell Biol. 131, 111-123

Qiao M, Macnaughton TB and Gowans EJ (1994) Adsorption and penetration of hepatitis B virus in anonpermissive cell line Virology 201, 356-363

Qiao M, Macnaughton TB and Gowans EJ (1994) Adsorption and penetration of hepatitis B virus in anonpermissive cell line Virology 201, 356-363

Rancourt C, Keyvaniamineh H, Sircar S, Labrecque P and Weber JM (1995) Proline 137 is critical foradenovirus protease encapsidation and activation but not enzyme activity Virol. 209, 167-173

Reichelt R, Holzenburg A, Buhle EJ, Jarnik M, Engel A and Aebi U (1990) Correlation between structure andmass distribution of the nuclear pore complex and of distinct pore complex components J. Cell Biol. 110,883-894

Rexach M and Blobel G (1995) Protein import into nuclei: association and dissociation reactions involvingtransport substrate, transport factors, and nucleoporins Cell 83, 683-692

Rickard JE and Kreis TE (1996) Clips For Organelle-Microtubule Interactions Trends in Cell Biology 6,178-183

Rigg RJ and Schaller H (1992) Duck hepatitis B virus infection of hepatocytes is not dependent on low pH JVirol 66, 2829-2836

Rodriguez E and Everitt E (1996) Adenovirus Uncoating and Nuclear Establishment Are Not Affected ByWeak Base Amines Journal of Virology 70, 3470-3477

27

Roizman B and Sears AE (1996) Herpes viruses and their replication. In Fundamental Virology, Fields BN,Knipe DM and Howley PM eds.), Lippincott-Raven, New York, pp. 1043-1107.

Schaefer S and Gerlich WH (1995) In vitro transformation by hepatitis B virus DNA Intervirology 38, 143-154

Schroer TA (1994) New insights into the interaction of cytoplasmic dynein with the actin-related protein,Arp1 J Cell Biol 127, 1-4

Seck T, Lingen M, Weise K and Falke D (1994) Evidence for a multistep mechanism for cell-cell fusion byherpes simplex virus with mutations in the syn 3 locus using heparin derivatives during fusion from withinArch Virol 136, 173-181

Shenk T (1996) Adenoviridae. In Fundamental Virology, Fields BN, Knipe DM and Howley PM eds.),Lippincott-Raven, New York, pp. 979-1016.

Shimura H, Umeno Y and Mimura G (1987) Effects of inhibitors of the cytoplasmic structures and functionson the early phase of infection of cultured cells with simian virus 40 Virol. 158, 34-43

Siomi H and Dreyfuss G (1995) A nuclear localization domain in the hnRNP A1 protein J Cell Biol 129,551-560

Sodeik B, Ebersold MW and Helenius A (1997) Microtubule-Mediated Transport Of Incoming HerpesSimplex Virus 1 Capsids to the Nucleus Journal of Cell Biology 136, 1007-1021

Spear PG (1993) Entry of alpha herpesviruses into cells Sem. Virol. 4, 167-180

Stang E, Kartenbeck J and Parton RG (1997) Major Histocompatibility Complex Class I Molecules MediateAssociation Of SV40 With Caveolae Molecular Biology of the Cell 8, 47-57

Stehle T, Gamblin SJ, Yan YW and Harrison SC (1996) The Structure Of Simian Virus 40 Refined At 3.1Angstrom Resolution Structure 4, 165-182

Stehle T and Harrison SC (1996) Crystal Structures Of Murine Polyomavirus In Complex With Straight-Chain and Branched-Chain Sialyloligosaccharide Receptor Fragments Structure 4, 183-194

Stehle T, Yan Y, Benjamin TL and Harrison SC (1994) Structure of murine polyomavirus complexed with anoligosaccharide receptor fragment Nature 369, 160-163

Stehno-Bittel L, Perez-Terzic C and Clapham DE (1995) Diffusion across the nuclear envelope inhibited bydepletion of the nuclear envelope calcium store Science 270, 1835-1838

28

Stevenson M (1996) Portals of entry: uncovering HIV nuclear transport pathways Trends Cell Biol. 6, 9-15

Stewart PL, Chiu CY, Huang S, Muir T, Zhao YM, Chait B, Mathias P and Nemerow GR (1997) Cryo-EmVisualization Of an Exposed Rgd Epitope On Adenovirus That Escapes Antibody Neutralization EMBOJournal 16, 1189-1198

Stewart PL, Fuller SD and Burnett RM (1993) Difference imaging of adenovirus: bridging the resolution gapbetween X-ray crystallography and electron microscopy EMBO J. 12, 2589-2599

Stralfors P (1997) Insulin second messengers Bioessays 19, 327-335

Summers MD (1971) Electron microscopic observations on granulosis virus entry, uncoating and replicationprocesses during infection of the midgut cells of Trichoplusia ni J Ultrastruct Res 35, 606-625

Svensson U (1985) Role of vesicles during Adenovirus 2 internalization into HeLa cells. J. Virol. 55, 442-449

Sweitzer TD and Hanover JA (1996) Calmodulin Activates Nuclear Protein Import - a Link Between SignalTransduction and Nuclear Transport Proceedings of the National Academy of Sciences of the United States ofAmerica 93, 14574-14579

Tognon M, Furlong D, Conley AJ and Roizman B (1981) Molecular genetics of herpes simplex virus. V.Characterization of a mutant defective in ability to form plaques at low temperatures and in a viral functionwhich prevents accumulation of coreless capsids at nuclear pores late in infection J. Virol. 40, 870-880

Tomko RP, Xu R and Philipson L (1997) HCAR and MCAR: The human and mouse cellular receptors forsubgroup C adenoviruses and group B coxsackieviruses Proc. Natl. Acad. Sci. USA 94, 3352-3356

Topp KS, Meade LB and LaVail JH (1994) Microtubule polarity in the peripheral processes of trigeminalganglion cells: relevance for the retrograde transport of herpes simplex virus J. Neurosci. 14, 318-325

Trono D (1995) HIV accessory proteins: leading roles for the supporting cast Cell 82, 189-192

Tullis GE, Burger LR and Pintel DJ (1993) The minor capsid protein VP1 of the autonomous parvovirusminute virus of mice is dispensable for encapsidation of progeny single-stranded DNA but is required forinfectivity J Virol 67, 131-141

Vallee RB and Sheetz MP (1996) Targeting of motor proteins Science 271, 1539-1544

Varga MJ, Weibull C and Everitt E (1991) Infectious entry pathway of Adenovirus type 2 J. Virol. 65, 6061-6070

29

Volkman LE and Goldsmith PA (1985) Mechanism of neutralization of budded Autographa californicanuclear polyhedrosis virus by a monoclonal antibody inhibition of entry by adsorpive endocytosis Virol. 143,185-195

Volkman LE and Goldsmith PA (1988) Resistance of the 64K protein of budded Autographa californicanuclear polyhedrosis virus to functional inactivation by proteolysis Virology 166, 285-289

Volkman LE and Zaal KJ (1990) Autographa californica M nuclear polyhedrosis virus: microtubules andreplication Virology 175, 292-302

Weatherbee JA, Luftig RB and Weihing RR (1977) Binding of adenovirus to microtubules. II. Depletion ofhigh-molecular-weight microtubule-associated protein content reduces specificity of in vitro binding J. Virol.21, 732-742

Weber JM (1995) The adenovirus endopeptidase and its role in virus infection. In Molecular repertoire ofadenoviruses, Doerfler W and Bohm P eds., , pp. 227-235.

Welch MD, Mallavarapu A, Rosenblatt J and Mitchison TJ (1997) Actin dynamics in vivo Curr Opin CellBiol 9, 54-61

Whitbeck JC, Peng C, Lou H, Xu RL, Willis SH, Deleon MP, Peng T, Nicola AV, Montgomery RI, WarnerMS, Soulika AM, Spruce LA, Moore WT, Lambris JD, Spear PG, Cohen GH and Eisenberg RJ (1997)Glycoprotein D Of Herpes Simplex Virus (Hsv) Binds Directly to Hvem, a Member Of the Tumor NecrosisFactor Receptor Superfamily and a Mediator Of Hsv Entry J. Virol. 71, 6083-6093

White JM (1990) Viral and cellular membrane fusion proteins Annu. Rev. Physiol. 52, 675-697

Whittaker G, Bui M and Helenius A (1996) The role of nuclear import and export in influenza virus infectionTrends Cell Biol. 6, 67-71

Wickham TJ, Filardo EJ, Cheresh DA and Nemerow GR (1994) Integrin αvβ5 selectively promotesadenovirus mediated cell membrane permeabilization J. Cell Biol. 127, 257-264

Wickham TJ, Mathias P, Cheresh DA and Nemerow GR (1993) Integrin-alpha-v-beta-3 and integrin-alpha-v-beta-5 promote adenovirus internalization but not virus attachment Cell 73, 309-319

Williams AF (1991) Cellular interactions. Out of the equilibrium Nature 352, 473-474

Wimmer E (1994) Cellular receptors for animal viruses, Cold Spring Harbor Laboratory Press, New York,pp. Pages.

30

Wistuba A, Kern A, Weger S, Grimm D and Kleinschmidt JA (1997) Subcellular compartmentalization ofadeno-associated virus type 2 assembly J Virol 71, 1341-1352

Xia D, Henry LJ, Gerard RD and Deisenhofer J (1995) Crystal structure of the receptor-binding domain ofadenovirus type 5 fiber protein at 1.7 A resolution Structure 2, 1259-1270

Yamada M and Kasamatsu H (1993) Role of nuclear pore complex in simian virus 40 nuclear targeting J.Virol. 67, 119-130

Yeh P and Perricaudet M (1997) Advances in adenoviral vectors: from genetic engineering to their biologyFASEB J. 11, 615-623

Zhao L-J and Padmanabhan R (1988) Nuclear transport of adenovirus DNA polymerase is facilitated byinteraction with preterminal protein Cell 55, 1005-1015

Zhou J, Gissmann L, Zentgraf H, Muller H, Picken M and Muller M (1995) Early Phase In the Infection OfCultured Cells With Papillomavirus Virions Virology 214, 167-176

zur Hausen H and de Villiers EM (1994) Human papillomaviruses Annu. Rev. Microbiol. 48, 427-447