The alien replicon: Artificial genetic constructs to direct the synthesis of transmissible self-replicating RNAs

Prospects & Overviews

The alien replicon: Artificial geneticconstructs to direct the synthesis oftransmissible self-replicating RNAs

In vivo synthesised heterologous (alien) RNA constructs are capable of

initiating self-replication following transmission to the host organism

Alex V. Kochetov1)2)

Artificial genetic constructs that direct the synthesis of self-

replicating RNA molecules are used widely to induce gene

silencing, for bioproduction, and for vaccination. Interest-

ingly, one variant of the self-replicon has not been discussed

in the literature: namely, transgenic organisms that syn-

thesise alien replicons. For example, plant cells may be

easily genetically modified to produce bacteriophages or

insect viruses. Alien replicon-producing organisms (ARPOs)

may serve as a unique tool for biocontrol or to selectively

influence the characteristics of a target organism. The ARPO

approachwould have tomeet strict biosafety criteria, and its

practical applications are problematic. However, a discus-

sion on ARPO applicability would be valuable to outline the

full set of options available in the bioengineering toolbox. In

this paper, RNA replicons for bioengineering are reviewed

briefly, and the ARPO approach is discussed.

Keywords:.biocontrol; heterologous; replicon; RNA-viruses

Introduction: The ability to self-replicatemakes genetic constructs more efficient

Genetic constructs that can self-replicate in susceptibleorganisms are used widely in both basic and applied research(e.g. for virus-induced gene silencing (VIGS) [1, 2], develop-ment of DNA or RNA vaccines [3–5], and the bioproduction ofrecombinant proteins [6–8]). These constructs are commonlybased on the self-replicons that originate in viruses with DNAor RNA genomes (only RNA replicons are discussed here).

RNA replicons are derived from either positive- ornegative-strand RNA viruses. Positive single-stranded RNAviral genomes function as mRNA when transfected into ahost-cell cytoplasm. The best-studied animal vectors of thistype are derived from alphavirus genomes (e.g. Sindbis,Semliki Forest, and Venezuelan equine encephalitis viruses)and flaviviruses (Kunjin virus, yellow fever virus, amongothers). Alphavirus (Togaviridae) genomes encode four non-structural proteins, nsP1-P4, which are translated directlyfrom the genomic RNA and interact with host factors to formreplicative enzyme complexes. These replicative complexessynthesise negative-strand RNA intermediates, new viralgenomes, and sub-genomic RNA, which encodes structuralproteins necessary for viral particle assembly (Fig. 1A) [3].

Truncated self-amplifying replicons are derived fromgenomic cDNA by replacing the region encoding the viralstructural proteins with a gene of interest (GOI; Fig. 1B). Insusceptible cells, this RNA is translated and self-amplified;however, cell-to-cell transmission of infectious RNA cannotoccur. Instead, high levels of the GOI-encoded proteins aretranslated from sub-genomic RNAs within the transfectedcells. Even naked full-sized genomic RNA may penetrate intocertain cells, initiate replication and result in the spread of aninfection that mimics the corresponding virus in nature(‘infectious RNA’ [9]). RNA replicons are commonly usedwhen

DOI 10.1002/bies.201400111

1) Institute of Cytology & Genetics, SB RAS, Novosibirsk, Russia2) Novosibirsk State University, Novosibirsk, Russia

Corresponding author:Alex V. KochetovE-mail: [email protected]

Abbreviations:ARPO, alien replicon-producing organism; GOI, gene of interest; ORF, openreading frame; SFV, Semliki Forest virus; TRV, Tobacco Rattle virus; VIGS,virus-induced gene silencing; VLP, virus-like particle.

www.bioessays-journal.com 1Bioessays 36: 0000–0000,� 2014 WILEY Periodicals, Inc.

Problems&Paradigms

packaged into virus-like particles (VLPs) because this methodof delivery is more advantageous. The VLP assembly may bearranged in trans in cell culture, where the correspondingstructural proteins are synthesised from separate transgenes.

Genetic constructs derived from negative-strand RNAviruses may also be used for bioengineering. However, thenegative-strand viral genomic RNA is not infectious. In thiscase, genomic RNA must be transcribed by a pre-existing viralRNA polymerase into positive-strand mRNA. Thus, theapplication of negative-strand virus-based RNA repliconsrequires preliminary virion or VLP production. For example,the vesicular stomatitis virus has a single-molecule negative-strand RNA genome that encodes five major proteins (G, L,phosphoprotein, M, and nucleoprotein). Deleting the ORF G-encoding single envelope glycoprotein produces a propaga-tion-incompetent virus. Thus, a VSV-based RNA replicon maycarry genes for the viral polymerase complex (N, P, L), geneM,and one or several GOIs. VLP production is arranged in transin cell culture, wherein the proteins N, P, L, and G aresynthesised, and the RNA replicon is transcribed fromindependent genetic constructs. The envelope glycoproteinG determines specific tissue- and cell-tropism. The use of theheterologous glycoprotein G gene provides this type ofreplicon-bearing VLP with modified cell tropism [10].

Self-replicating RNAs efficientlyvaccinate animals

There are different types of nucleic acid-based vaccines.Plasmid DNA vaccines (bearing an antigen-encoding trans-gene controlled by an appropriate host promoter) have beenevaluated in animal models of diseases (e.g. [5, 11–13]).However, DNA vaccines typically induce immune responses oflower magnitude than conventional vaccines. It has also beendemonstrated that intra-muscular injection of mRNA in

mammals results in the local production of the encodedreporter protein [14] and may induce an immune responseagainst the encoded antigen [15]. This type of vaccinationdisplays certain advantages: typically, the construct is small,simple (it only comprises antigen-encoding ORFs bordered byuntranslated regions and a poly(A)-tail), and biologicallysafer. Also, mRNA needs only to be delivered into the host-cellcytoplasm for translation into protein to occur. In contrast,DNA must enter the nucleus to be transcribed into mRNA,which is then transported back into the cytoplasm fortranslation. Finally, the plasmid DNA may occasionallyintegrate into the host genome, whereas the integration ofRNA is impossible [15].

NakedmRNA delivery via the cell membrane likely uses anactive transport mechanism [16] involving scavenger recep-tors [17]. Upon internalisation, some mRNA molecules occurin the cytoplasm where they are translated into protein.However, mRNA is typically unstable in vivo; antigens may betranslated transiently and at a relatively low level, whichnecessitates repeated administration of high doses of RNA.Development of new techniques will likely further improvethe utility of mRNA-based vaccines (e.g. the use of lipidnanoparticles increases RNA stability and delivery ratesignificantly [18]).

One of the most attractive applications of animal RNAreplicons is for vaccination. Replicon-based RNA vaccines aresignificantly more potent than non-amplifying mRNA vac-cines; due to autonomous RNA replication, they can drivehigh levels of recombinant antigen expression. Researchershave also shown that double-stranded RNA intermediatesgenerated during alphavirus replicon activity may induce astrong innate antiviral response that increases vaccineefficiency considerably [19, 20]. For example, in a recentcomparison of experimental vaccines against human cyto-megalovirus that were based on plasmid DNA, alphavirusRNA replicons, or antigenic peptides demonstrated that onlythe RNA replicon vaccine elicited both humoral and cellularimmunity [21].

Replicon-based RNA vaccines may be designed in differentways. Antigens from highly pathogenic or immunosuppres-sive viruses may be expressed using a well-characterised,attenuated vector virus that serves as a vehicle for the vaccine.However, RNA viruses are highly prone to mutations, andpropagation-competent viral vectors may acquire enhancedvirulence. Thus, RNA replicons used for vaccination aretypically introduced using disabled viral vectors that are bothavirulent and unable to revert to virulence. In susceptiblecells, these RNAs are translated and self-amplified; however,cell-to-cell transmission of infectious RNA cannot occur(Fig. 1B; these replicons are referred to as ‘propagation-incompetent’, ‘non-transmissible’, ‘single-round infectiousparticles’, or ‘single-cycle’, among others).

Injection of naked RNA replicon vectors may raise humoraland/or cell-mediated immune responses to the antigenencoded [22]. However, RNA replicon delivery efficiency maybe increased substantially by packaging within VLPs [23],which protects the replicon RNA physically from degradationby environmental factors and yields a particular type ofcell tropism. VLP proteins are also immunogenic, and thisapproach may be limited by anti-vector immunity, as was

nsP1-nsP4 Capsid, E2/E1A)

nsP1-nsP4 transgene26S sgProm

B)

26S sgProm

Figure 1. Schematic illustration of alphavirus-based RNA-replicons.A: The 50-part of the alphavirus genome encodes four non-structuralproteins (nsP1-nsP4). These proteins are translated from genomicRNA in the cytoplasm of the host cell and form a replicationcomplex for further amplification steps. The 26S sub-genomicpromoter (26S sgProm) directs the transcription of sub-genomicmRNA encoding the structural proteins (capsid, E1 and E2 glyco-proteins) required for virion assembly and cell-to-cell transmission.RNA is 50-capped and 30-polyadenylated (to serve as mRNA in hostcells), and typically, all of these processes (replication, transcription,translation, virion assembly) occur within the cytoplasm. B: A typicalRNA-replicon derived from the alphavirus genome with the capsidand E2/E1 genes replaced with a transgene (gene of interest, GOI).

A. V. Kochetov Prospects & Overviews....

2 Bioessays 36: 0000–0000,� 2014 WILEY Periodicals, Inc.

Problems&Paradigms

discussed previously for adenoviruses [24–26], Vacciniavirus [27, 28], and Venezuelan equine encephalitis virus [29].

RNA replicons may be delivered as DNA constructs (‘DNA-layered RNA replicons’). In this case, the plasmid containscDNA of the RNA replicon that is controlled by a hostpromoter. Following transfection, the DNA is transcribed inthe nucleus into replicon mRNA, which is exported into thecytoplasm where translation and amplification occur [30].DNA-layered RNA replicons have the advantage that large-scale antigen production is feasible; however, they also havethe DNA vaccines’ safety problems. DNA-layered alphavirus-based RNA replicons have also been referred to as ‘suicidalDNA vaccines’ [31] because the transfected cells may undergoapoptosis, which further enhances the high levels of humoraland cell-mediated immunity [20].

Numerous reports on self-replicating RNA vaccines areavailable in the literature (for review, see [3, 32]). Recentexamples of alphavirus-derived RNA replicons include experi-mental vaccines against the bovine diarrhoea virus [33],HIV-1 [34], Ebola [35], dengue [36], and Peste des petitsruminants viruses [37]. In some cases, DNA-layered RNAreplicons were constructed based on propagation-deficientattenuated variants of the pathogenic virus: recent examplesinclude the disarmed Japanese encephalitis virus repliconcontrolled by the cytomegalovirus promoter [38] and thetruncated replicon of the Porcine reproductive and respiratorysyndrome virus [39].

Animal RNA replicons may be used foroncolytic virotherapy

Oncolytic viruses are therapeutically useful anticancer virusesthat will selectively infect and damage cancerous tissueswithout causing significant harm to normal tissues. Someviruses show a particular tropism toward tumour cells, othersmay bemodified to enhance their oncolytic properties [40, 41].Due to the strong apoptosis-inducing ability observed ininfected mammalian cells, alphavirus RNA replicons havebeen applied in tumour therapy [42]. The most popularapproach has been intra-tumour injection of alphavirusvectors that carry reporter and/or therapeutic genes [43,44]. An interesting class of DNA-layered RNA vaccines isintroduced via hybrid vectors, which are considered particu-larly promising for oncolytic virotherapy. For example, theVaccinia virus has been modified to encode both thepropagation-deficient Semliki Forest virus replicon and,separately, SFV structural genes. Infection with this recombi-nant virus generated single-round infectious SFV VLPs thatwere released into the medium and that infected surroundingtumour cells [45].

In the adenovirus/SFV hybrid-vector system, an SFV RNAreplicon was inserted into the adenovirus genome under thecontrol of the alpha-fetoprotein promoter, which enabled atumour-specific transcription pattern. The transgene wasexpressed under the control of the SFV sub-genomic promoterwithin the same expression unit. In vivo, these vectorsinduced apoptosis in a hepatocellular carcinoma animalmodel, initiated by SFV replication [46]. Researchers also

noted that adenovirus/alphavirus hybrid vectors mighttransduce malignant haematopoietic cells [47]. In general,adenovirus/alphavirus hybrid vectors have attracted consid-erable attention [48, 49]. Alphavirus RNA replicons may alsobe used as a vehicle to deliver genomes of other viruses; forexample, cell cultures infected with an SFV RNA repliconcontaining the Moloney murine leukaemia virus genomerather than its own structural genes may produce infectiousretrovirus particles [50].

Plant RNA replicons are powerful toolsfor bioengineering

Many plant viruses have RNA-positive genomes and may beused easily to construct RNA replicons in a manner similar toanimal alphaviruses (Fig. 1). Essentially, this approach may beillustrated as a plasmid-containing full-sized genomic cDNA ofan RNA-positive single-stranded virus that is controlled by anappropriate promoter; if transfected into cells of a susceptibleorganism, it initiates infection (virus production and spread-ing). For example, the plasmid pTY-S [51] contains an almostintact cDNA of monopartite RNA-positive turnip yellow mosaicvirus that is controlled by a plant promoter. The insertion ofa small inverted repeat targeted to the host gene underinvestigation into pTY-S induced efficient RNA interferencein susceptible plants. Silencing simply requires that plants beinoculated via abrasion with a few micrograms of intactplasmid because even low-level occasional transcriptionenables the synthesis of self-amplifying infectious viralRNA [51]. Similar infectious vectors were developed usingtobacco mosaic virus (TMV) [52], potato virus X [53], turnipmosaic virus [54], and tobacco necrosis virus A [55], amongother viruses. In other cases, the vectors contained truncatedviral genomes and supported self-replication inside thetransfected cell but not transmission to other cells ororganisms. For example, the vector TRBO contains cDNA fromthe TMV that is controlled by the 35S promoter; however, thecoat protein ORF is replaced with a GOI. This replicon cannotspread systemically but can direct the high-level local synthesisof a recombinant protein in agroinfiltrated leaves [56].

Plant self-replicated genetic constructs are used widely inbiotechnology and for basic research. Numerous studies haveused viral RNA replicons to produce a protein of interest.Due to high production levels (up to 5 g/kg), low cost, andbiological safety (for reviews, see [7, 8]), plants are attractiveas potential biofactories. A primary direction of the researchconcerns antigen production for human and animal immu-nisation, and the concept of ‘edible vaccines’ has also beenwidely discussed in the literature (for reviews, see [57, 58]).Coat proteins from many viruses produced in plants are self-assembled into VLPs and may be used both for efficientvaccination and as nanomaterials (e.g. [59]). Another highlyvaluable application of plant self-replicated genetic con-structs is VIGS. This technology has been exploited widely toinvestigate functions of plant genes [2].

The tobacco rattle virus (TRV)-based vector system isdescribed here as an example of a plant self-replicatinggenetic construct (Fig. 2).

....Prospects & Overviews A. V. Kochetov

3Bioessays 36: 0000–0000,� 2014 WILEY Periodicals, Inc.

Problems&Paradigms

TRV is a two-part single-stranded RNA-positive virus [60].RNA1 encodes two replicase proteins, amovement protein anda cysteine-rich silencing suppressor protein (16k). Thereplicase proteins are translated directly from RNA1, whereasthe movement and 16k proteins are translated from sub-genomic RNA. RNA2 is amplified by the RNA1-encodedreplicase and encodes the coat protein and two non-structuralproteins involved in virus plant-to-plant transmission bynematodes, but it does not influence replication and cell-to-cell transmission within the infected plant. RNA1 may self-replicate and move systemically in plants even without RNA2.Thus, RNA2 may be modified to bear either transgenes orRNAi-inducing antisense segment/inverted repeats [61]. Twovectors suitable for agrobacterial transformation should beused together: TRV1, which expresses unmodified RNA1(Fig. 2A), and TRV2, which contains RNA2 cDNA with non-essential genes replaced by a multiple cloning site or theGateway1 insertion site (Fig. 2B). A mixture of TRV1 and TRV2agrobacterial strains is used to transform by inoculatingleaves using a needleless syringe or via the agrodrenchmethod or by pricking the stem with a toothpick [62]. TRVhas a wide range of host plant species, and the repliconvectors have become a popular tool for RNAi-mediated targetgene silencing. In the case of Nicotiana benthamiana, thisprotocol requires only four weeks for implementation, whichis much more rapid than typical genetic transformationtechniques [61, 62]. If RNA2 contains a transgene rather thanan RNAi-inducing construct, the foreign protein will beexpressed efficiently (particularly in roots [63]).

Vector systems based on the plant DNA viruses alsoprovide a wide range of opportunities for bioproduction andgene silencing [64–67].

Alien replicon-producing organisms

The characteristic feature of the majority of DNA-layeredRNA replicons is that they are transferred directly into cells ofthe host organism. Following transmission, the replicon-bearing mRNA is transcribed by RNA polymerase II, exportedinto the cytoplasm, and translated; the synthesised proteins(together with the host factors involved) facilitate the

self-replication. However, in non-host organism cells, thisprocess is often inefficient: the mRNA may be transcribed andeven translated but not replicated because this processrequires fine-tuning between the viral and host factors. A fewinteresting exceptions are known: for example, flock housevirus (FHV) is a non-enveloped, icosahedral insect virus with agenome that comprises two single-stranded, positive-senseRNA molecules, and FHV genomic RNA may replicate in plantcells. A DNA-layered FHV RNA replicon facilitates infectiousvirus particle synthesis in transgenic plants [68].

However, it is possible to separate the organisms in whicha replicon’s primary synthesis occurs from organisms inwhich further replication/action occurs. Many RNA-positivesingle-stranded viral genomes include specific packing signals(pac-sites) that mediate their interactions with coat-proteinmolecules and virion assembly. Thus, if cells from the non-host organism express a genetic construct that facilitates theintracellular synthesis of both viral genomic RNA and the coatprotein, virus particle assembly may occur, and contact withthe host organism may result in viral transfection followedby productive infection. Robust and selective interactionbetween the RNA-replicon and the corresponding coat proteinis an important pre-requisite for this process. If a virusmeets these criteria, it may be considered a candidate forsynthesis in the non-host organism. These ‘alien replicon-producing organisms’ (ARPOs) may be used as potentialtools for bioengineering and biocontrol. Although the useof ARPOs for practical purposes is not easy and certainlydemands expertise when evaluating biosafety, it may bevaluable to outline their utility for theoretical bioengineering.In this review, I discuss this approach by taking plants asexamples of non-host organisms that produce various alienreplicons for bacteria, animals, or other plant species.

ARPOs: How to make them

An example of a simple genetic construct for ARPOs is shownin Fig. 3A. Therein, cDNA from a genome of single-strandedRNA-positive virus is considered an alien replicon variant; itis controlled by a plant promoter. Another element of theconstruct is a gene for a coat protein to pack the RNA repliconinto virus or VLPs. Many virus genomes contain specificsignals that mediate the interactions with their coat proteinsrequired for virion assembly. In the nucleus of the plant cell,this construct will be transcribed by RNA polymerase II,and two types of mRNA molecules will be exported to thecytoplasm: a viral RNA replicon and a coat protein mRNA.Occasionally, expression levels of heterologous genes intransgenic plants are low due to the presence of spurioussplicing or polyadenylation sites that disrupt the integrity ofopen reading frames. However, the nucleotide sequences ofalien genes may be modified to reach reasonable expressionlevels [7]. In the case of ARPOs, these concerns are withrespect to the transgene encoding the viral coat protein(Fig. 3A). Another mRNA represents a viral replicon, and itstranslation or replication inside plant cells is not required forthe formation of the VLP and may even be harmful.

Indeed, this scheme is the basis of ARPO construction, andits operation depends upon the viral replicon features. In

LB p35S REPLICASE MP 16k Rz nos RB

LB p35S CP MCS Rz nos RB

A)

B)

Figure 2. Schematic illustration of the tobacco rattle virus-basedreplicon vector. A: RNA1 encodes replicase, the movement protein(MP), and the 16 kD cysteine-rich protein. B: RNA2 encodes thecoat protein (CP) and contains a multiple cloning site (MCS). Bothconstructs are placed into T-regions of separate plasmid vectorsunder the control of the 35S CaMV promoter, and they also have aself-cleaving ribozyme (Rz), nos 30-terminator, and border repeats(LB, RB) for agrobacterial transformation.



Problems&Paradigms

some cases, the replicon mRNAmay not be synthesised due toincorrect splicing by plant nuclear factors. HeterologousmRNA may also be unstable in plant cells due to its intrinsicfeatures. These issues cannot be predicted ab initio due to thelack of preliminary data. However, because genomes of manyviruses contain pac-sites for selective assembly into VLPs,they may be considered candidate replicons for ARPOconstruction.

Transgenic plants with genetic constructs that includealien replicons may be obtained using conventional transfor-mation methods. Agroinoculation or particle bombardmentalso makes local transient expression possible. RNA may bepacked into virions either in the nucleus (if a coat protein has anuclear localisation signal, which some viruses carry) orcytoplasm (Fig. 3B). In the latter case, RNA replicons will becapped and may be polyadenylated, which renders themdirectly translatable in eukaryotic cells. A heterologous coatprotein may also be used if the self-replicating RNA contains acorresponding pack-site (these hybrid virions may displaysome advantages). The proper replicon synthesis pattern

(quantity, tissue specificity, induction inresponse to external stimuli, among others)may be controlled by using an appropriateplant promoter (Fig. 3A). Interactionsbetween transgenic plants and target hostorganisms may result in the transfer ofthe replicon, which, in some cases, may befacilitated by the intrinsic features of thevirion. Within the host cells, the transferredmRNA will be translated, and the synthes-ised proteins will initiate the amplificationprocess in a manner that is typical of theoriginal virus. If the RNA replicon wasdesigned to be propagation-competent(e.g. a full-sized virus genome), it may befurther disseminated throughout the hostpopulation.

For any ARPO, primary production ofthe alien replicon may be low; however, if it

penetrates into susceptible cells of the host organism andreplication is initiated, quantities will increase rapidly(Fig. 3C; the exact characteristics of this process dependupon the replicon’s intrinsic features). This property providesan opportunity to selectively influence natural populations ofsusceptible organisms that cannot be achieved using otherapproaches. Various examples of potential ARPOs arediscussed briefly below.

Genetic constructs produced in plantcells may replicate in bacteria

Multiple phages with single-stranded RNA-positive genomes(e.g. from leviviruses (MS2, R17, fr, GA) or alloleviviruses (Qb,SP)) may be used for ARPO construction. The MS2 phage is agood example of their typical organisation. Its genomic RNA isapproximately 3.2 kb and encodes four proteins: the coatprotein, the lysis protein, the maturation protein, and a viral

Promoter 1 genomic cDNA of RNA-positive virus Promoter 2 coat protein ORF

1

Spreading in popula�ons of target host organisms (insects, mammals, other plant species)

spreading

Local synthesis to protect plant against pathogenic bacteria or fungi

C)

A)

B)

2

3

4

Figure 3. Alien replicon-producing organisms (ARPOs). A: An example of a geneticconstruct encoding an alien replicon. Genomic cDNA of a virus with a single-strandedRNA-positive genome (intact or modified) is placed under the control of Promoter 1.The ORF for the coat protein is cloned separately under the control of Promoter 2.B: Schematic presentation of a plant cell expressing an alien replicon. 1: Geneticconstruct (A) introduced into the plant nuclear genome. 2: mRNA transcribed from thegenetic construct (Promoter 2) directs the synthesis of the coat protein. 3: Viral genomicRNA is transcribed from the genetic construct (Promoter 1). 4: Interaction of viral genomicRNA with coat proteins results in the assembly of infectious virus particles. This step maytake place either in the cytoplasm or the nucleus (depending upon the properties of theviral replicon used for ARPO construction: if its coat protein contains a functional nuclearlocalisation signal, the VLP will form in the nucleus). C: Alien replicons may be designedin two principally different variants: transmissible (i.e. they can infect other host organismsand spread rapidly to the entire population) and non-transmissible (i.e. only the hostorganism contacted with the ARPO will be infected, and alien replicon spreading will berestricted by the extent of the ARPO). Thus, either the local protection of a producingplant against pathogenic microorganisms and phytophages or the transmission of thereplicon to the entire population of target (host) organism(s) interacting with the ARPO ispossible.



Problems&Paradigms

replicase subunit. The coat protein recognises a small stem-loop structure. This pac-site initiates virion assembly andensures the selective encapsidation of phage genomic RNA.Each virion also contains a single copy of the maturationprotein, which is essential for successful infection.

The coat proteins of many phages may form VLPs even ifthe corresponding transgenes are expressed in eukaryoticSaccharomyces cerevisiae cells [69]. Heterologous mRNAswith MS2 pac-sites also interact with the MS2 coat protein inS. cerevisiae [70] and plant cells [71]. This approach wassuccessfully applied for the production of protected (‘arm-oured’) translatable mRNAs, which were further used as RNAvaccines for mammals [72, 73]; another interesting applicationof MS2 VLPs is specific delivery of microRNAs intomammaliancells [74, 75]. It may be assumed that a genetic construct withfull-sized MS2 genomic cDNA and capsid and maturationprotein ORFs that are controlled separately by appropriatepromoters may direct the synthesis of fully infectious phageparticles in plant cells. In turn, contact between the plant-produced bacteriophages and susceptible microorganismsmay initiate infection even if the phage content in ARPO cellsis low in the beginning.

This type of ARPO may be used for several purposes.First, some phages in this group show a broad host range(PPR1, M) [76, 77] and may propagate in different strains ofEscherichia, Salmonella, Klebsiella, Proteus, and Serratia,among others. Phages are considered a promising tool forcontrolling animal and human antibiotic-resistant infec-tions [78–80]. If a virulent phage (or cocktail of severalphages) is produced in an edible plant specially designedfor medicinal purposes, the plant may be used as a foodsupplement for antibacterial treatment.

Another potential application includes improved plantresistance against specific bacterial pathogens. Bacterio-phages have been considered a useful instrument forbiocontrol (e.g. against Xanthomonas pruni, Ralstonia sol-anacearum, and Pseudomonas tolaasii) [78]. Local, induciblebacteriophage production in plant cells surrounding patho-gen invasion sites may enhance the efficiency of naturallyoccurring defence mechanisms (Fig. 3C).

Plants may produce replicons that areinfectious to other plant species

Viruses may spread systemically in susceptible plants butnot in resistant cultivars or non-host species. DNA-layeredRNA replicons are frequently used in laboratories to initiateinfection in host plants if viruses cannot be transmittedvia mechanical inoculation. For example, cotton leaf rolldwarf virus cDNA, controlled by a CaMV35S promoter, wasagroinfiltrated into cotton or N. benthamiana leaves, andit initiated systemic infection that was successfully transmit-ted to non-infected plants via its specific vector Aphisgossypii [81]. Similarly, agroinfiltrated plants with a geneticconstruct that included beet necrotic virus yellow vein viruscDNA were systemically infected, and the virus wastransmitted to healthy plants via Polymyxa betae [82]. Theplants bearing these genetic constructs were also influenced

by the replicon that was produced (i.e. they suffered fromviral disease). However, these plants may also be used forbioengineering purposes. For example, self-replicating trans-missible genetic constructs based on tobacco necrosis virusA [55], TRV [61, 63], or apple latent spherical virus (ASLV) [83]may infect a broad range of plant species and do not producepronounced disease symptoms; however, they can efficientlyinduce specific RNA interference or initiate recombinantprotein overproduction. In particular, ASLV variants thatincorporate sequence segments from pathogenic tospovi-ruses [83] or cucumoviruses [84] have been proposed as ‘ALSVvector vaccines’ that induce cross-protection in infectedplants via target-specific RNA interference (not an example ofan ‘ARPO’; however, the theory is sufficiently similar, andthey may be designated ‘alien siRNA-producing viruses’).

A replicon that influences a target plant’s secondarymetabolism characteristics is one example of ARPO use. Theopium poppy is cultivated illegally for heroin production insome countries, and elimination of the growing fields may beproblematic. In theory, the best solution to this problem maybe to selectively decrease the morphine alkaloid content inPapaver somnifera plants grown in these regions, preferablywith no visible symptoms. This mechanism is technicallypossible because VIGS-mediated decreases in codeine O-demethylase or thebaine 6-O-demethylase selectively reducemorphine content three- and two-fold, respectively [85]. Aself-replicating transmissible genetic construct that sup-presses the genes in the morphine metabolic pathway andthat spreads to the illegal opium poppy areas of cultivationmay render heroin production unprofitable. The constructmay be based on the TRV replicon (Fig. 2), which has beenused successfully to arrange VIGS in Papaver somniferum [85,86]. TRV-induced VIGS was stable over a long period of timeand was transmitted via seeds to the next generation ofplants [1]. This virus may infect a broad range of plant species(in many cases, the infection was almost symptomless) and istransmitted via nematodes (Trichodoridae), mechanically, orvia seeds. TRV may be a promising candidate for this type ofARPO; however, its infectivity in natural populations ofPapaver somniferum has not been tested. The design of theTRV2 genetic construct (Fig. 2B) may include promoters thatare active in roots and pollen grains to increase disseminationefficiency to non-infected plants using both nematode vectorsand cross-pollination. Other candidate replicon variants maybe based on viruses that infect Papaver somnifera in nature(e.g. tomato spotted wilt virus or turnip mosaic virus).

In this theoretical discussion, transmissible replicon-producing transgenic plants distributed throughout the illegalfields of opium poppies may serve as a tool to selectivelydecrease morphine levels in affected plants. Of course, thistype of ARPO may harm legal morphine production; however,the development of virus-resistant plant cultivars mayovercome this weakness. Biosafety is always a concern forreplicon-based technologies; however, here, we discuss onlytheoretical bioengineering aspects.

Another example of the potential utility of ARPOs is in thearea of allergies to certain tree pollens (birch, pines, peach,among others). Currently, pollen production may only beprevented by eliminating the allergen-producing trees fromthe inhabited area. ARPOs can be used to produce RNA-



Problems&Paradigms

replicons that induce highly selective suppression of synthesisof allergenic proteins, thus providing another solution to thisproblem.

Plants may produce genetic constructsthat replicate in animals

Infectious RNAs of many animal viruses with single-strandedpositive RNA genomes may be synthesised in vitro usingcDNAs controlled by T7 promoters (e.g. dengue virus cDNAcloned into the high-copy plasmid BlueScript II [87]). Even aninfectious virus may be synthesised fully in vitro, asdemonstrated using the poliovirus [88]. Theoretically, animalviruses may be synthesised in plants using the geneticconstructs described above: cDNAs from genomic RNA andcoat protein ORF(s) controlled by plant promoters (Fig. 3A).The simplest method for viral transmission is successful if theproducing plant belongs to the host animal’s diet and the viralreplicon can penetrate through the gastrointestinal tract.

Interestingly, mammals appear vulnerable to this alienreplicon infiltration method. VLP-protected mRNAs intro-duced into an animal organism can penetrate into the cells,and the protein synthesised by the cellular translationmachinery may serve as an antigen to induce an immuneresponse. These RNA vaccines have been discussed widely asa prospective technology (see above; [3–5, 89]). Orallydelivered VLPs may be transported to Peyer’s patches,lamina propria, macrophages, and the spleen. This defaultroute does not depend strongly on VLP type, and the majorityof ‘armoured’ mRNA variants are likely translated. Thus,based on an appropriate VLP-based mRNA construct design,it may be replicated in transfected mammalian cells toproduce either protein or an miRNA of interest at consider-able levels.

An interesting example of hybrid plant virion use has beenpresented. The SFV replicon-containing vector was modifiedto include the TMV pac-site (�300 nucleotides in size). VLPswere assembled in vitro by mixing the purified TMV coatprotein with SFV replicon-containing mRNA. This geneticconstruct was movement deficient but could enter mousecells following subcutaneous introduction; the mRNA wasthen released, translated and efficiently replicated [90]. Asimilar effect may be possible if the replicon-containingmRNA is co-expressed with a TMV coat protein within plantcells and penetrates an animal organism via the gastro-intestinal tract.

In theory, this technology may also be applied forbiocontrol. Insect or mammal overgrowth in natural pop-ulations occurs occasionally and may cause serious damage.For example, Lymantria dispar overpopulation is harmful toforest regions in Eurasia. ARPOs can produce virus-basedreplicons that selectively suppress the reproduction-control-ling genes of a target species. In the case of Lymantria dispar,specifically designed ‘guard’ trees may be placed randomlythroughout a risky region. Low-level inducible synthesis of ahighly virulent RNA replicon that specifically limits thereproduction rate of an infected target species may diminishthe exhausting effects of its population overgrowth. The

population size of some mammals (rabbit, mouse, rat) may becontrolled using a similar method.

Host-induced gene silencing (HIGS) has now been widelydiscussed as a prospective bioengineering tool. It has beenobserved that siRNA synthesised in plants can spread tophytophages and switch off target gene expression, henceinhibiting functions vital for the life of the phytophage. Severalsuccessful HIGS approaches have been generated againstpathogenic fungi [91], parasitic nematodes [92], and herbivo-rous insects [93]. Notably, TRV DNA-layered RNA replicons(Fig. 2) were used to generate high levels of siRNA in some ofthese investigations [92, 93]. The ARPOs described above are adevelopment of the HIGS approach: an alien RNA replicon thatsynthesises siRNA/miRNA, not in the producing plant but inthe host animal cells,may increase the power of this technologysubstantially and may work as a selective biocontrol agent.

Conclusion

The use of transmissible RNA replicons may provide anefficient but risky method of influencing the naturalpopulations of target species. ARPO efficiency results fromthe combination of a viral replicon with an opportunity toregulate its dissemination over the long-term. It may beapplied for highly selective biocontrol, metabolic adjustment,and vaccination, among other uses. Risks of ARPO applicationare also based on the intrinsic properties of the replicon. It iswidely considered that viral self-replicons are prone tomutations, and some mutations may increase pathogenicityor change the host preferences. The spread of ARPOs mayinfluence the target species populations in distant regionswhere it is not required and may even be harmful. Targetedorganisms may also acquire mutations that help avoid thereplicon’s effects [94]. Despite all of these characteristics thatshould be considered and that require caution, there is noclear evidence of damage resulting from the application ofbioengineered organisms (e.g. [95]). Risks of ARPOs may bediminished by thorough planning and making them sterile toprevent their uncontrolled spreading. In some situations,using risky techniques may be the only method of preventingintolerable consequences. For example, a natural populationof a target organism may come under threat of extinction dueto the rise of a new pathogen (as is currently the case withhoneybees). In this case, ARPOsmay be used for the long-termspread of RNA vaccines.

Another point concerns the behaviour of viral replicons inthe natural non-host population. Field investigations typicallyconcentrate on viruses that strongly affect the host phenotypeand fitness, and the role of local amplification of ‘alien’replicons in non-host organisms in virus transmission, storageand spreading may be underestimated. In my opinion, adiscussion on these topics is valuable for bioengineering,biotechnology, molecular evolution, and virology.

AcknowledgementsI am grateful to Anatoly Kochetov who inspired this researchand also to the RAS program ‘Molecular and Cellular Biology’and RFBR 14-04-0103614 for financial support.



Problems&Paradigms

References

1. Senthil-Kumar M, Mysore KS. 2011. Virus-induced gene silencing canpersist for more than 2 years and also be transmitted to progenyseedlings in Nicotiana benthamiana and tomato. Plant Biotechnol J 9:797–806.

2. Lange M, Yellina AL, Orashakova S, Becker A. 2013. Virus-inducedgene silencing (VIGS) in plants: an overview of target species and thevirus-derived vector systems. Methods Mol Biol 975: 1–14.

3. Geall AJ, Mandl CW, Ulmer JB. 2013. RNA: the new revolution in nucleicacid vaccines. Semin Immunol 25: 152–9.

4. Parks CL, Picker LJ, King CR. 2013. Development of replication-competent viral vectors for HIV vaccine delivery. Curr Opin HIV AIDS 8:402–11.

5. Ulmer JB, Mason PW, Geall A, Mandl CW. 2012. RNA-based vaccines.Vaccine 30: 4414–8.

6. Werner S, BreusO, Symonenko Y,Marillonnet S, et al. 2011. High-levelrecombinant protein expression in transgenic plants by using a double-inducible viral vector. Proc Natl Acad Sci USA 108: 14061–6.

7. Egelkrout E, Rajan V, Howard JA. 2012. Overproduction of recombinantproteins in plants. Plant Sci 184: 83–101.

8. Melnik S, Stoger E. 2013. Green factories for biopharmaceuticals. CurrMed Chem 20: 1038–46.

9. Mandl CW, Aberle JH, Aberle SW, Holzmann H, et al. 1998. In vitro-synthesized infectious RNA as an attenuated live vaccine in a flavivirusmodel. Nat Med 4: 1438–40.

10. Takada A, Robison C, Goto H, Sanchez A, et al. 1997. A system forfunctional analysis of Ebola virus glycoprotein. Proc Natl Acad Sci USA94: 14764–9.

11. Davis BS, Chang GJ, Cropp B, Roehrig JT, et al. 2001. West Nile virusrecombinant DNA vaccine protects mouse and horse from viruschallenge and expresses in vitro a noninfectious recombinant antigenthat can be used in enzyme-linked immunosorbent assays. J Virol 75:4040–7.

12. Garver KA, LaPatra SE, Kurath G. 2005. Efficacy of an infectioushematopoietic necrosis (IHN) virus DNA vaccine in Chinook Oncorhyn-chus tshawytscha and sockeye O. nerka salmon. Dis Aquat Organ 64:13–22.

13. Grosenbaugh DA, Leard AT, Bergman PJ, Klein MK, et al. 2011. Safetyand efficacy of a xenogeneic DNA vaccine encoding for human tyrosinaseas adjunctive treatment for oral malignant melanoma in dogs followingsurgical excision of the primary tumor. Am J Vet Res 72: 1631–8.

14. Wolff JA, Malone RW, Williams P, Chong W, et al. 1990. Direct genetransfer into mouse muscle in vivo. Science 247: 1465–8.

15. Schlake T, Thess A, Fotin-Mleczek M, Kallen KJ. 2012. DevelopingmRNA-vaccine technologies. RNA Biol 9: 1319–30.

16. Probst J, Weide B, Scheel B, Pichler BJ, et al. 2007. Spontaneouscellular uptake of exogenous messenger RNA in vivo is nucleic acid-specific, saturable and ion dependent. Gene Ther 14: 1175–80.

17. Lorenz C, Fotin-Mleczek M, Roth G, Becker C, et al. 2011. Proteinexpression from exogenous mRNA: uptake by receptor-mediatedendocytosis and trafficking via the lysosomal pathway. RNA Biol 8:627–36.

18. Geall AJ, Verma A, Otten GR, Shaw CA, et al. 2012. Nonviral delivery ofself-amplifying RNA vaccines. Proc Natl Acad Sci USA 109: 14604–9.

19. MatsumotoM, Oshiumi H, Seya T. 2011. Antiviral responses induced bythe TLR3 pathway. Rev Med Virol 21: 67–77.

20. Jensen S, Thomsen AR. 2012. Sensing of RNA viruses: a review ofinnate immune receptors involved in recognizing RNA virus invasion. JVirol 86: 2900–10.

21. Lilja AE, Mason PW. 2012. The next generation recombinant humancytomegalovirus vaccine candidates-beyond gB. Vaccine 30: 6980–90.

22. Johansson DX, Ljungberg K, Kakoulidou M, Liljestrom P. 2012.Intradermal electroporation of naked replicon RNA elicits strong immuneresponses. PLoS One 7: e29732.

23. Frolov I, Frolova E, Schlesinger S. 1997. Sindbis virus replicons andSindbis virus: assembly of chimeras and of particles deficient in virusRNA. J Virol 71: 2819–29.

24. McCoy K, Tatsis N, Korioth-Schmitz B, Lasaro MO, et al. 2007. Effectof preexisting immunity to adenovirus human serotype 5 antigens on theimmune responses of nonhuman primates to vaccine regimens based onhuman- or chimpanzee-derived adenovirus vectors. J Virol 81: 6594–604.

25. Dharmapuri S, Peruzzi D, Aurisicchio L. 2009. Engineered adenovirusserotypes for overcoming anti-vector immunity. Expert Opin Biol Ther 9:1279–87.

26. Betts G, Poyntz H, Stylianou E, Reyes-Sandoval A, et al. 2012.Optimising immunogenicity with viral vectors: mixing MVA and HAdV-5

expressing the mycobacterial antigen Ag85A in a single injection. PLoSOne 7: e50447.

27. Lodmell DL, Ewalt LC. 2000. Rabies vaccination: comparison ofneutralizing antibody responses after priming and boosting with differentcombinations of DNA, inactivated virus, or recombinant vaccinia virusvaccines. Vaccine 18: 2394–8.

28. Sharpe S, Polyanskaya N, Dennis M, Sutter G, et al. 2001. Induction ofsimian immunodeficiency virus (SIV)-specific CTL in rhesus macaques byvaccination with modified vaccinia virus Ankara expressing SIV trans-genes: influence of pre-existing antivector immunity. J Gen Virol 82:2215–23.

29. Ljungberg K,Whitmore AC, Fluet ME, Moran TP, et al. 2007. Increasedimmunogenicity of a DNA-launched Venezuelan equine encephalitisvirus-based replicon DNA vaccine. J Virol 81: 13412–23.

30. Dubensky TW, Jr, Driver DA, Polo JM, Belli BA, et al. 1996. Sindbisvirus DNA-based expression vectors: utility for in vitro and in vivo genetransfer. J Virol 70: 508–19.

31. Berglund P, Smerdou C, Fleeton MN, Tubulekas I, et al. 1998.Enhancing immune responses using suicidal DNA vaccines. NatBiotechnol 16: 562–5.

32. Vander Veen RL, Harris DL, Kamrud KI. 2012. Alphavirus repliconvaccines. Anim Health Res Rev 13: 1–9.

33. Loy JD, Gander J, Mogler M, Vander Veen R, et al. 2013. Developmentand evaluation of a replicon particle vaccine expressing the E2glycoprotein of bovine viral diarrhea virus (BVDV) in cattle. Virol J 10: 35.

34. Center RJ, Miller A, Wheatley AK, Campbell SM, et al. 2013. Utility ofthe Sindbis replicon system as an Env-targeted HIV vaccine. Vaccine 31:2260–6.

35. Herbert AS, Kuehne AI, Barth JF, Ortiz RA, et al. 2013. Venezuelanequine encephalitis virus replicon particle vaccine protects nonhumanprimates from intramuscular and aerosol challengewith ebolavirus. J Virol87: 4952–64.

36. White LJ, Sariol CA, Mattocks MD, Wahala MPBW, et al. 2013. Analphavirus vector-based tetravalent dengue vaccine induces a rapid andprotective immune response in macaques that differs qualitatively fromimmunity induced by live virus infection. J Virol 87: 3409–24.

37. Wang Y, Liu G, Shi L, Li W, et al. 2013. Immune responses in micevaccinated with a suicidal DNA vaccine expressing the hemagglutininglycoprotein from the peste des petits ruminants virus. J Virol Meth 193:525–30.

38. Suzuki R, Ishikawa T, Konishi E, Matsuda M, et al. 2014. Production ofsingle-round infectious chimeric flaviviruses with DNA-based Japaneseencephalitis virus replicon. J Gen Virol 95: 60–5.

39. Pujhari S, Baig TT, Hansra S, Zakhartchouk AN. 2013. Development ofa DNA-launched replicon as a vaccine for porcine reproductive andrespiratory syndrome virus. Virus Res 173: 321–6.

40. Russell SJ, Peng KW, Bell JC. 2012. Oncolyticvirotherapy. NatBiotechnol 30: 658–70.

41. Elsedawy NB, Russell SJ. 2013. Oncolytic vaccines. Expert RevVaccines 12: 1155–72.

42. Zhang L, Wang Y, Xiao Y, Wang Y, et al. 2014. Enhancement ofantitumor immunity using a DNA-based replicon vaccine derived fromSemliki Forest virus. PLoS One 9: e90551.

43. Lyons JA, Sheahan BJ, Galbraith SE, Mehra R, et al. 2007. Inhibition ofangiogenesis by a Semliki Forest virus vector expressing VEGFR-2reduces tumour growth and metastasis in mice. Gene Ther 14: 503–13.

44. Zhang W, Hagedorn C, Schulz E, Lipps HJ, et al. 2014. Viral hybrid-vectors for delivery of autonomous replicons. Curr Gene Ther 14: 10–23.

45. Sanchez-Puig JM, Lorenzo MM, Blasco R. 2013. A vaccinia virusrecombinant transcribing an alphavirus replicon and expressing alpha-virus structural proteins leads to packaging of alphavirus infectious singlecycle particles. PLoS One 8: e75574.

46. Guan M, Rodriguez-Madoz JR, Alzuguren P, Gomar C, et al. 2006.Increased efficacy and safety in the treatment of experimental liver cancerwith a novel adenovirus-alphavirus hybrid vector.Cancer Res 66: 1620–9.

47. Yang Y, Xiao F, Lu Z, Li Z, et al. 2013. Development of a novel adenovirus-alphavirus hybrid vector with RNA replicon features for malignanthematopoietic cell transduction. Cancer Gene Ther 20: 429–36.

48. Osada T, Morse MA, Hobeika A, Lyerly HK. 2012. Novel recombinantalphaviral and adenoviral vectors for cancer immunotherapy. SeminOncol 39: 305–10.

49. Sun Y, Tian DY, Li S, MengQL, et al. 2013. Comprehensive evaluation ofthe adenovirus/alphavirus-replicon chimeric vector-based vaccine rAdV-SFV-E2 against classical swine fever. Vaccine 31: 538–44.

50. Li KJ, Garoff H. 1996. Production of infectious recombinant Moloneymurine leukemia virus particles in BHK cells using Semliki Forest virus-derived RNA expression vectors. Proc Natl Acad Sci USA 93: 11658–63.



Problems&Paradigms

51. Jupin I. 2013. A protocol for VIGS in Arabidopsis thaliana using a one-step TYMV-derived vector. Methods Mol Biol 975: 197–210.

52. Lindbo JA. 2007. High-efficiency protein expression in plants fromagroinfection-compatible Tobacco mosaic virus expression vectors.BMC Biotechnol 7: 52.

53. Lacorte C, Ribeiro SG, Lohuis D, Goldbach R, et al. 2010. Potato virus Xand Tobacco mosaic virus-based vectors compatible with the Gatewaycloning system. J Virol Methods 164: 7–13.

54. Chen CC, Chen TC, Raja JA, Chang CA, et al. 2007. Effectiveness andstability of heterologous proteins expressed in plants by Turnip mosaicvirus vector at five different insertion sites. Virus Res 130: 210–27.

55. Zhang Y, Li J, Pu H, Jin J, et al. 2010. Development of Tobacco necrosisvirus A as a vector for efficient and stable expression of FMDV VP1peptides. Plant Biotechnol J 8: 506–23.

56. Lindbo JA. 2007. TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant Physiol 145: 1232–40.

57. Jain A, Saini V, Kohli DV. 2013. Edible transgenic plant vaccines fordifferent diseases. Curr Pharm Biotechnol 14: 594–614.

58. Hefferon K. 2013. Plant-derived pharmaceuticals for the developingworld. Biotechnol J 8: 1193–202.

59. Love AJ, Makarov V, Yaminsky I, Kalinina NO, et al. 2014. The use oftobacco mosaic virus and cowpea mosaic virus for the production ofnovel metal nanomaterials. Virology 449: 133–9.

60. MacFarlane SA. 2010. Tobraviruses – plant pathogens and tools forbiotechnology. Mol Plant Pathol 11: 577–83.

61. Bachan S, Dinesh-Kumar SP. 2012. Tobacco rattle virus (TRV)-basedvirus-induced gene silencing. Methods Mol Biol 894: 83–92.

62. Senthil-Kumar M, Mysore KS. 2014. Tobacco rattle virus-based virus-induced gene silencing in Nicotiana benthamiana.Nat Protoc 9: 1549–62.

63. Larsen JS,CurtisWR. 2012. RNA viral vectors for improvedAgrobacterium-mediated transient expression of heterologous proteins in Nicotianabenthamiana cell suspensions and hairy roots. BMC Biotechnol 12: 21.

64. Peretz Y, Mozes-Koch R, Akad F, Tanne E, et al. 2007. A universalexpression/silencing vector in plants. Plant Physiol 145: 1251–63.

65. Chen Q, He J, Phoolcharoen W, Mason HS. 2011. Geminiviral vectorsbased on bean yellow dwarf virus for production of vaccine antigens andmonoclonal antibodies in plants. Hum Vaccin 7: 331–8.

66. Lai H, He J, Engle M, Diamond MS, et al. 2012. Robust production ofvirus-like particles and monoclonal antibodies with geminiviral repliconvectors in lettuce. Plant Biotechnol J 10: 95–104.

67. Rybicki EP, Martin DP. 2014. Virus-derived ssDNA vectors for theexpression of foreign proteins in plants. Curr Top Microbiol Immunol 375:19–45.

68. Annamalai P, Rofail F, Demason DA, Rao AL. 2008. Replication-coupled packaging mechanism in positive-strand RNA viruses: synchro-nized coexpression of functional multigenome RNA components of ananimal and a plant virus in Nicotiana benthamiana cells by agroinfiltration.J Virol 82: 1484–95.

69. Freivalds J, Kotelovica S, Voronkova T, Ose V, et al. 2014. Yeast-expressed bacteriophage-like particles for the packaging of nano-materials. Mol Biotechnol 56: 102–10.

70. Legendre D, Fastrez J. 2005. Production in Saccharomyces cerevisiaeof MS2 virus-like particles packaging functional heterologous mRNAs. JBiotechnol 117: 183–94.

71. Zhang F, Simon AE. 2003. A novel procedure for the localization of viralRNAs in protoplasts and whole plants. Plant J 35: 665–73.

72. Sun S, Li W, Sun Y, Pan Y, et al. 2011. A new RNA vaccine platformbased on MS2 virus-like particles produced in Saccharomycescerevisiae. Biochem Biophys Res Commun 407: 124–8.

73. Li J, Sun Y, Jia T, Zhang R, et al. 2014. Messenger RNA vaccine basedon recombinant MS2 virus-like particles against prostate cancer. Int JCancer 134: 1683–94.

74. Pan Y, Jia T, Zhang Y, Zhang K, et al. 2012. MS2 VLP-based delivery ofmicroRNA-146a inhibits autoantibody production in lupus-pronemice. IntJ Nanomed 7: 5957–67.

75. Pan Y, Zhang Y, Jia T, Zhang K, et al. 2012. Development of amicroRNAdelivery system based on bacteriophage MS2 virus-like particles. FEBS J279: 1198–208.

76. Ruokoranta TM, Grahn AM, Ravantti JJ, Poranen MM, et al. 2006.Complete genome sequence of the broad host range single-strandedRNA phage PRR1 places it in the Levivirus genus with characteristicsshared with Alloleviviruses. J Virol 80: 9326–30.

77. Rumnieks J, Tars K. 2012. Diversity of pili-specific bacteriophages:genome sequence of IncM plasmid-dependent RNA phage M. BMCMicrobiol 12: 277.

78. Haq IU, Chaudhry WN, Akhtar MN, Andleeb S, et al. 2012.Bacteriophages and their implications on future biotechnology: a review.Virol J 9: 9.

79. Chan BK, Abedon ST, Loc-Carrillo C. 2013. Phage cocktails and thefuture of phage therapy. Future Microbiol 8: 769–83.

80. Sulakvelidze A. 2013. Using lytic bacteriophages to eliminate orsignificantly reduce contamination of food by foodborne bacterialpathogens. J Sci Food Agric 93: 3137–46.

81. Delfosse VC, Casse MF, Agrofoglio YC, Kresic IB, et al. 2013.Agroinoculation of a full-length cDNA clone of cotton leafroll dwarf virus(CLRDV) results in systemic infection in cotton and the model plantNicotiana benthamiana. Virus Res 175: 64–70.

82. Delbianco A, Lanzoni C, Klein E, Rubies Autonell C, et al. 2013.Agroinoculation of Beet necrotic yellow vein virus cDNA clones results inplant systemic infection and efficient Polymyxa betae transmission. MolPlant Pathol 14: 422–8.

83. Taki A, Yamagishi N, Yoshikawa N. 2013. Development of apple latentspherical virus-based vaccines against three tospoviruses. Virus Res 176:251–8.

84. Tamura A, Kato T, Taki A, Sone M, et al. 2013. Preventive and curativeeffects of Apple latent spherical virus vectors harboring part of the targetvirus genome against potyvirus and cucumovirus infections. Virology 446:314–24.

85. Wijekoon CP, Facchini PJ. 2012. Systematic knockdown of morphinepathway enzymes in opium poppy using virus-induced gene silencing.Plant J 69: 1052–63.

86. Hileman LC, Drea S, Martino G, Litt A, et al. 2005. Virus-induced genesilencing is an effective tool for assaying gene function in the basaleudicot species Papaver somniferum (opium poppy). Plant J 44: 334–41.

87. Sriburi R, Keelapang P, Duangchinda T, Pruksakorn S, et al. 2001.Construction of infectious dengue 2 virus cDNA clones using high copynumber plasmid. J Virol Methods 92: 71–82.

88. Cello J, Mueller S. 2012. Production of infectious poliovirus fromsynthetic viral genomes. Methods Mol Biol 852: 181–93.

89. Zimmer G. 2010. RNA replicons – a new approach for influenza virusimmunoprophylaxis. Viruses 2: 413–34.

90. Smith ML, Corbo T, Bernales J, Lindbo JA, et al. 2007. Assembly oftrans-encapsidated recombinant viral vectors engineered from Tobaccomosaic virus and Semliki Forest virus and their evaluation asimmunogens. Virology 358: 321–33.

91. Panwar V, McCallum B, Bakkeren G. 2013. Host-induced genesilencing of wheat leaf rust fungus Puccinia triticina pathogenicitygenes mediated by the Barley stripe mosaic virus. Plant Mol Biol 8: 595–608.

92. Dubreuil G, Magliano M, Dubrana MP, Lozano J, et al. 2009. Tobaccorattle virusmediates gene silencing in a plant parasitic root-knot nematode.J Exp Bot 60: 4041–50.

93. Kumar P, Pandit SS, Baldwin IT. 2012. Tobacco rattle virus vector: arapid and transient means of silencing Manduca sexta genes by plantmediated RNA interference. PLoS One 7: e31347.

94. Stern A, Sorek R. 2011. The phage-host arms race: shaping the evolutionof microbes. BioEssays 33: 43–51.

95. de Lorenzo V. 2010. Environmental biosafety in the age of syntheticbiology: do we really need a radical new approach? BioEssays 32:926–31.



Problems&Paradigms

Documents

The alien replicon: Artificial genetic constructs to direct the synthesis of transmissible self-replicating RNAs