How RNA viruses maintain their genome integrity

Review How RNA viruses maintain their genome integrity

John N. Barr1 and Rachel Fearns2

Correspondence

Rachel Fearns

[email protected]

1Institute of Molecular and Cellular Biology, University of Leeds, Leeds, UK

2Department of Microbiology, Boston University School of Medicine, Boston, MA, USA

RNA genomes are vulnerable to corruption by a range of activities, including inaccurate replication

by the error-prone replicase, damage from environmental factors, and attack by nucleases and

other RNA-modifying enzymes that comprise the cellular intrinsic or innate immune response.

Damage to coding regions and loss of critical cis-acting signals inevitably impair genome fitness;

as a consequence, RNA viruses have evolved a variety of mechanisms to protect their genome

integrity. These include mechanisms to promote replicase fidelity, recombination activities that

allow exchange of sequences between different RNA templates, and mechanisms to repair the

genome termini. In this article, we review examples of these processes from a range of RNA

viruses to showcase the diverse approaches that viruses have evolved to maintain their genome

sequence integrity, focusing first on mechanisms that viruses use to protect their entire genome,

and then concentrating on mechanisms that allow protection of the genome termini, which are

especially vulnerable. In addition, we discuss examples in which it might be beneficial for a virus to

‘lose’ its genomic termini and reduce its replication efficiency.

Maintenance mechanisms to protect the entirevirus genome

RNA virus genome replication is performed by the viralreplicase complex, which for most viruses is likely to be anassembly of multiple viral and cellular proteins. Thecatalytic subunit of this complex is referred to as theRNA-dependent RNA polymerase (RdRp). RdRps arenotoriously error-prone and so RNA virus genomes aresubject to potentially catastrophic alteration from theirown error-prone RNA-synthesis machinery. In addition,some non-segmented, negative-strand RNA viruses mightbe susceptible to insertions within the coding region of thegenome as a consequence of their editing programme. Viralgenomes may also be susceptible to physical or chemicaldamage by environmental factors (Fig. 1). Below, wedescribe examples of mechanisms that viruses have evolvedto overcome these insults.

RdRp proofreading

The error rate of the viral RdRp is estimated to be between1.561023 bp21 (Qb phage) and 7.261025 bp21 (influ-enza virus) (Drake, 1993). This relaxed polymerase fidelityis an important facet of RNA virus biology, providing asource of sequence diversity that can allow virus quasis-pecies to form, enabling the virus to adapt successfully tochanging environments. Indeed, it has been shown that amutant RdRp with increased fidelity, whilst able toreplicate efficiently in cell culture, was unable to replicateefficiently in the complex environment of an animal hostbecause of its inability to yield virus variants (Vignuzzi et

al., 2006). However, the flip side of the coin is thatpolymerase error can also lead to the generation of non-viable templates that reduce overall viral fitness. The term‘error catastrophe’ has been coined to describe theoutcome of an RdRp error rate at which too many non-viable templates are generated and a virus populationbecomes unsustainable, and it is believed that many viralRdRps operate close to this threshold (Crotty & Andino,2002). Conventional wisdom holds that the RdRp errorrate constrains viral RNA genome sizes to a relatively lowupper limit. However, there is a significant range in RNAvirus genome length, with the filoviruses and someparamyxoviruses having genome lengths of 18–19 kb,more than 7-fold longer than the genomes of the smallestRNA viruses, and the coronaviruses being even larger, withmaximum genome sizes between 27 and 32 kb. Viruseswith longer genome lengths probably require RdRps withinherently greater fidelity, which could be conferredpassively by RdRp structure, with other factors such asgenome structure also playing a role (Castro et al., 2005;Duffy et al., 2008).

Interestingly, recent evidence suggests that coronavirusesmight also have an active mechanism to promote fidelity.The nsp14 protein of these viruses possesses sequencemotifs that are analogous to the active-site motifs ofcellular exonuclease enzymes, and the nsp14 protein ofsevere acute respiratory syndrome (SARS)-related corona-virus has been shown to have 39A59 exonuclease activity invitro (Chen et al., 2007; Minskaia et al., 2006). Evidencethat this protein functions as a proofreading enzyme comesfrom the fact that mutant viruses that have a defective

Journal of General Virology (2010), 91, 1373–1387 DOI 10.1099/vir.0.020818-0

020818 G 2010 SGM Printed in Great Britain 1373

nsp14 protein are more error-prone than wild-type virusby a factor of at least 15-fold (Eckerle et al., 2007). Thesedata suggest that nsp14 acts as an exonuclease to removeand repair misincorporated nucleotides, thus helping tomaintain the sequence integrity of the large coronavirusRNA genomes.

RNA editing and the ‘rule of six’

Some non-segmented, negative-stranded RNA virusespossess a specialized ‘editing’ sequence that can allow theRdRp to sometimes add non-templated nucleotides duringmRNA transcription. Because the RdRp sometimes copiesthe template faithfully and sometimes adds non-templatednucleotides, this results in generation of qualitativelydistinct transcripts from the same gene (Cattaneo et al.,1989; Galinski et al., 1992; Sanchez et al., 1996; Thomaset al., 1988; Vidal et al., 1990). This mechanism allows theexpression of multiple polypeptides from a single tran-scriptional unit and is essential for the virus to express itsfull complement of proteins. However, if non-templatedadditions were to occur during genome replication, thiscould lead to accumulation of defective genomes, whichcould be lethal for the virus. Interestingly, most (but notall) viruses that have an RNA-editing function share a strictrequirement for their genome nucleotide length to bedivisible by six, a phenomenon known as the ‘rule of six’(Calain & Roux, 1993; Kolakofsky et al., 1998). In theseviruses, the viral nucleocapsid protein (N) encapsidates thereplicative RNA in a 59A39 direction as the RNA is beingsynthesized and remains associated with the genomic and

antigenomic RNA throughout the virus replication cycle.The ‘rule of six’ is thought to reflect the stoichiometry ofthe RNA nucleotides to the N protein (Egelman et al.,1989). The viral RdRp recognizes RNA sequences withinthe context of the nucleocapsid structure, and thepromoter sequences must be phased correctly in relationto each N protein monomer for efficient virus replicationto occur (Vulliemoz & Roux, 2001). These observations ledto a model proposed by Kolakofsky et al. (2005), which isthat, if non-templated insertions were to occur duringgenome replication, generating genomes of non-hexamericlength, the consequent disruption of N–RNA phase in thepromoter region would preclude these mutant replicationproducts from acting as templates for further rounds ofreplication (Fig. 2). If this model is correct, then the ‘rule ofsix’ is a non-catalytic ‘screening’ mechanism to eliminategenomes containing non-templated insertions from thegenome pool and to help maintain genomic integrity in thevirus population.

Repair of alkylation damage to genomic RNA

Virus genomes may also be susceptible to chemicalmodification. One such modification is alkylation, theaddition of carbon chains to N and O atoms in RNA bases,in the form of either methylation (addition of a singlecarbon) or addition of longer carbon chains. Alkylationcould inhibit genome expression and replication and mightcause base-mispairing to occur, resulting in an increasedmutation rate. Alkylating agents can be found in theenvironment and inside cells, and prokaryotes and

Fig. 1. Summary of the mechanisms involved in genome maintenance and repair. Mutations (indicated by red lightningsymbols) can occur either internally on a viral RNA genome or at the extreme termini. The figure shows mechanisms by whichmutations at these two regions on the genome could occur and possible mechanisms by which the different types of mutationcould be repaired. Details are described in the text.

J. Barr and R. Fearns

1374 Journal of General Virology 91

eukaryotes have evolved mechanisms to protect theirgenomic DNA from their effects. One of these mechanismsinvolves a protein called AlkB, which has homologues in allmulticellular organisms, as well as in some bacteria andfungi, and which has been shown to repair damage to RNA,in addition to DNA (Aas et al., 2003). Bioinformaticanalysis revealed that a number of different positive-strandRNA plant viruses contain an AlkB domain, and functionalstudies have shown that some of these domains can repairRNA damage by oxidative demethylation (Bratlie &Drablos, 2005; van den Born et al., 2008). These findingssuggest that some viruses have acquired this protectivemechanism and that it has a role in the virus replicationcycle. Interestingly, the presence of the AlkB domains doesnot correlate with specific virus taxonomic lineages,suggesting that the gene was acquired relatively recently,and it has been suggested that it exists within viruses thatare highly exposed to alkylating environments, such aspesticides and/or the phloem of woody or perennial plants(Bratlie & Drablos, 2005; van den Born et al., 2008).

Recombination in RNA viruses

RNA recombination is a property of positive- and negative-stranded RNA viruses that infect plant (Bujarski & Kaesberg,1986), animal (Copper et al., 1974) and bacterial(Munishkin et al., 1988) hosts. Recombination in a viralcontext generally occurs when a replicating RdRp stopscopying one RNA strand and transfers to another (Copperet al., 1974). When the transfer sites share commonsequences, the recombination event is said to be homologous;conversely, when the sites are unrelated, the recombinationevent is non-homologous. Recent evidence suggests that

recombination of the tripartite, positive-stranded RNAvirus brome mosaic virus (BMV) is exceedingly common.By analysing the progeny of co-infections with markedgenomes, observed and calculated BMV recombinationfrequencies implied that each replicating RNA moleculerecombined at least once during the amplification of itslineage, and thus few viral progeny genomes containednucleotides copied from a single parental template(Urbanowicz et al., 2005).

In addition to being a source of sequence diversity, RNArecombination has been shown to be a mechanism that alsoallows genome repair, a property that was recognized earlyon in studies of virus evolution, and was predicted to allowRNA viruses to repair defective or weakened genomes inthe face of intense selective pressure. Recombination eventscould allow restoration of genomes damaged by a numberof insults, including RdRp error, chemical modificationand damage from UV light (Fig. 1). Genome repair byrecombination has been reported for several viruses andcan be extremely efficient. Bacteriophage MS2 possesses acritical secondary-structure element at an internal locationwithin the genome, between the maturation and coatprotein genes, that is involved in regulating coat proteinexpression. Disruption of this structural element bydeleting 19 nt, including the Shine–Dalgarno sequence,reduced virus titres by a massive 10 orders of magnitude,demonstrating the critical nature of this motif. However,despite the removal of such a large sequence, revertantscapable of amplifying to high virus titres arose withinovernight cultures, and analysis of the revertant MS2 RNAsequence showed that the structural element had beenrepaired and the Shine–Dalgarno sequence restored.Sequence analysis of the revertant suggested that repair

Fig. 2. The ‘rule of six’ and genome replication in the subfamily Paramyxovirinae. (a) Accurate genome replication yields anantigenome RNA that is the exact complement of the genome, which places the RNA promoter sequence at the 39 end of theantigenome in the correct context relative to the associated N protein (red ovals). This allows the RdRp (grey) to bind specificnucleotides in the promoter (in upper case) to initiate genome RNA synthesis. (b) If the RdRp stutters at the transcriptionalediting site during genome replication, it will insert non-templated residues in the RNA product (in this example, 1 nt). Thisresults in an antigenome in which the RNA sequence is displaced by 1 nt relative to the N protein, thus preventing the RdRpfrom recognizing the promoter efficiently.

How RNA viruses maintain their genome integrity

http://vir.sgmjournals.org 1375

was mediated through recruitment of additional nucleo-tides through a non-homologous recombination event(Olsthoorn & van Duin, 1996). Additional examples ofinternal sequence repair are the repair of 87 missingnucleotides of the mouse hepatitis virus nucleocapsid gene(Koetzner et al., 1992) and the repair of missingnucleotides of the bacteriophage Qb replicase gene(Palasingam & Shaklee, 1992). In both instances, themissing nucleotides were supplied through homologousrecombination from RNA templates supplied in trans and,whilst these systems may not reproduce the conditions ofnatural infections, they clearly highlight the capability ofthe respective viral RdRps to perform recombination-mediated repair given the presence of suitable donor andacceptor templates. The RdRp of the positive-strandedSindbis virus is also thought to be capable of bothhomologous and non-homologous recombination, asdemonstrated by the production of replication-competentgenomes from donor and acceptor templates that wereindividually replication-incompetent (Raju et al., 1995). Ina similar way, homologous recombination of two partiallydeleted RNA3 variants by the cowpea chlorotic mottle virusRdRp was shown to be responsible for rapid and frequentgeneration of an intact RNA3 sequence (Allison et al.,1990). It is also worth noting that recombination can occurvery close to the end of templates to repair terminalsequences. An experiment with mutated bunyavirustemplates showed that its RdRp was capable of correctingpromoter deletions of 2 or 15 nt. This repair wasdependent on the presence of an intact promoter suppliedin tandem, which was lost during the repair process. Thesefindings suggested that the bunyavirus RdRp could initiateon the external intact promoter and then ‘jump’ to thedamaged internal promoter sequence, thus generating acorrected product (Walter & Barr, 2010). Whilst this is anartificial situation, it suggests that the bunyavirus RdRpwould have the capability to repair damaged termini,provided that there was a source of intact templates for itto initiate on. Taken together, these experiments serve todemonstrate the promiscuous nature of some viral RdRpsduring genome replication and the potential benefit thatmight occur.

These varied examples of recombination have one aspect incommon: all are mediated by the viral RdRp and therecombination event occurs co-transcriptionally duringcopying of the RNA template. However, recent evidencewith poliovirus now indicates that recombination canoccur in the absence of active polymerase. By introducingpurified poliovirus genomes split into two fragments,either with or without sequence overlap, intact genomeswere recovered, despite neither fragment possessing anintact polymerase-encoding region. Although the mech-anism responsible remains elusive, the absence of viralpolymerase involvement strongly implies a role for host-cell components (Gmyl et al., 1999, 2003). Whether thisnon-replicative recombination acts during the course ofnatural infections is unknown. However, this mechanism

possesses the potential not only to repair truncatedgenomes, but also to act as a potent source of sequencevariation through combining both host and viral RNAsequences.

It should be noted that, whilst some RNA virusesdemonstrate promiscuous recombination, in others it is arare event. Template jumping or switching by the RdRp ofnon-segmented, negative-strand RNA viruses can occur, asdemonstrated by the production of defective interferingparticles during high-titre virus amplification (Huang &Baltimore, 1970; Lazzarini et al., 1981). There are alsoexamples of sequence duplications arising in circulatingviruses, consistent with the idea that the RdRp can ‘jump’either within or between templates (McClure et al., 1992;Trento et al., 2003). In addition, the observation ofsignificant phylogenetic incongruence in the genes ofEbola, mumps, Hantaan and Newcastle disease virusesalso lends support to the idea that recombination may playa role in negative-strand RNA virus evolution (Chare et al.,2003; Han et al., 2008a; Wittmann et al., 2007). However,there are very few examples of recombination-mediatedrepair for this group of viruses. An experiment aimedspecifically at testing the capacity for repair was performedwith respiratory syncytial virus. Despite experimentalconditions that were optimized deliberately to detectrecombination, only one functional recombinant viruswas generated in six co-infection experiments (Spann et al.,2003). Similarly, sequence analysis of circulating strains ofinfluenza virus, a segmented, negative-strand RNA virus,demonstrated that intragenic recombination occurs onlyrarely (Boni et al., 2008; Han et al., 2008b). Theinfrequency of intragenic recombination in the negative-strand RNA viruses could be a consequence of theirgenome template structure, in which the RNA remainsencapsidated throughout the replication cycle; however,there are examples of positive-strand RNA viruses that alsodemonstrate negligible levels of recombination (Taucheret al., 2010). Thus, efficient RNA recombination might bean activity that a subset of viruses has evolved that providesthem with the opportunity for genetic variation andgenomic repair.

Mechanisms to protect and restore the genometermini

The terminal sequences of RNA virus genomes areparticularly vulnerable to deletion or degradation. RNAviruses have evolved sophisticated mechanisms to avoidtruncation of terminal sequences during replicationinitiation and termination (Poranen et al., 2008; Tayon etal., 2001; van Dijk et al., 2004). However, despite thesemeasures, the termini of virus genomes remain susceptibleto host cell-mediated RNA degradation, either throughspecific antiviral host proteins (Bick et al., 2003; Silverman,2007) or due to the components of the normal host-cellRNA-biosynthesis machinery (reviewed by Houseley &Tollervey, 2009). Virus genomes possess various adapta-



tions helping them to evade these nuclease activities. Forexample, some positive-strand RNA virus genomes have acovalently bound cap, poly(A) tail and/or protein at theirtermini, and the genomes of negative-strand RNA virusesare sequestered within a helical nucleocapsid throughoutthe infection cycle. In addition, many RNA viruses havebeen shown to replicate in membraneous compartmentsthat might offer protection from nucleases (Mackenzie,2005). As a second line of defence, RNA viruses haveevolved means to repair their genome termini in the eventthat they are damaged (Fig. 1). Examples of terminal-repairmechanisms span numerous virus taxonomic lineages(Table 1), indicating that possession of a terminal-repairactivity is a fundamental element of RNA virus molecularbiology. The sections below describe examples of theserepair mechanisms.

Repair as a consequence of the initiation process

Because virus RNA genomes are linear, they requirereplication mechanisms that allow either initiation oppo-site the 39-terminal nucleotide of the template or recreationof terminal nucleotides during the initiation process. RNAviruses utilize a variety of strategies to achieve this,including protein-primed initiation, prime/realignmechanisms (in which the RdRp initiates internally onthe template and then realigns the nascent RNA andutilizes it as a primer), and RdRps structured to ensureinitiation opposite the 39 terminus of a single-strandedtemplate with a strong preference for the correct initiatingNTP (reviewed by van Dijk et al., 2004). In some cases,these highly coordinated mechanisms not only facilitateaccurate replication, but might also offer the possibility ofrepairing termini lacking a small number of nucleotides.For example, for several viruses, such as the picornavirusescoxsackie B3 virus (CB3V) and poliovirus and thepotyviruses potato virus Y and plum pox virus, it hasbeen shown that small deletions of 1 or 2 nt from genometermini are restored to wild-type sequence (Harmon et al.,1991; Jakab et al., 1997; Klump et al., 1990; Simon-Buela etal., 2000). Whilst the mechanism(s) for repair in thesecases has not been established and could involve one of themechanisms described in the sections below, the small sizeof the deletions could potentially allow repair as aconsequence of replication initiation. For example, picor-navirus RNA replication begins with uridylylation of a viralprotein, VPg, to create a molecule VPg–pU–pU, which actsas a primer for RNA synthesis initiation. Typically, theprimer anneals to adenylate residues at the 39 terminus ofthe template; however, there are multiple factors that areimportant for positioning the replicase complex (Liu et al.,2009). Thus, it is possible that the primer can still functionto initiate RNA replication, even if it cannot base-pair withthe template. This would restore the missing nucleotidesand allow rapid amplification of the repaired genome. Avirus that initiates using a prime/realign mechanism hasalso been shown to be able to repair the terminus of itsreplication product. Alteration or deletion of the extreme

39 residue of an influenza virus template resulted insubsequent restoration of the wild-type nucleotide.Evidence suggests that, in this case, the influenza virusRdRp initiates internally at position 4 and generates adinucleotide primer complementary to positions 4 and 5,and then the RdRp and primer realign to recreate an intact59 end (see Table 1 for sequence information) (Deng et al.,2006).

Primer-mediated repair using abortive transcripts

One means that the viral RdRp can use to repair damagedtermini is to use genetic material derived from intact viraltermini or from the termini of associated satellite RNAs.An example of this type of repair mechanism is primer-mediated repair. Initiation of RNA synthesis is a complexprocess involving several distinct stages, including a stagein which the RdRp transitions from an initiating to anelongating complex. If the RdRp fails to enter theelongation mode successfully, this results in the productionof a short oligonucleotide abortive transcript, of approxi-mately 3–12 nt, in a process known as abortive cycling(Carpousis & Gralla, 1980). Abortive initiations are afrequent event in normal RNA synthesis, with abortivetranscripts reaching 10- to 100-fold molar excesses overfull-length transcripts in some cases (Nagy et al., 1997).Primer-mediated repair involves initiating RNA replicationusing abortive transcripts generated from an intact viralRNA as primers to repair a damaged RNA.

Repair using abortive transcripts has been shown to occurfor a satellite RNA of turnip crinkle virus (TCV)(Carpenter & Simon, 1996; Nagy et al., 1997). SatelliteRNA C (satC) is one of several small RNAs that areassociated with the TCV genome and replicated by the viralRdRp. The 39 end of satC shares sequences with the 39

terminus of the TCV genome that are required for efficientRNA replication, including a stable stem–loop structurefollowed by the sequence 59-CUGCCC-39. If a satC RNAlacking the terminal 6 nt of this sequence was introducedinto plants together with wild-type TCV, satC was restoredefficiently to its wild-type sequence. Marker mutationsshowed that the source of restored nucleotides was theTCV genome. The TCV RdRp was shown to generateabundant short (4–8 nt) abortive transcripts and wascapable of using these in vitro to extend templates withpartial terminal deletions (Fig. 3). The exact mechanism bywhich primer-mediated repair occurs is unclear, but itappears to involve specific interactions between thestructures at the 39 end of the RNA and the RdRp, andbetween the RdRp and the oligoribonucleotide primers.The stem–loop structure at the 39 end of satC needed to beintact, indicating that the RdRp required a binding site onthe truncated RNA to be able to use it as a template andmediate repair. Interestingly, no base-pairing between the39 end of the satC RNA and the oligoribonucleotide primerwas necessary, but repair could only occur using certainoligoribonucleotide sequences (Nagy et al., 1997). These



Table 1. Examples of terminal repair in RNA viruses, with suggestions of possible repair mechanisms

Virus; genus (family) Nature of

terminus investigated

Terminal mutations Possible repair

mechanism(s)

Reference(s)

Coxsackievirus B3;

Enterovirus

(Picornaviridae)

39 oriR and poly(A) tail Deletion/substitution

of oriR and poly(A) tail

Viral/cellular terminal

transferase

van Ooij et al. (2006)

Hepatitis A virus;

Hepatovirus

(Picornaviridae)

39 poly(A) tail Deletion of poly(A) tail Viral/cellular terminal

transferase

Kusov et al. (2005)

Beet necrotic yellow vein

virus; Benyvirus

(unassigned)

39 poly(A) tail Deletion of poly(A) tail Viral/cellular terminal

transferase

Jupin et al. (1990)

Clover yellow vein virus;

Potyvirus (Potyviridae)

39 poly(A) tail Deletion/substitution of

poly(A) tail


transferase

Tacahashi & Uyeda (1999)

White clover mosaic virus;

Potexvirus (Flexiviridae)

39 AAUAAA+poly(A) tail Deletion of poly(A) tail Cellular terminal

transferase

Guilford et al. (1991)

Cucumber mosaic virus

satellite RNA;

Cucumovirus

(Bromoviridae)

39-UCCUCCGCAGGACCC-

OH

Deletion of up to 7 nt Viral terminal

transferase or viral

non-templated

polymerization

Burgyan & Garcia-Arenal

(1998)

Brome mosaic virus;

Bromovirus

(Bromoviridae)

39 tRNA-like Deletion/substitution

of 3 nt

Cellular tRNA

nucleotidyltransferase

Hema et al. (2005);

Rao et al. (1989)

Hepatitis C virus;

Hepacivirus (Flaviviridae)

39 polypyrimidine and

hairpin structure

Deletions extending into

hairpin structure


transferase

van Leeuwen et al. (2006)

Dengue virus type 2;

Flavivirus (Flaviviridae)

39 terminal stem–loop Substitution/deletion of

terminal nucleotides

Viral replicase non-

templated polymerization;

viral/cellular terminal

transferase

Teramoto et al. (2008)

Sindbis virus; Alphavirus

(Togaviridae)

39 conserved sequence

element and poly(A) tail

Deletions in the conserved

sequence element and/or

poly(A) tail


transferase

George & Raju (2000);

Hill et al. (1997);

Raju et al. (1999)

23S RNA virus; Narnavirus

(Narnaviridae)

39 stem–loop and CCC-OH Deletion/substitution of

terminal 1 or 2 nt

Cellular tRNA

nucleotidyltransferase

Fujimura & Esteban (2004)

Cymbidium ringspot virus;

Tombusvirus

(Tombusviridae)

39-GCAGCCC-OH Deletion of terminal 3 nt Viral replicase or

virus or host terminal

transferase

Dalmay et al. (1993)

Turnip crinkle virus

satellite RNA C;

Carmovirus

(Tombusviridae)

39-terminal stem–

loop+CUGCCC-OH

Deletion of terminal 6 nt Primers generated by

abortive initiation

Nagy et al. (1997)


satellite RNA C;

Carmovirus

(Tombusviridae)

39-terminal stem–

loop+CUGCCC-OH

Deletion of terminal 4–5 nt Viral replicase

polymerization of

non-templated bases

Guan & Simon (2000)


satellite RNA D;

Carmovirus

(Tombusviridae)

39-terminal stem–

loop+CUGCCC-OH

Deletion of up to 11 nt Primers generated by

abortive initiation

Carpenter & Simon (1996)

Coxsackievirus B3;

Enterovirus

(Picornaviridae)

59-UUAAAACAGC Deletion of terminal 2 nt VPg uridylylation

acting as a primer

Klump et al. (1990)

Poliovirus; Enterovirus

(Picornaviridae)

59-UUAAAACAGC Deletion of terminal 2 nt VPg uridylylation

acting as a primer

Harmon et al. (1991)

Potato virus Y; Potyvirus

(Potyviridae)

59 cap and AAATTAAAAC Deletion of terminal 1 nt VPg adenylylation

acting as a primer

Jakab et al. (1997)

Plum pox virus; Potyvirus

(Potyviridae)

59 cap and GAAAA Substitution/deletion of

terminal 1 or 2 nt

VPg adenylylation

acting as a primer

Simon-Buela et al. (2000)



findings suggest that this repair mechanism involvesbinding of the TCV RdRp to the defective template RNA,recruiting oligoribonucleotides of specific sequences intoits active site and using these to initiate RNA synthesis.

These findings suggest that the strategy of utilizing a poolof abortive transcripts as primers may be a useful way toovercome the problem of terminal deletions. To ourknowledge, repair of TCV-associated RNAs represents theonly described example of primer-mediated templaterepair for an RNA virus to date, but RdRps of positive-,

negative- and double-stranded RNA viruses have beenshown to generate abortive transcripts (Dupuy et al., 1999;Farsetta et al., 2000; Klumpp et al., 1998; Sun & Kao, 1997)and short oligonucleotides can be used as primers toinitiate RNA synthesis in several virus systems (Chen &Patton, 2000; Garcin & Kolakofsky, 1992; Honda et al.,1986; Kao & Sun, 1996; Kawakami et al., 1981; Nomaguchiet al., 2003). Interestingly, in some of these cases, it wasshown that the RdRp demonstrated specificity for primersof the correct sequence or specific length (Chen & Patton,2000; Kao & Sun, 1996; Kawakami et al., 1981). Thus, itmight be a common feature of viral RdRps to have apreference for binding particular oligoribonucleotides intheir substrate pocket during the initiation phase of RNAsynthesis, allowing them to repair defective templates.

Non-templated polymerization by the viral replicase togenerate random primers

In another series of experiments studying TCV satC RNArepair, templates with shorter deletions of 3–5 nt wereexamined. It was found that these deletions were alsorepaired to create RNA that was similar in length to thewild-type satC RNA, but, in this case, the satC terminuswas not restored consistently to its wild-type sequence, butrather consisted of apparently random sequence, indicatingthat an alternative repair mechanism was employed (Guan& Simon, 2000). If the 39 terminus of the deleted satC wasfused to non-specific sequence, the non-specific sequencewas removed and replaced with random sequence ofappropriate length. This occurred in the context of an invitro replicase reaction, suggesting that this repair occurredin a single step. Guan & Simon (2000) provide a model forthese results in which the TCV RdRp generates a smallrandom RNA independently of the template, then uses thisRNA as a primer to initiate RNA synthesis at the 39 end ofthe satC RNA sequence, positioning the primer using thestem–loop structure near the 39 end of the satC RNA.

If the model proposed by Guan & Simon (2000) is correct,it raises the question of how the replicase generates RNAprimers if there is no viable promoter sequence for it toinitiate on. A possible explanation comes from studies withthe replicase of the RNA phage Qb. Some studies havesuggested that the Qb replicase is capable of generatingRNA de novo in the absence of a template (Biebricher et al.,1986; Sumper & Luce, 1975). Other studies have suggestedthat the Qb replicase is not truly template-independent,but rather can readily utilize any RNA as a template, even if

Table 1. cont.

Virus; genus (family) Nature of

terminus investigated

Terminal mutations Possible repair

mechanism(s)

Reference(s)

Influenza A virus;

Influenzavirus A

(Orthomyxoviridae)

59-AGUAGAAA Substitution/deletion of

terminal 1 nt

Prime/realign initiation Deng et al. (2006).

Fig. 3. Mechanism for TCV primer-mediated repair. (a) The TCVgenome (black line) and satC RNA (red line) share common 39-terminal sequences. In this scenario, the genome is a template forabortive transcripts, which are generated from the 39-terminalsequences during replication initiation; the satC RNA 39 terminusis degraded. (b) An abortive transcript generated from the intactTCV genome can be used to prime initiation from a truncated satCRNA template and be extended by the TCV RdRp to generate arestored complement of the satC RNA.



the RNA is only present at low levels as a contaminant, andthat, whilst the Qb replicase can initiate RNA synthesisrandomly, it does not enter a stable elongation mode ifinitiation occurs in the absence of the appropriatepromoter (Chetverin et al., 1991; Hill & Blumenthal,1983; Ugarov et al., 2003). Thus, an explanation for theTCV-mediated repair is that, in the absence of anappropriate promoter sequence, the viral replicase gen-erates RNA in a template-independent manner or initiatesrandomly on any RNA available, thus generating a pool ofrandom oligonucleotides, some of which could fit appro-priately with the RdRp to be utilized as primers to initiatereplication, similarly to the mechanism described above.Once the RdRp has generated a ‘patched’ RNA that is ableto support even a low level of replication, the RNA wouldhave the opportunity to evolve towards a wild-typesequence over multiple replication cycles.

TCV satC RNA is not the only example of an RNA virus(or virus-associated RNA) acquiring heterologous sequenceat its termini. Evidence for additional non-templatedresidues has been found for the positive-strand RNAviruses cucumber mosaic virus (Burgyan & Garcia-Arenal,1998) and dengue virus (Teramoto et al., 2008), and thenegative-strand RNA viruses Borna disease virus (BDV),lymphocytic choriomeningitis virus (LCMV) and Hantaanhantavirus (Meyer & Schmaljohn, 2000; Meyer &Southern, 1994; Schneider et al., 2005). Although themechanism by which the additional nucleotides wereadded to these virus genomes is not known and couldinvolve a terminal transferase activity, such as thatdescribed below, the random nature of the additionalsequence is consistent with the non-templated initiationmechanism described for TCV.

Terminal transferase and poly(A) tail repairmechanisms

Many positive-stranded RNA viruses possess a 39 poly(A)tract, which serves roles in translation, RNA stability andgenome replication. These poly(A) tails are heterogeneous,but must be of a minimum length to support efficient virusreplication, and there are data that point to the existence ofa viral mechanism that restores adenylates that are lostfrom the 39 end of the poly(A) tail during normal genomereplication (van Ooij et al., 2006). There is also evidencethat completely deleted poly(A) tails can be repaired. Forexample, an engineered clone of cowpea mosaic virusregained a deleted poly(A) tail in vivo (Eggen et al., 1989)and similar examples exist for a number of other viruses(Chen & Frey, 1999; Guilford et al., 1991; Hill et al., 1997;Kusov et al., 2005; Riechmann et al., 1990; Tacahashi &Uyeda, 1999). It is possible that the poly(A) tails arerestored by a cellular poly(A) polymerase activity and, insome cases, there is evidence for this: white clover mosaicvirus can recover a deleted poly(A) tail, but this isdependent on an AAUAAA motif, which is a signalsequence for cellular poly(A) polymerase (Fitzgerald &

Shenk, 1981; Guilford et al., 1991). Alternatively, someviruses have been shown to possess virus-encoded terminaladenyltransferase activity that enables them to transferpoly(A) sequences to the 39 end of their templates (Neufeldet al., 1994; Tomar et al., 2006).

Interestingly, some flaviviruses that do not possess poly(A)tails have also been shown to encode terminal transferaseactivity (Behrens et al., 1996; Ranjith-Kumar et al., 2001).In vitro studies of hepatitis C virus terminal transferaseactivity showed that, in this case, the specificity of theadded nucleotide is guided by the 39-terminal sequence ofthe template RNA, and the terminal transferase activityallows the RdRp to add a terminal cytidylate residue to amutated 39 terminus and restore template function(Ranjith-Kumar et al., 2001). Indeed, terminal transferaseactivity is apparently widespread, having been reported forother positive-strand RNA viruses, a double-stranded RNAvirus and a negative-strand RNA virus (Fullerton et al.,2007; Poranen et al., 2008; Rohayem et al., 2006;Smallwood & Moyer, 1993), suggesting that it might be acommon mechanism to facilitate terminal repair.

The mechanism for viral terminal transferase activity hasnot been elucidated completely. The activity is dependenton the active site of the RdRp (Poranen et al., 2008;Ranjith-Kumar et al., 2001; Tomar et al., 2006), which isnot surprising, as cellular ribonucleotidyltransferases use asimilar catalytic motif (Martin & Keller, 2007). However,this raises the question of how does an RdRp catalyseterminal transferase activity. The RdRp would normallyposition the 39 end of template RNA in its templatechannel, add nucleotides onto the 39 end of the nascentRNA in its active site and then extrude nascent RNA in a59A39 direction via its exit channel. If the RdRp bound tothe defective template in its usual orientation, the 39 end ofthe RNA would not be positioned appropriately relative tothe catalytic site for nucleotide additions. Studies on theRdRp/terminal transferase of bacteriophage w6 suggest thatthe most likely explanation is that, for viral terminaltransferase activity to occur, the RdRp subunit becomesoriented in the opposite direction with respect to the RNAthan it would be for template-directed RdRp activity anddraws the 39 end of the RNA through its exit channel intothe catalytic site, so that the 39 terminus of the RNA ispositioned appropriately in the active site for catalysis tooccur (Fig. 4) (Poranen et al., 2008). However, it remainsto be determined why terminal transferase activity can bespecific for a particular NTP substrate, or how the terminalsequence of the template RNA can determine the specificityof the added nucleotides. Regardless of the specificmechanism, it seems likely that, if all of the elements forinitiation of template-directed polymerization were pre-sent, including all of the 39-terminal signals, the RdRpwould be most likely to bind the template in theorientation illustrated in Fig. 4(a) and initiate RNAreplication. However, if 39-terminal signals were not intact,the RdRp would be unable to form a stable initiationcomplex, increasing its propensity to adopt a terminal



transferase orientation until the RNA has been repairedsufficiently (Fig. 4c).

A common but uncharacterized mechanism of repair: apossible role for the cellular RNA-degradationmachinery

Some polyadenylated virus genomes that were shown toundergo repair to restore the poly(A) tail were found tohave acquired novel U-rich sequences. This phenomenonwas first described for beet necrotic yellow vein virus. Inthis case, if the poly(A) tail was removed completely,progeny viruses contained a novel heterogeneous U-richregion adjacent to the viral sequence, followed by a poly(A)tail (Jupin et al., 1990). Similar results have been observedfor Sindbis virus (Raju et al., 1999), coxsackie B virus (vanOoij et al., 2006) and hepatitis C virus (van Leeuwen et al.,2006). The mechanism that generates the heterogeneousU-rich linker followed by the poly(A) tail is so farundetermined and it has been suggested that it mightinvolve a virus-mediated activity, such as virus-encodedterminal transferase, or internal initiation followed byprimer jumping (Raju et al., 1999). However, the fact that

this type of repair has been observed for a diverse range ofviruses, some of which have been shown to possess onlyadenyltransferase activity (with no evidence for uridyl-transferase activity), is intriguing and suggests that thisrepair mechanism might involve cellular enzymes.

A possible candidate for the U-rich insertions is the cellularRNA-decay machinery. A growing body of evidence hasidentified relationships between replication complexes of anumber of plant and animal viruses and cytoplasmic RNA-processing sites, such as processing bodies and stressgranules (Beckham & Parker, 2008). This suggests a closeinvolvement between the cellular degradation machineryand virus replication sites. A group of newly discoveredfactors in mRNA turnover are cellular poly(U)polymerases; the poly(U) tail added by these enzymesstimulates removal of the 59 cap, promoting mRNAdegradation (Song & Kiledjian, 2007; Wickens & Kwak,2008). Poly(U) polymerases are widespread, from yeast tohumans, and they are non-specific in their RNA substrate(Guschina & Benecke, 2008; Wickens & Kwak, 2008); thus,they have the potential to act on a variety of viral RNAs.Poly(U) polymerases can also be relaxed in their nucleotidespecificity: there is an example of a poly(U) RdRp that canswitch between adding U or A residues (Mellman et al.,2008). Thus, one possible explanation for how virusgenomes acquire non-viral U-rich sequences is that atruncated viral RNA [lacking its poly(A) tail] becomestailed by poly(U) polymerase to mark it for degradation(Fig. 5). Some viruses might have evolved mechanisms thatallow them to then intercept the degradation pathway andpromote polyadenylation, either by using a virus-encodedterminal transferase activity, as described above, or byprovoking the poly(U) polymerase to switch NTP specificityto ATP. Addition of the poly(A) tail would be expected toconfer stability to the virus genome and, in combinationwith cis-acting signals elsewhere in the virus genome, mightbe sufficient to enable genome replication and propagation.

Repair of 39 termini by tRNA mimicry

The final mechanism of terminal repair almost certainlyinvolves a cellular enzyme and is facilitated by virusmimicry of cellular tRNAs. A 39-terminal modificationfound in the RNA genomes of many plant RNA viruses isamino acylation to charge the 39 terminus of the genomeRNA with the amino acids valine, histidine or tyrosine. Themechanism by which amino acylation of viral genomesoccurs is related to tRNA amino acylation: these viruseshave 39-terminal RNA sequences that can adopt a highlyorganized secondary/tertiary structure akin to that of host-cell tRNAs, allowing them to be charged by the respectiveamino acyl tRNA synthetase (reviewed by Dreher, 2009).The similarity between the virus genome and a tRNAstructure not only allows acylation to occur, but also offersan opportunity for 39-terminal sequence repair.

Cellular tRNAs have a characteristic cloverleaf structurewith an unpaired CCA-OH motif at the 39 terminus. The

Fig. 4. Putative model for the RdRp terminal transferasemechanism. (a) Replication initiation involves assembly of thereplicase complex, including the RdRp, onto the template RNA. Inthe case of de novo initiation at the 39 terminus, the 39 end of thetemplate is located in the active site. (b) During RNA synthesis, the39 OH group of the nascent strand is positioned appropriately inthe active site for addition of the next nucleotide, and the newlysynthesized RNA is extruded through an exit channel. (c) Forterminal transferase activity, it is proposed that the RdRp and theRNA ‘template’ are rotated relative to each other. In this case, theRNA enters via the exit channel, positioning the 39 OH groupappropriately in the active site for further nucleotides to be added.



CCA motif is not encoded by the eukaryotic genome, butrather is added post-transcriptionally by the cellularenzyme tRNA nucleotidyltransferase, which also functionsto maintain the tRNA 39 terminus in the face of constantnuclease degradation. The tRNA nucleotidyltransferasefunctions independently of a nucleic acid template, withnucleotide selection apparently being guided by pocketswithin the protein structure, created by conformationalchanges following addition of each nucleotide (reviewed byMartin & Keller, 2007). The 39 terminus of the BMVgenome ends in CC, but is modified to CCA upon entryinto the cell. The similarity between viral 39 tRNAstructures and authentic cellular tRNAs is thought toafford the virus genome the ability to bind to tRNAnucleotidyltransferase, allowing this enzyme to restore the39 A residue in the same manner as it would repair acellular tRNA (Joshi et al., 1983). This tRNA mimicryalso provides an opportunity for repair of genomes fromwhich the 39-terminal cytidylates have been removed.Experiments with BMV have shown that genomes contain-ing 39-terminal deletions to the CCA motif are repaired towild-type sequence very rapidly in cells (Hema et al., 2005;Rao et al., 1989). This repair activity is not dependent onthe other virus genome segments acting as a template(Hema et al., 2005) and does not occur in reactionsperformed in vitro using purified replicase (Miller et al.,1986). Thus, the most likely mechanism for repair isaddition of the CCA motif by tRNA nucleotidyltransferase.

Potential benefits of terminal deletions: truncatedgenomes and viral persistence

Many RNA viruses that cause acute infections rely on rapidand efficient multiplication to enable virus spread to newhost cells before clearance by the host immune system. Thisviral strategy requires maintenance of a genome sequence

that is able to support sufficient levels of virus replication,and this requirement probably contributes to the selectionpressure that drives development of the repair activitiesdescribed above. A number of RNA viruses are alsoassociated with persistent infections, particularly in certaintissues or organisms. During persistent infection, muta-tions that arise can play an important role. For example,mutations in the coding region of hepatitis C virus canallow the virus to evade immune attack (Burke & Cox,2010). In addition, persistence is also frequently associatedwith truncations in terminal cis-acting sequences. Indeed,the high prevalence of terminally deleted genomes in somepersistent virus infections suggests that terminal deletionsmight be involved directly in the establishment and/ormaintenance of the persistent state.

Examples of associations between terminal truncationsand persistent infections

LCMV, a member of the family Arenaviridae, is asegmented, negative-sense RNA virus. LCMV infection ofnewborn mice progresses from an initial acute phase to apersistent phase associated with a sustained low level ofvirion production. Extended time-course experiments incell culture (Meyer & Southern, 1994) or mice (Meyer &Southern, 1997) showed that both of the viral genomesegments were corrupted by short terminal deletionsand nucleotide additions of up to 4 nt. The abundanceof these corrupted genomes increased throughout thetime-course to the extent that, by 28 days post-infection,between 60 and 70 % of all genome sequences exhibiteddeletions. Interestingly these genomic sequence changesappeared to be matched by complementary changes in thecorresponding antigenomic strands, suggesting that thesealtered RNAs were replication-competent; however,they were deficient in mRNA transcription (Meyer &Southern, 1997). The presence of replication-competent, but

Fig. 5. Model for the acquisition of a non-viralpoly(U) sequence. According to this model,following removal or loss of the poly(A) tail atthe 39 end of the viral genome RNA, cellularpoly(U) polymerase uridylates the truncatedRNA to mark it for degradation. In some cases,viral proteins might be able to intervene in thisprocess, either by altering the activity of thepoly(U) polymerase, so that it adds adenylateresidues onto the 39 terminus, or by enablingthe viral RdRp to polyadenylate the RNA byterminal transferase activity.



transcription-incompetent, genome segments is consistentwith the diminished viral protein expression and low yield ofinfectious virus that are characteristics of the persistentinfection. Terminally deleted genomes are also a feature ofSeoul virus, a member of the genus Hantavirus, which isanother segmented, negative-sense RNA virus that estab-lishes long-term persistent infections in rodent hosts (Meyer& Schmaljohn, 2000). Extended time-course experiments incell culture revealed that the abundance of genome segmentswith terminal deletions exhibited a cyclical pattern, risingand falling throughout the duration of the experiment. Theabundance of viral RNA and virus progeny mirrored theproportion of truncated genomes, and thus stronglyimplicated the deletions in maintaining the persistent state(Meyer & Schmaljohn, 2000).

Terminal sequence deletions also have been shown to playa role in the transition between acute and persistentinfections of a positive-strand RNA virus. CB3V, a humanenterovirus, is frequently associated with an acute myo-carditis that can often lead to heart failure. CB3V genomescan be detected in tissue from previously infectedindividuals, yet infectious viruses have been almostimpossible to isolate, suggesting that CB3V may be capableof viral persistence. Evidence for a persistence mechanismcame from sequence analysis, which showed that CB3Vgenomes within infected tissues possessed 59-terminaldeletions of between 7 and 49 nt (Kim et al., 2005). Itwas found that there was a correlation between thepresence of complete or truncated termini, and acuteversus persistent infection, respectively (Kim et al., 2005).More recent work has detected the presence of terminallydeleted genomes in human tissue collected from a fatal caseof myocarditis, suggesting that there is significance forterminal deletions in the context of a human infection(Chapman et al., 2008).

Evidence for a specific mechanism for terminaltruncation

The mechanism by which these viruses acquire truncationsto their termini might differ. In the case of CB3V, terminaldeletions arise more rapidly in either intact heart tissues orprimary myocardial cell cultures than in other cells,suggesting that the cellular environment has a stronginfluence (Kim et al., 2005). However, one virus associatedwith persistent virus infections appears to have evolved aspecific strategy for truncating its termini. BDV canestablish a persistent infection in cell-culture systems andalso brain cells of infected animals. This virus is a memberof the order Mononegavirales, members of which typicallypossess genomes that exhibit terminal complementarity.However, the 59 termini of the viral RNA and cRNAs ofBDV isolates extracted from infected cells and animals arerecessed by 4 nt compared with the 39 termini (Rosarioet al., 2005; Schneider et al., 2005). Experiments haveshown that recombinant virus with perfectly complement-ary 39 and 59 ends quickly reverts to having recessed 59

termini, similar to the naturally occurring virus (Schneideret al., 2005). The high level of precision of this terminaltruncation suggests that it occurs by a specific mechanism,possibly involving replication initiation at an internallylocated nucleotide. Although that mechanism has not yetbeen elucidated, it apparently involves an activity thatleaves a monophosphate moiety at the 59 terminus, insteadof the 59 triphosphate that would typically be found at the59 end of viral RNA. Gene-expression studies indicate thattrimmed genomes are efficient templates for transcription,but inefficient templates for replication, thus limiting thegeneration of infectious particles. In addition, the 59

monophosphate moiety and the recessed 59 terminus ofgenomic and antigenomic strands enable the viral RNA toavoid detection by retinoic acid-induced gene 1 (RIG-I)(Habjan et al., 2008). This raises the interesting possibilitythat, in addition to modulating virus replication and geneexpression, BDV genome trimming may be a mechanismto facilitate virus persistence by evading cellular antiviralresponses.

Concluding remarks

The studies described above demonstrate a range ofmechanisms that exist to enable maintenance and restora-tion of genome integrity in RNA viruses. These studiesreveal that, despite their often fastidious mechanisms toensure accurate replication initiation and termination, viralRdRps are sufficiently flexible to accommodate alternativemodes of initiation and elongation, enabling terminalrepair, terminal transferase activity and recombination. Forany given virus, the behaviour of the RdRp at the 39 end ofa template might be impacted by the nature of the 39-terminal sequence. It seems likely that, if the genometermini are intact and all promoter and accessorysequences required for replication initiation are present,the RdRp will engage preferentially in accurate replicationinitiation. However, if key terminal sequences are missing,the replicase complex might not be able to assemblecorrectly, releasing the RdRp to perform ‘abnormal’actions, such as non-templated polymerization or terminaltransferase activity. Once a genome has been repaired, theRdRp could revert to its ‘normal’ function of template-dependent polymerization. Viruses that replicate inparticularly harsh environments or which have no passivedefences to protect their genomes may have replicases thataccommodate alternative initiation mechanisms to facil-itate terminal repair more readily, and fundamentaldifferences in virus genome and replicase architectureprobably affect the propensity for RNA recombination. It isstriking that most examples of RNA virus genome repairinvolve positive-strand viruses, whose genomes might bemore vulnerable than those of the double-stranded ornegative-sense viruses, in which the genomes are seques-tered in protein capsids during the entire cycle of infection.The data also suggest that formation of truncated genomes,whilst hindering virus replication kinetics, might allow oraid some viruses to become persistent, which ultimately



could aid their propagation within the host population.Thus, the capacity for genome repair could be animportant factor in virus pathogenesis.

Acknowledgements

We thank Elke Muhlberger, Sean Whelan and Sarah Noton for

constructive comments on the manuscript.

References

Aas, P. A., Otterlei, M., Falnes, P. O., Vagbo, C. B., Skorpen, F.,Akbari, M., Sundheim, O., Bjoras, M., Slupphaug, G. & other authors(2003). Human and bacterial oxidative demethylases repair alkylation

damage in both RNA and DNA. Nature 421, 859–863.

Allison, R., Thompson, C. & Ahlquist, P. (1990). Regeneration of a

functional RNA virus genome by recombination between deletion

mutants and requirement for cowpea chlorotic mottle virus 3a and

coat genes for systemic infection. Proc Natl Acad Sci U S A 87, 1820–

1824.

Beckham, C. J. & Parker, R. (2008). P bodies, stress granules, and

viral life cycles. Cell Host Microbe 3, 206–212.

Behrens, S. E., Tomei, L. & De Francesco, R. (1996). Identification

and properties of the RNA-dependent RNA polymerase of hepatitis C

virus. EMBO J 15, 12–22.

Bick, M. J., Carroll, J. W., Gao, G., Goff, S. P., Rice, C. M. &MacDonald, M. R. (2003). Expression of the zinc-finger antiviral

protein inhibits alphavirus replication. J Virol 77, 11555–11562.

Biebricher, C. K., Eigen, M. & Luce, R. (1986). Template-free RNA

synthesis by Qb replicase. Nature 321, 89–91.

Boni, M. F., Zhou, Y., Taubenberger, J. K. & Holmes, E. C. (2008).Homologous recombination is very rare or absent in human influenza

A virus. J Virol 82, 4807–4811.

Bratlie, M. S. & Drablos, F. (2005). Bioinformatic mapping of AlkB

homology domains in viruses. BMC Genomics 6, 1.

Bujarski, J. J. & Kaesberg, P. (1986). Genetic recombination between

RNA components of a multipartite plant virus. Nature 321, 528–

531.

Burgyan, J. & Garcia-Arenal, F. (1998). Template-independent repair

of the 39 end of cucumber mosaic virus satellite RNA controlled by

RNAs 1 and 2 of helper virus. J Virol 72, 5061–5066.

Burke, K. P. & Cox, A. L. (2010). Hepatitis C virus evasion of adaptive

immune responses: a model for viral persistence. Immunol Res (in

press). doi:10.1007/s12026-009-8152-3

Calain, P. & Roux, L. (1993). The rule of six, a basic feature for

efficient replication of Sendai virus defective interfering RNA. J Virol

67, 4822–4830.

Carpenter, C. D. & Simon, A. E. (1996). In vivo repair of 39-end

deletions in a TCV satellite RNA may involve two abortive synthesis

and priming events. Virology 226, 153–160.

Carpousis, A. J. & Gralla, J. D. (1980). Cycling of ribonucleic acid

polymerase to produce oligonucleotides during initiation in vitro at

the lac UV5 promoter. Biochemistry 19, 3245–3253.

Castro, C., Arnold, J. J. & Cameron, C. E. (2005). Incorporation

fidelity of the viral RNA-dependent RNA polymerase: a kinetic,

thermodynamic and structural perspective. Virus Res 107, 141–149.

Cattaneo, R., Kaelin, K., Baczko, K. & Billeter, M. A. (1989). Measles

virus editing provides an additional cysteine-rich protein. Cell 56,

759–764.

Chapman, N. M., Kim, K. S., Drescher, K. M., Oka, K. & Tracy, S.(2008). 59 Terminal deletions in the genome of a coxsackievirus B2strain occurred naturally in human heart. Virology 375, 480–491.

Chare, E. R., Gould, E. A. & Holmes, E. C. (2003). Phylogeneticanalysis reveals a low rate of homologous recombination in negative-sense RNA viruses. J Gen Virol 84, 2691–2703.

Chen, M. H. & Frey, T. K. (1999). Mutagenic analysis of the 39 cis-acting elements of the rubella virus genome. J Virol 73, 3386–3403.

Chen, D. & Patton, J. T. (2000). De novo synthesis of minus strandRNA by the rotavirus RNA polymerase in a cell-free system involves anovel mechanism of initiation. RNA 6, 1455–1467.

Chen, P., Jiang, M., Hu, T., Liu, Q., Chen, X. S. & Guo, D. (2007).Biochemical characterization of exoribonuclease encoded by SARScoronavirus. J Biochem Mol Biol 40, 649–655.

Chetverin, A. B., Chetverina, H. V. & Munishkin, A. V. (1991). On thenature of spontaneous RNA synthesis by Qb replicase. J Mol Biol 222,3–9.

Copper, P. D., Steiner-Pryor, A., Scotti, P. D. & Delong, D. (1974). Onthe nature of poliovirus genetic recombinants. J Gen Virol 23, 41–49.

Crotty, S. & Andino, R. (2002). Implications of high RNA virusmutation rates: lethal mutagenesis and the antiviral drug ribavirin.Microbes Infect 4, 1301–1307.

Dalmay, T., Russo, M. & Burgyan, J. (1993). Repair in vivo of altered39 terminus of Cymbidium ringspot tombusvirus RNA. Virology 192,551–555.

Deng, T., Vreede, F. T. & Brownlee, G. G. (2006). Different de novoinitiation strategies are used by influenza virus RNA polymerase on itscRNA and viral RNA promoters during viral RNA replication. J Virol80, 2337–2348.

Drake, J. W. (1993). Rates of spontaneous mutation among RNAviruses. Proc Natl Acad Sci U S A 90, 4171–4175.

Dreher, T. W. (2009). Role of tRNA-like structures in controlling plantvirus replication. Virus Res 139, 217–229.

Duffy, S., Shackelton, L. A. & Holmes, E. C. (2008). Rates ofevolutionary change in viruses: patterns and determinants. Nat RevGenet 9, 267–276.

Dupuy, L. C., Dobson, S., Bitko, V. & Barik, S. (1999). Casein kinase 2-mediated phosphorylation of respiratory syncytial virus phosphopro-tein P is essential for the transcription elongation activity of the viralpolymerase; phosphorylation by casein kinase 1 occurs mainly atSer(215) and is without effect. J Virol 73, 8384–8392.

Eckerle, L. D., Lu, X., Sperry, S. M., Choi, L. & Denison, M. R. (2007).High fidelity of murine hepatitis virus replication is decreased innsp14 exoribonuclease mutants. J Virol 81, 12135–12144.

Egelman, E. H., Wu, S. S., Amrein, M., Portner, A. & Murti, G. (1989).The Sendai virus nucleocapsid exists in at least four different helicalstates. J Virol 63, 2233–2243.

Eggen, R., Verver, J., Wellink, J., De Jong, A., Goldbach, R. & vanKammen, A. (1989). Improvements of the infectivity of in vitrotranscripts from cloned cowpea mosaic virus cDNA: impact ofterminal nucleotide sequences. Virology 173, 447–455.

Farsetta, D. L., Chandran, K. & Nibert, M. L. (2000). Transcriptionalactivities of reovirus RNA polymerase in recoated cores. Initiationand elongation are regulated by separate mechanisms. J Biol Chem275, 39693–39701.

Fitzgerald, M. & Shenk, T. (1981). The sequence 59-AAUAAA-39

forms parts of the recognition site for polyadenylation of late SV40mRNAs. Cell 24, 251–260.

Fujimura, T. & Esteban, R. (2004). Bipartite 39-cis-acting signal forreplication in yeast 23 S RNA virus and its repair. J Biol Chem 279,13215–13223.



Fullerton, S. W., Blaschke, M., Coutard, B., Gebhardt, J., Gorbalenya, A.,Canard, B., Tucker, P. A. & Rohayem, J. (2007). Structural and

functional characterization of sapovirus RNA-dependent RNA poly-

merase. J Virol 81, 1858–1871.

Galinski, M. S., Troy, R. M. & Banerjee, A. K. (1992). RNA editing in

the phosphoprotein gene of the human parainfluenza virus type 3.

Virology 186, 543–550.

Garcin, D. & Kolakofsky, D. (1992). Tacaribe arenavirus RNA

synthesis in vitro is primer dependent and suggests an unusual model

for the initiation of genome replication. J Virol 66, 1370–1376.

George, J. & Raju, R. (2000). Alphavirus RNA genome repair and

evolution: molecular characterization of infectious Sindbis virus

isolates lacking a known conserved motif at the 39 end of the genome.

J Virol 74, 9776–9785.

Gmyl, A. P., Belousov, E. V., Maslova, S. V., Khitrina, E. V., Chetverin,

A. B. & Agol, V. I. (1999). Nonreplicative RNA recombination in

poliovirus. J Virol 73, 8958–8965.

Gmyl, A. P., Korshenko, S. A., Belousov, E. V., Khitrina, E. V. & Agol,

V. I. (2003). Nonreplicative homologous RNA recombination:

promiscuous joining of RNA pieces? RNA 9, 1221–1231.

Guan, H. & Simon, A. E. (2000). Polymerization of nontemplate bases

before transcription initiation at the 39 ends of templates by an RNA-

dependent RNA polymerase: an activity involved in 39 end repair of

viral RNAs. Proc Natl Acad Sci U S A 97, 12451–12456.

Guilford, P. J., Beck, D. L. & Forster, R. L. (1991). Influence of the

poly(A) tail and putative polyadenylation signal on the infectivity of

white clover mosaic potexvirus. Virology 182, 61–67.

Guschina, E. & Benecke, B. J. (2008). Specific and non-specific

mammalian RNA terminal uridylyl transferases. Biochim Biophys Acta

1779, 281–285.

Habjan, M., Andersson, I., Klingstrom, J., Schumann, M., Martin, A.,

Zimmermann, P., Wagner, V., Pichlmair, A., Schneider, U. & otherauthors (2008). Processing of genome 59 termini as a strategy of

negative-strand RNA viruses to avoid RIG-I-dependent interferon

induction. PLoS One 3, e2032.

Han, G. Z., He, C. Q., Ding, N. Z. & Ma, L. Y. (2008a). Identification of

a natural multi-recombinant of Newcastle disease virus. Virology 371,

54–60.

Han, G. Z., Liu, X. P. & Li, S. S. (2008b). Homologous recombination

is unlikely to play a major role in influenza B virus evolution. Virol J

5, 65.

Harmon, S. A., Richards, O. C., Summers, D. F. & Ehrenfeld, E.(1991). The 59-terminal nucleotides of hepatitis A virus RNA, but not

poliovirus RNA, are required for infectivity. J Virol 65, 2757–2760.

Hema, M., Gopinath, K. & Kao, C. (2005). Repair of the tRNA-like

CCA sequence in a multipartite positive-strand RNA virus. J Virol 79,

1417–1427.

Hill, D. & Blumenthal, T. (1983). Does Qb replicase synthesize RNA in

the absence of template? Nature 301, 350–352.

Hill, K. R., Hajjou, M., Hu, J. Y. & Raju, R. (1997). RNA–RNA

recombination in Sindbis virus: roles of the 39 conserved motif,

poly(A) tail, and nonviral sequences of template RNAs in polymerase

recognition and template switching. J Virol 71, 2693–2704.

Honda, A., Mizumoto, K. & Ishihama, A. (1986). RNA polymerase of

influenza virus. Dinucleotide-primed initiation of transcription at

specific positions on viral RNA. J Biol Chem 261, 5987–5991.

Houseley, J. & Tollervey, D. (2009). The many pathways of RNA

degradation. Cell 136, 763–776.

Huang, A. S. & Baltimore, D. (1970). Defective viral particles and viral

disease processes. Nature 226, 325–327.

Jakab, G., Droz, E., Brigneti, G., Baulcombe, D. & Malnoe, P. (1997).Infectious in vivo and in vitro transcripts from a full-length cDNAclone of PVY-N605, a Swiss necrotic isolate of potato virus Y. J Gen

Virol 78, 3141–3145.

Joshi, R. L., Joshi, S., Chapeville, F. & Haenni, A. L. (1983). tRNA-like

structures of plant viral RNAs: conformational requirements for

adenylation and aminoacylation. EMBO J 2, 1123–1127.

Jupin, I., Bouzoubaa, S., Richards, K., Jonard, G. & Guilley, H. (1990).Multiplication of beet necrotic yellow vein virus RNA 3 lacking a 39

poly(A) tail is accompanied by reappearance of the poly(A) tail and anovel short U-rich tract preceding it. Virology 178, 281–284.

Kao, C. C. & Sun, J. H. (1996). Initiation of minus-strand RNAsynthesis by the brome mosaic virus RNA-dependent RNA poly-

merase: use of oligoribonucleotide primers. J Virol 70, 6826–6830.

Kawakami, K., Ishihama, A., Ohtsuka, E., Tanaka, T., Takashima, H.& Ikehara, M. (1981). RNA polymerase of influenza virus. II.

Influence of oligonucleotide chain length on the priming activity of

RNA synthesis by virion-associated RNA polymerase. J Biochem 89,1759–1768.

Kim, K. S., Tracy, S., Tapprich, W., Bailey, J., Lee, C. K., Kim, K., Barry,W. H. & Chapman, N. M. (2005). 59-Terminal deletions occur incoxsackievirus B3 during replication in murine hearts and cardiac

myocyte cultures and correlate with encapsidation of negative-strand

viral RNA. J Virol 79, 7024–7041.

Klump, W. M., Bergmann, I., Muller, B. C., Ameis, D. & Kandolf, R.(1990). Complete nucleotide sequence of infectious coxsackievirus B3cDNA: two initial 59 uridine residues are regained during plus-strand

RNA synthesis. J Virol 64, 1573–1583.

Klumpp, K., Ford, M. J. & Ruigrok, R. W. (1998). Variation in ATPrequirement during influenza virus transcription. J Gen Virol 79,

1033–1045.

Koetzner, C. A., Parker, M. M., Ricard, C. S., Sturman, L. S. &Masters, P. S. (1992). Repair and mutagenesis of the genome of a

deletion mutant of the coronavirus mouse hepatitis virus by targetedRNA recombination. J Virol 66, 1841–1848.

Kolakofsky, D., Pelet, T., Garcin, D., Hausmann, S., Curran, J. &Roux, L. (1998). Paramyxovirus RNA synthesis and the requirementfor hexamer genome length: the rule of six revisited. J Virol 72, 891–

899.

Kolakofsky, D., Roux, L., Garcin, D. & Ruigrok, R. W. (2005).Paramyxovirus mRNA editing, the ‘rule of six’ and error catastrophe:

a hypothesis. J Gen Virol 86, 1869–1877.

Kusov, Y. Y., Gosert, R. & Gauss-Muller, V. (2005). Replication and in

vivo repair of the hepatitis A virus genome lacking the poly(A) tail.

J Gen Virol 86, 1363–1368.

Lazzarini, R. A., Keene, J. D. & Schubert, M. (1981). The origins of

defective interfering particles of the negative-strand RNA viruses. Cell26, 145–154.

Liu, Y., Wimmer, E. & Paul, A. V. (2009). Cis-acting RNA elements in

human and animal plus-strand RNA viruses. Biochim Biophys Acta1789, 495–517.

Mackenzie, J. (2005). Wrapping things up about virus RNAreplication. Traffic 6, 967–977.

Martin, G. & Keller, W. (2007). RNA-specific ribonucleotidyl

transferases. RNA 13, 1834–1849.

McClure, M. A., Thibault, K. J., Hatalski, C. G. & Lipkin, W. I. (1992).Sequence similarity between Borna disease virus p40 and a duplicateddomain within the paramyxovirus and rhabdovirus polymerase

proteins. J Virol 66, 6572–6577.

Mellman, D. L., Gonzales, M. L., Song, C., Barlow, C. A., Wang, P.,Kendziorski, C. & Anderson, R. A. (2008). A PtdIns4,5P2-regulated



nuclear poly(A) polymerase controls expression of select mRNAs.Nature 451, 1013–1017.

Meyer, B. J. & Schmaljohn, C. (2000). Accumulation of terminallydeleted RNAs may play a role in Seoul virus persistence. J Virol 74,1321–1331.

Meyer, B. J. & Southern, P. J. (1994). Sequence heterogeneity in thetermini of lymphocytic choriomeningitis virus genomic and anti-genomic RNAs. J Virol 68, 7659–7664.

Meyer, B. J. & Southern, P. J. (1997). A novel type of defective viralgenome suggests a unique strategy to establish and maintainpersistent lymphocytic choriomeningitis virus infections. J Virol 71,6757–6764.

Miller, W. A., Bujarski, J. J., Dreher, T. W. & Hall, T. C. (1986). Minus-strand initiation by brome mosaic virus replicase within the 39 tRNA-like structure of native and modified RNA templates. J Mol Biol 187,537–546.

Minskaia, E., Hertzig, T., Gorbalenya, A. E., Campanacci, V.,Cambillau, C., Canard, B. & Ziebuhr, J. (2006). Discovery of anRNA virus 39A59 exoribonuclease that is critically involved incoronavirus RNA synthesis. Proc Natl Acad Sci U S A 103, 5108–5113.

Munishkin, A. V., Voronin, L. A. & Chetverin, A. B. (1988). An in vivorecombinant RNA capable of autocatalytic synthesis by Qb replicase.Nature 333, 473–475.

Nagy, P. D., Carpenter, C. D. & Simon, A. E. (1997). A novel 39-endrepair mechanism in an RNA virus. Proc Natl Acad Sci U S A 94,1113–1118.

Neufeld, K. L., Galarza, J. M., Richards, O. C., Summers, D. F. &Ehrenfeld, E. (1994). Identification of terminal adenylyl transferaseactivity of the poliovirus polymerase 3Dpol. J Virol 68, 5811–5818.

Nomaguchi, M., Ackermann, M., Yon, C., You, S. & Padmanabhan, R.(2003). De novo synthesis of negative-strand RNA by dengue virusRNA-dependent RNA polymerase in vitro: nucleotide, primer, andtemplate parameters. J Virol 77, 8831–8842.

Olsthoorn, R. C. & van Duin, J. (1996). Evolutionary reconstruction ofa hairpin deleted from the genome of an RNA virus. Proc Natl AcadSci U S A 93, 12256–12261.

Palasingam, K. & Shaklee, P. N. (1992). Reversion of Qb RNA phagemutants by homologous RNA recombination. J Virol 66, 2435–2442.

Poranen, M. M., Koivunen, M. R. & Bamford, D. H. (2008).Nontemplated terminal nucleotidyltransferase activity of double-stranded RNA bacteriophage w6 RNA-dependent RNA polymerase.J Virol 82, 9254–9264.

Raju, R., Subramaniam, S. V. & Hajjou, M. (1995). Genesis of Sindbisvirus by in vivo recombination of nonreplicative RNA precursors.J Virol 69, 7391–7401.

Raju, R., Hajjou, M., Hill, K. R., Botta, V. & Botta, S. (1999). In vivoaddition of poly(A) tail and AU-rich sequences to the 39 terminus ofthe Sindbis virus RNA genome: a novel 39-end repair pathway. J Virol73, 2410–2419.

Ranjith-Kumar, C. T., Gajewski, J., Gutshall, L., Maley, D., Sarisky,R. T. & Kao, C. C. (2001). Terminal nucleotidyl transferase activity ofrecombinant Flaviviridae RNA-dependent RNA polymerases:implication for viral RNA synthesis. J Virol 75, 8615–8623.

Rao, A. L., Dreher, T. W., Marsh, L. E. & Hall, T. C. (1989). Telomericfunction of the tRNA-like structure of brome mosaic virus RNA. ProcNatl Acad Sci U S A 86, 5335–5339.

Riechmann, J. L., Lain, S. & Garcia, J. A. (1990). Infectious in vitrotranscripts from a plum pox potyvirus cDNA clone. Virology 177,710–716.

Rohayem, J., Jager, K., Robel, I., Scheffler, U., Temme, A. & Rudolph, W.(2006). Characterization of norovirus 3Dpol RNA-dependent RNA

polymerase activity and initiation of RNA synthesis. J Gen Virol 87,

2621–2630.

Rosario, D., Perez, M. & de la Torre, J. C. (2005). Functional

characterization of the genomic promoter of Borna disease virus

(BDV): implications of 39-terminal sequence heterogeneity for BDV

persistence. J Virol 79, 6544–6550.

Sanchez, A., Trappier, S. G., Mahy, B. W., Peters, C. J. & Nichol, S. T.(1996). The virion glycoproteins of Ebola viruses are encoded in two

reading frames and are expressed through transcriptional editing. Proc

Natl Acad Sci U S A 93, 3602–3607.

Schneider, U., Schwemmle, M. & Staeheli, P. (2005). Genome

trimming: a unique strategy for replication control employed by

Borna disease virus. Proc Natl Acad Sci U S A 102, 3441–3446.

Silverman, R. H. (2007). Viral encounters with 29,59-oligoadenylate

synthetase and RNase L during the interferon antiviral response.

J Virol 81, 12720–12729.

Simon-Buela, L., Osaba, L., Garcia, J. A. & Lopez-Moya, J. J. (2000).Preservation of 59-end integrity of a potyvirus genomic RNA is not

dependent on template specificity. Virology 269, 377–382.

Smallwood, S. & Moyer, S. A. (1993). Promoter analysis of the

vesicular stomatitis virus RNA polymerase. Virology 192, 254–263.

Song, M. G. & Kiledjian, M. (2007). 39 Terminal oligo U-tract-

mediated stimulation of decapping. RNA 13, 2356–2365.

Spann, K. M., Collins, P. L. & Teng, M. N. (2003). Genetic

recombination during coinfection of two mutants of human

respiratory syncytial virus. J Virol 77, 11201–11211.

Sumper, M. & Luce, R. (1975). Evidence for de novo production of

self-replicating and environmentally adapted RNA structures by

bacteriophage Qb replicase. Proc Natl Acad Sci U S A 72, 162–166.

Sun, J. H. & Kao, C. C. (1997). Characterization of RNA products

associated with or aborted by a viral RNA-dependent RNA

polymerase. Virology 236, 348–353.

Tacahashi, Y. & Uyeda, I. (1999). Restoration of the 39 end of

potyvirus RNA derived from poly(A)-deficient infectious cDNA

clones. Virology 265, 147–152.

Taucher, C., Berger, A. & Mandl, C. W. (2010). A trans-complement-

ing recombination trap demonstrates a low propensity of flaviviruses

for intermolecular recombination. J Virol 84, 599–611.

Tayon, R., Jr, Kim, M. J. & Kao, C. C. (2001). Completion of RNA

synthesis by viral RNA replicases. Nucleic Acids Res 29, 3576–3582.

Teramoto, T., Kohno, Y., Mattoo, P., Markoff, L., Falgout, B. &Padmanabhan, R. (2008). Genome 39-end repair in dengue virus type

2. RNA 14, 2645–2656.

Thomas, S. M., Lamb, R. A. & Paterson, R. G. (1988). Two mRNAs

that differ by two nontemplated nucleotides encode the amino

coterminal proteins P and V of the paramyxovirus SV5. Cell 54, 891–

902.

Tomar, S., Hardy, R. W., Smith, J. L. & Kuhn, R. J. (2006). Catalytic

core of alphavirus nonstructural protein nsP4 possesses terminal

adenylyltransferase activity. J Virol 80, 9962–9969.

Trento, A., Galiano, M., Videla, C., Carballal, G., Garcia-Barreno, B.,Melero, J. A. & Palomo, C. (2003). Major changes in the G protein of

human respiratory syncytial virus isolates introduced by a duplication

of 60 nucleotides. J Gen Virol 84, 3115–3120.

Ugarov, V. I., Demidenko, A. A. & Chetverin, A. B. (2003). Qb

replicase discriminates between legitimate and illegitimate templates

by having different mechanisms of initiation. J Biol Chem 278, 44139–

44146.

Urbanowicz, A., Alejska, M., Formanowicz, P., Blazewicz, J.,Figlerowicz, M. & Bujarski, J. J. (2005). Homologous crossovers



among molecules of brome mosaic bromovirus RNA1 or RNA2

segments in vivo. J Virol 79, 5732–5742.

van den Born, E., Omelchenko, M. V., Bekkelund, A., Leihne, V.,Koonin, E. V., Dolja, V. V. & Falnes, P. O. (2008). Viral AlkB proteins

repair RNA damage by oxidative demethylation. Nucleic Acids Res 36,

5451–5461.

van Dijk, A. A., Makeyev, E. V. & Bamford, D. H. (2004). Initiation of

viral RNA-dependent RNA polymerization. J Gen Virol 85, 1077–

1093.

van Leeuwen, H. C., Liefhebber, J. M. & Spaan, W. J. (2006). Repair

and polyadenylation of a naturally occurring hepatitis C virus 39

nontranslated region – shorter variant in selectable replicon cell lines.

J Virol 80, 4336–4343.

van Ooij, M. J., Polacek, C., Glaudemans, D. H., Kuijpers, J., vanKuppeveld, F. J., Andino, R., Agol, V. I. & Melchers, W. J. (2006).Polyadenylation of genomic RNA and initiation of antigenomic RNA

in a positive-strand RNA virus are controlled by the same cis-element.

Nucleic Acids Res 34, 2953–2965.

Vidal, S., Curran, J. & Kolakofsky, D. (1990). Editing of the Sendaivirus P/C mRNA by G insertion occurs during mRNA synthesis via avirus-encoded activity. J Virol 64, 239–246.

Vignuzzi, M., Stone, J. K., Arnold, J. J., Cameron, C. E. & Andino, R.(2006). Quasispecies diversity determines pathogenesis throughcooperative interactions in a viral population. Nature 439, 344–348.

Vulliemoz, D. & Roux, L. (2001). ‘Rule of six’: how does the Sendaivirus RNA polymerase keep count? J Virol 75, 4506–4518.

Walter, C. T. & Barr, J. N. (2010). Bunyamwera virus can repairboth insertions and deletions during RNA replication. RNA(in press). doi:10.1261/rna.1962010

Wickens, M. & Kwak, J. E. (2008). Molecular biology. A tail tale for U.Science 319, 1344–1345.

Wittmann, T. J., Biek, R., Hassanin, A., Rouquet, P., Reed, P., Yaba, P.,Pourrut, X., Real, L. A., Gonzalez, J. P. & Leroy, E. M. (2007). Isolatesof Zaire ebolavirus from wild apes reveal genetic lineage andrecombinants. Proc Natl Acad Sci U S A 104, 17123–17127.



How RNA viruses maintain their genome integrity

Documents

Transcript of How RNA viruses maintain their genome integrity