Recombination in Enteroviruses is a Biphasic Replicative
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Recombination in Enteroviruses is a Biphasic Replicative Process Involving the Generation of Greater-than Genome Length 'Imprecise' Intermediates Andrew Woodman, University of Warwick, BBSRC funded studentship A.Woodman@warwick.ac.uk Acknowledgements • Supervisor – Professor David. J. Evans www.evanslab.org.uk – – • Kym Lowry (previous PhD student) Fadi Alnaji BBSRC funded studentship Human enteroviruses (HEV) • Picornavirus • SS +sense RNA genome • One ORF, protein processing • Modular genome • 4 distinct phylogenetic groups (A-D) • Polio = type species (group C) Recombinants and recombination • A significant evolutionary mechanism – ‘Copy choice’ replicative model (Kirkegaard & Baltimore,1986) Image adapted from Flint et al., 2004 Recombination between Polioviruses and Co-Circulating Coxsackie A Viruses: Role in the Emergence of Pathogenic Vaccine-Derived Polioviruses Sophie Jegouic1, Marie-Line Joffret1, Claire Blanchard1, Franck B. Riquet1, Céline Perret1, Isabelle Pelletier1, Florence Colbere-Garapin1, Mala Rakoto-Andrianarivelo2, Francis Delpeyroux1* 1 Institut Pasteur, Unité de Biologie des Virus Entériques, Paris, France, 2 Institut Pasteur de Madagascar, Unité de Virologie Médicale, Antananarivo, Madagascar Abstract Ten outbreaks of poliomyelitis caused by pathogenic circulating vaccine-derived polioviruses (cVDPVs) have recently been reported in different regions of the world. Two of these outbreaks occurred in Madagascar. Most cVDPVs were recombinants of mutated poliovaccine strains and other unidentified enteroviruses of species C. We previously reported that a type 2 cVDPV isolated during an outbreak in Madagascar was co-circulating with coxsackieviruses A17 (CA17) and that sequences in the 39 half of the cVDPV and CA17 genomes were related. The goal of this study was to investigate whether these CA17 isolates can act as recombination partners of poliovirus and subsequently to evaluate the major effects of recombination events on the phenotype of the recombinants. We first cloned the infectious cDNA of a Madagascar CA17 isolate. We then generated recombinant constructs combining the genetic material of this CA17 isolate with that of the type Recombination system PV3 VP2 VP3 VP1 2A 2B 2C CR 3C 3D 3A 3C 3D luc 3A 3C 3D VP1 3A 3C 3D VP4 3A 3’ N 5’ N CR CR E A VPg PV1 / PV3 luc VP2 VP3 2A 2B 2C Crossover region Co-transfect RNA to permissive cells Serial passage B • System allows comparisonClone of byviable intra and intertypic recombination Growth limit dilution analysis PCR and sequence analysis Further analysis VP2 VP1 Crossover Crossover region region 3A 3C 3D Recombination in practice Crossover region Co-transfect Co-transfect RNA RNA to to permissive permissive cells cells Clone Clone by by Co-transfectlimit RNA dilution limit dilution to permissive cells Growth Growth analysis analysis Clone by limit dilution Growth PCR and analysis sequence analysis Further analysis PCR and sequence analysis C D Serial passage B Serial Serial passage passage B VP3 Further analysis * 1000 D • round of replication Plaque directly onto HeLa cells/RD cells pfu/ml + + 100 * 1000 + 10 pT7/SL3 + + 100 GuHCl pT7Rep3-L + + + + PV3/PV3 PV1/PV3 10 pT7/SL3 + + + GuHCl + PV3/PV3 PV1/PV3 • Co-transfect rodent cells (L929) which lack the poliovirus receptor – single pT7Rep3-L + pfu/ml pfu/ml C R N C VP2 VP3 VP1 2A 2B 2C 3A 3C 3D 3A 3C 3D luc 3A 3C 3D VP1 3A 3C 3D Recombinant characterisation VP4 VPg luc • RT-PCR of recombinant virus isolated from plaques or from biological cloning 3’ 5’ N C R C R E A VP2 VP3 2A 2B 2C Crossover region Co-transfect RNA to permissive cells Clone by limit dilution Intertypic recombinant (PV3/1) isolates primarily ‘imprecise’ with additional sequence at the cross-over Growth analysis PCR and Further analysis sequence Recombinant isolates N Pos analysis C D * 1000 pT7Rep3-L pT7/SL3 GuHCl + + + + + + + pfu/ml • Serial passage B 100 10 PV3/PV3 PV1/PV3 Intertypic recombinant junctions • 150+ PV3/1 recombinant isolates sequenced to date • Form two clusters – All in frame, insert size from 3 nt to 321 nt – Span VP1/2A and 2A/2B junctions – Sequence and structure independent Resolved recombinants – all precise • Four primary recombinants serially passaged in HeLa cells • Additional sequence lost • Intermediate sized products • Resolved recombinants all wild type in genome length M C Neg p2 p5 p7 Replicative or Non-replicative? • ‘Copy-choice’ mechanism proposes that template switching occurs during anti-sense RNA synthesis and is therefore replicative Nonreplicative homologous RNA recombination: Promiscuous joining of RNA pieces? ANATOLY P. GMYL,1 SERGEY A. KORSHENKO,1 EVGENY V. BELOUSOV,1 ELENA V. KHITRINA,1 and VADIM I. AGOL1,2 1 M.P. Chumakov Institute of Poliomyelitis & Viral Encephalitides, Russian Academy of Medical Sciences, Moscow 142782, Russia M.V. Lomonosov Moscow State University, Moscow 119899, Russia 2 ABSTRACT • Biologically important joining of RNA pieces in cells, as exemplified by splicing and some classes of RNA editing, is posttranscriptional, whereas in RNA viruses it is generally believed to occur during viral RNA polymerase-dependent RNA synthesis. Here, we demonstrate the assembly of precise genome of an RNA virus (poliovirus) from its cotransfected fragments, which does not require specific RNA sequences, takes place before generation of the viral RNA polymerase, and occurs in different ways: Apparently unrestricted ligation of the terminal nucleotides, joining of any one of the two entire fragments with the relevant internal nucleotide of its partner, or internal crossovers within the overlapping sequence. Incorporation of the entire 5! or 3! partners into the recombinant RNA is activated by the presence of terminal 3!-phosphate and 5!-OH, respectively. Such postreplicative reactions, fundamentally differing from the known site-specific and structurally demanding cellular RNA rearrangements, might contribute to the origin and evolution of RNA viruses and could generate new RNA species during all stages of biological evolution. Manipulation of replicase (3Dpol) fidelity – Nucleoside mutagens that reduce fidelity Keywords: Evolution; poliovirus; RNA ligation; RNA recombination – Single site mutation to increase fidelity INTRODUCTION A variety of important biological processes involve covalent rearrangements of RNA molecules, such as joining of noncontiguous segments of the same RNA molecule or of segments of different RNA species. Two fundamentally distinct mechanisms, nonreplicative (posttranscriptional) and replicative/transcriptional, are used to accomplish this goal. The former operates with cellular RNAs and is exemplified by various types of splicing (Gonzalez et al. 1999; Reed 2000; Doudna and Cech 2002; Singh 2002) and insertion/ deletion RNA editing (Simpson et al. 2003). Common features of these processes are their site specificity and strict diverse processes as recombination (Lai 1992; Nagy and Simon 1997; Worobey and Holmes 1999), discontinuous transcription in nidoviruses, for example, coronaviruses (Lai and Holmes 2001), and acquisition of capped 5"-segments of host mRNA by transcripts of several families of viruses with negative-strand RNA genomes, for example, influenza virus (Lamb and Krug 2001). All of these processes, distinctive from the rearrangements of cellular RNAs, are believed to occur during the viral RNA polymerase-dependent RNA synthesis. Another distinction of viral RNA rearrangements, at least as far as RNA recombination in many viruses is concerned, is much more relaxed structural requirements. Intertypic recombination + Ribavirin !!!!!!!!Negative!!!!!!!!!!!!!!!!!!untreated!!!!!!!!!!!!!!50μM!!!!!!!!!!!!!! !!!!!!!!!!!!!!!!!!100μM!!!!!!!!!!!!!!!!!!!200μM!!!!!!!!!!!!!!!!400μM! " 400! 350! 300! 250! 200! 150! 100! 50! 0! ** * untreated! 50μM! Recombination! PV3!replication! 100μM! 200μM! 400μM! Ribavirin&concentration& ! Error bars indicate standard deviation.** P<0.01, * P<0.05. One tailed T-Test with treated samples compared to untreated • Similar phenomenon with Intratypic recombination experiments pRLucWT"/"SL3"recombination" in"the"presence"of"ribavirin" "yield"" treated)" " " " Virus&yield&& (%&of&untreated)& PV3&/&PV1&recombination&in&the& presence&of&ribavirin& 400! 300! !!!**! !*! Replicase fidelity and recombination • Glycine to Serine change at position 64 of the 3Dpol region increases replicase fidelity – • (Pfeiffer & Kirkegaard, 2003; Vignuzzi et al., 2006) Change in sequence has little or no effect upon replication pRLucWT - v - pRLuc-G64S PV3/1 recombination – Fidelity mutant G64S 1.00E+04 1.00E+03 G64S 1.00E+02 pRlucWT 1.00E+01 1.00E+00 0 2.5 5 Time (Hrs) 7.5 Virus yield (% of WT) Luciferase activity (RLU) 1.00E+05 160 140 120 100 80 60 40 20 0 ** pRLucWT / SL3 pRLuc-G64S / SL3-G64S RNA partners Error bars indicate standard deviation.** P<0.01 Non-replicative recombination VP3 VP1 2A 2B R 2C 3C 3D 3C 3C 3D 3D 3A 3C 3D luc 3A 3C 3D VP1 luc 3A 3A 3C 3C 3D 3D 3A 3C 3D VPg C VP2 VP3 luc VP1 2A 2B 2A 2B 2C 2C VP4 VPg luc VP2 • VP3 2A 2B 2C Crossover region Truncated partners VP2 VP3 VP1 Crossover ‘Zone’Bof recombination remains the same as replicative system region to permissive cells Co-transfect RNA B Clone by limit dilution Co-transfect RNA to permissive cells Clone by limit dilution Growth analysis PCR and Growth sequence analysis analysis Serial passag Serial passage • 3A 3A 3’ N 5’ N C R C R VP4 3A R VP2 E A 3’ N C 5’ N C R C R E A Further analysis Virus&yield&& (%&of&untreated)& 180! 160! 140! 120! 100! 80! 60! 40! 20! 0! Replicative -v- Non replicative PV3/3! PV3/3!+!Ribavirin! RNA&partners& • Non-replicative 2 logs lower thanPV3/3&Truncated&partners&(G64S)& replicative counterpart PV3/3&recombination& Replicative&and&nonBreplicative& Virus&yield&& (%&of&replicative)& 140! Virus&yield& &(%&of&wild&type)& ! ! 120! 100! • Ribavirin and G64S mutation 80! have60!little or no effect upon 40! non-replicative recombination 20! 120! 100! 80! 60! 40! 20! 0! pT7rep3L!/!SL3! 0! Truncated!PV3/3! Truncated!Partners! RNA&partners& ! Truncated!PV3/3!(G64S)! RNA&partners& ! ! PV3/3&Truncated&partners&(G64S)& 140! Virus&yield& &(%&of&wild&type)& Virus&yield&& (%&of&untreated)& Truncated&PV3/3& +/B&100uM&Ribavirin& 180! 160! 140! 120! 100! 80! 60! 40! 20! 0! 120! 100! 80! 60! 40! 20! PV3/3! 0! PV3/3!+!Ribavirin! Truncated!PV3/3! RNA&partners& ! ! ! ! Truncated&PV3/3& +/B&100uM&Ribavirin& 120! 80! ield&& eated)& eld&& cative)& PV3/3&recombination& Replicative&and&nonBreplicative& 100! Truncated!PV3/3!(G64S)! RNA&partners& 180! 160! 140! 120! 3393 +27 : CTTTGGGCATCAGAAgaccacatatggctt : 3367 3390 +78 : GGGCTTTGGGCATCAtggaccaggggtgga : 3313 Evolution through duplication? R C N VP2 VP3 3A VP1 3C 3’ 5’ N C R C R E B 3D VP1 2A 2B 2C PV3 luc 2A 2B 2C PV1 R E Cluster 2 C Cluster 1 C 2A 2B 2C PV3 luc 2A 2B 2C PV1 C R E VP1 Avihepatovirus (Duck Hepatitis A) Aphthovirus (FMDV) Summary • We propose that recombination is a biphasic replicative process • Initial sequence independent cross-over is followed by secondary resolution events where all additional sequence is lost • Additionally, a drug that inhibits RNA co-localisation (Nocodazole) also inhibits recombination frequency • Imprecise recombination may account for duplications seen in other picornavirus genomes
