Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing
Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.
Vyšlo v časopise:
Infidelity of SARS-CoV Nsp14-Exonuclease Mutant Virus Replication Is Revealed by Complete Genome Sequencing. PLoS Pathog 6(5): e32767. doi:10.1371/journal.ppat.1000896
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.ppat.1000896
Souhrn
Most RNA viruses lack the mechanisms to recognize and correct mutations that arise during genome replication, resulting in quasispecies diversity that is required for pathogenesis and adaptation. However, it is not known how viruses encoding large viral RNA genomes such as the Coronaviridae (26 to 32 kb) balance the requirements for genome stability and quasispecies diversity. Further, the limits of replication infidelity during replication of large RNA genomes and how decreased fidelity impacts virus fitness over time are not known. Our previous work demonstrated that genetic inactivation of the coronavirus exoribonuclease (ExoN) in nonstructural protein 14 (nsp14) of murine hepatitis virus results in a 15-fold decrease in replication fidelity. However, it is not known whether nsp14-ExoN is required for replication fidelity of all coronaviruses, nor the impact of decreased fidelity on genome diversity and fitness during replication and passage. We report here the engineering and recovery of nsp14-ExoN mutant viruses of severe acute respiratory syndrome coronavirus (SARS-CoV) that have stable growth defects and demonstrate a 21-fold increase in mutation frequency during replication in culture. Analysis of complete genome sequences from SARS-ExoN mutant viral clones revealed unique mutation sets in every genome examined from the same round of replication and a total of 100 unique mutations across the genome. Using novel bioinformatic tools and deep sequencing across the full-length genome following 10 population passages in vitro, we demonstrate retention of ExoN mutations and continued increased diversity and mutational load compared to wild-type SARS-CoV. The results define a novel genetic and bioinformatics model for introduction and identification of multi-allelic mutations in replication competent viruses that will be powerful tools for testing the effects of decreased fidelity and increased quasispecies diversity on viral replication, pathogenesis, and evolution.
Zdroje
1. DrakeJW
HollandJJ
1999 Mutation rates among RNA viruses. Proc Natl Acad Sci U S A 96 13910 13913
2. BullJJ
MeyersLA
LachmannM
2005 Quasispecies made simple. PLoS Comput Biol 1 e61 doi:10.1371/journal.pcbi.0010061
3. DomingoE
SaboD
TaniguchiT
WeissmannC
1978 Nucleotide sequence heterogeneity of an RNA phage population. Cell 13 735 744
4. DopazoJ
SobrinoF
PalmaEL
DomingoE
MoyaA
1988 Gene encoding capsid protein VP1 of foot-and-mouth disease virus: a quasispecies model of molecular evolution. Proc Natl Acad Sci U S A 85 6811 6815
5. PfeifferJK
KirkegaardK
2005 Increased fidelity reduces poliovirus fitness and virulence under selective pressure in mice. PLoS Pathog 1 e11 doi:10.1371/journal.ppat.0010011
6. VignuzziM
StoneJK
ArnoldJJ
CameronCE
AndinoR
2006 Quasispecies diversity determines pathogenesis through cooperative interactions in a viral population. Nature 439 344 348
7. VignuzziM
WendtE
AndinoR
2008 Engineering attenuated virus vaccines by controlling replication fidelity. Nat Med 14 154 161
8. CrottyS
MaagD
ArnoldJJ
ZhongW
LauJY
2000 The broad-spectrum antiviral ribonucleoside ribavirin is an RNA virus mutagen. Nat Med 6 1375 1379
9. CrottyS
CameronCE
AndinoR
2001 RNA virus error catastrophe: direct molecular test by using ribavirin. Proc Natl Acad Sci U S A 98 6895 6900
10. BullJJ
SanjuanR
WilkeCO
2007 Theory of lethal mutagenesis for viruses. J Virol 81 2930 2939
11. GorbalenyaAE
EnjuanesL
ZiebuhrJ
SnijderEJ
2006 Nidovirales: evolving the largest RNA virus genome. Virus Res 117 17 37
12. BhardwajK
GuarinoL
KaoCC
2004 The severe acute respiratory syndrome coronavirus Nsp15 protein is an endoribonuclease that prefers manganese as a cofactor. J Virol 78 12218 12224
13. BrockwaySM
ClayCT
LuXT
DenisonMR
2003 Characterization of the expression, intracellular localization, and replication complex association of the putative mouse hepatitis virus RNA-dependent RNA polymerase. J Virol 77 10515 10527
14. ChenY
CaiH
PanJ
XiangN
TienP
2009 Functional screen reveals SARS coronavirus nonstructural protein nsp14 as a novel cap N7 methyltransferase. Proc Natl Acad Sci U S A 106 3484 3489
15. ImbertI
GuillemotJC
BourhisJM
BussettaC
CoutardB
2006 A second, non-canonical RNA-dependent RNA polymerase in SARS coronavirus. Embo J 25 4933 4942
16. MinskaiaE
HertzigT
GorbalenyaAE
CampanacciV
CambillauC
2006 Discovery of an RNA virus 3'->5' exoribonuclease that is critically involved in coronavirus RNA synthesis. Proc Natl Acad Sci U S A 103 5108 5113
17. SnijderEJ
BredenbeekPJ
DobbeJC
ThielV
ZiebuhrJ
2003 Unique and conserved features of genome and proteome of SARS-coronavirus, an early split-off from the coronavirus group 2 lineage. J Mol Biol 331 991 1004
18. MoserMJ
HolleyWR
ChatterjeeA
MianIS
1997 The proofreading domain of Escherichia coli DNA polymerase I and other DNA and/or RNA exonuclease domains. Nucleic Acids Res 25 5110 5118
19. ChenP
JiangM
HuT
LiuQ
ChenXS
2007 Biochemical characterization of exoribonuclease encoded by SARS coronavirus. J Biochem Mol Biol 40 649 655
20. EckerleLD
LuX
SperrySM
ChoiL
DenisonMR
2007 High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J Virol 81 12135 12144
21. GrahamRL
SimsAC
BrockwaySM
BaricRS
DenisonMR
2005 The nsp2 replicase proteins of murine hepatitis virus and severe acute respiratory syndrome coronavirus are dispensable for viral replication. J Virol 79 13399 13411
22. SimsAC
YountB
BurkettSE
BaricRS
PicklesRJ
2006 SARS CoV replication and pathogenesis in human airway epithelial cultures. Adv Exp Med Biol 581 535 538
23. YountB
CurtisKM
FritzEA
HensleyLE
JahrlingPB
2003 Reverse genetics with a full-length infectious cDNA of severe acute respiratory syndrome coronavirus. Proc Natl Acad Sci U S A 100 12995 13000
24. LiK
BrownleyA
StockwellTB
BeesonK
McIntoshTC
2008 Novel computational methods for increasing PCR primer design effectiveness in directed sequencing. BMC Bioinformatics 9 191
25. BentleyDR
BalasubramanianS
SwerdlowHP
SmithGP
MiltonJ
2008 Accurate whole human genome sequencing using reversible terminator chemistry. Nature 456 53 59
26. WeeseD
EmdeAK
RauschT
DoringA
ReinertK
2009 RazerS–fast read mapping with sensitivity control. Genome Res 19 1646 1654
27. SillitoeI
OrengoCA
2003 Protein structure comparison.
OrengoCA
JonesDT
ThorntonJM
Bioinformatics: genes, proteins and computers. 1st ed Cromwell, UK Cromwell Press 81 101
28. HollandJJ
de la TorreJC
ClarkeDK
DuarteE
1991 Quantitation of relative fitness and great adaptability of clonal populations of RNA viruses. J Virol 65 2960 2967
29. NovellaIS
BallLA
WertzGW
2004 Fitness analyses of vesicular stomatitis strains with rearranged genomes reveal replicative disadvantages. J Virol 78 9837 9841
30. HarismendyO
FrazerK
2009 Method for improving sequence coverage uniformity of targeted genomic intervals amplified by LR-PCR using Illumina GA sequencing-by-synthesis technology. Biotechniques 46 229 231
31. QiJ
ZhaoF
BuboltzA
SchusterSC
2009 inGAP: an integrated next-generation genome analysis pipeline. Bioinformatics
32. KnoopsK
KikkertM
WormSH
Zevenhoven-DobbeJC
van der MeerY
2008 SARS-coronavirus replication is supported by a reticulovesicular network of modified endoplasmic reticulum. PLoS Biol 6 e226 doi:10.1371/journal.pbio.0060226
33. von BrunnA
TeepeC
SimpsonJC
PepperkokR
FriedelCC
2007 Analysis of intraviral protein-protein interactions of the SARS coronavirus ORFeome. PLoS ONE 2 e459 doi:10.1371/journal.pone.0000459
34. van HemertMJ
van den WormSH
KnoopsK
MommaasAM
GorbalenyaAE
2008 SARS-coronavirus replication/transcription complexes are membrane-protected and need a host factor for activity in vitro. PLoS Pathog 4 e1000054 doi:10.1371/journal.ppat.1000054
35. ScottJ
Thompson-MayberryP
LahmamsiS
KingCJ
McShanWM
2008 Phage-associated mutator phenotype in group A streptococcus. J Bacteriol 190 6290 6301
36. RobertsA
DemingD
PaddockCD
ChengA
YountB
2007 A mouse-adapted SARS-coronavirus causes disease and mortality in BALB/c mice. PLoS Pathog 3 e5 doi:10.1371/journal.ppat.0030005
37. AriasA
LazaroE
EscarmisC
DomingoE
2001 Molecular intermediates of fitness gain of an RNA virus: characterization of a mutant spectrum by biological and molecular cloning. J Gen Virol 82 1049 1060
38. JerzakGV
BernardK
KramerLD
ShiPY
EbelGD
2007 The West Nile virus mutant spectrum is host-dependant and a determinant of mortality in mice. Virology 360 469 476
39. VabretA
DinaJ
MourezT
GouarinS
PetitjeanJ
2006 Inter- and intra-variant genetic heterogeneity of human coronavirus OC43 strains in France. J Gen Virol 87 3349 3353
40. TsibrisAM
KorberB
ArnaoutR
RussC
LoCC
2009 Quantitative deep sequencing reveals dynamic HIV-1 escape and large population shifts during CCR5 antagonist therapy in vivo. PLoS ONE 4 e5683 doi:10.1371/journal.pone.0005683
41. von WagnerM
LeeJH
RusterB
KronenbergerB
SarrazinC
2003 Dynamics of hepatitis C virus quasispecies turnover during interferon-alpha treatment. J Viral Hepat 10 413 422
42. WangC
MitsuyaY
GharizadehB
RonaghiM
ShaferRW
2007 Characterization of mutation spectra with ultra-deep pyrosequencing: application to HIV-1 drug resistance. Genome Res 17 1195 1201
43. AlmazanF
DediegoML
GalanC
EscorsD
AlvarezE
2006 Construction of a severe acute respiratory syndrome coronavirus infectious cDNA clone and a replicon to study coronavirus RNA synthesis. J Virol 80 10900 10906
44. ChungDH
SunY
ParkerWB
ArterburnJB
BartolucciA
2007 Ribavirin reveals a lethal threshold of allowable mutation frequency for Hantaan virus. J Virol 81 11722 11729
45. VoNV
YoungKC
LaiMM
2003 Mutagenic and inhibitory effects of ribavirin on hepatitis C virus RNA polymerase. Biochemistry 42 10462 10471
46. ArnoldJJ
VignuzziM
StoneJK
AndinoR
CameronCE
2005 Remote site control of an active site fidelity checkpoint in a viral RNA-dependent RNA polymerase. J Biol Chem 280 25706 25716
47. PfeifferJK
KirkegaardK
2003 A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity. Proc Natl Acad Sci U S A 100 7289 7294
48. VignuzziM
StoneJK
AndinoR
2005 Ribavirin and lethal mutagenesis of poliovirus: molecular mechanisms, resistance and biological implications. Virus Res 107 173 181
49. ChineseSMEC
2004 Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China. Science 303 1666 1669
50. YehSH
WangHY
TsaiCY
KaoCL
YangJY
2004 Characterization of severe acute respiratory syndrome coronavirus genomes in Taiwan: molecular epidemiology and genome evolution. Proc Natl Acad Sci U S A 101 2542 2547
51. HollandJJ
DomingoE
de la TorreJC
SteinhauerDA
1990 Mutation frequencies at defined single codon sites in vesicular stomatitis virus and poliovirus can be increased only slightly by chemical mutagenesis. J Virol 64 3960 3962
52. PeralesC
AgudoR
TejeroH
ManrubiaSC
DomingoE
2009 Potential benefits of sequential inhibitor-mutagen treatments of RNA virus infections. PLoS Pathog 5 e1000658
53. BeckerMM
GrahamRL
DonaldsonEF
RockxB
SimsAC
2008 Synthetic recombinant bat SARS-like coronavirus is infectious in cultured cells and in mice. Proc Natl Acad Sci U S A 105 19944 19949
54. CuevasJM
DuffyS
SanjuanR
2009 Point mutation rate of bacteriophage PhiX174. Genetics 183 747 749
55. DrakeJW
1991 A constant rate of spontaneous mutation in DNA-based microbes. Proc Natl Acad Sci U S A 88 7160 7164
56. PoonLL
LeungCS
ChanKH
YuenKY
GuanY
2005 Recurrent mutations associated with isolation and passage of SARS coronavirus in cells from non-human primates. J Med Virol 76 435 440
57. YountB
RobertsRS
SimsAC
DemingD
FriemanMB
2005 Severe acute respiratory syndrome coronavirus group-specific open reading frames encode nonessential functions for replication in cell cultures and mice. J Virol 79 14909 14922
58. SawickiSG
SawickiDL
1995 Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands. Adv Exp Med Biol 380 499 506
59. SawickiSG
SawickiDL
2005 Coronavirus transcription: a perspective. Curr Top Microbiol Immunol 287 31 55
60. YountB
RobertsRS
LindesmithL
BaricRS
2006 Rewiring the severe acute respiratory syndrome coronavirus (SARS-CoV) transcription circuit: engineering a recombination-resistant genome. Proc Natl Acad Sci U S A 103 12546 12551
61. ZunigaS
SolaI
AlonsoS
EnjuanesL
2004 Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J Virol 78 980 994
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Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
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