Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs

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The gene expression of plus-strand RNA viruses with a polycistronic genome depends on translation and replication of the genomic mRNA, as well as synthesis of subgenomic (sg) mRNAs. Arteriviruses and coronaviruses, distantly related members of the nidovirus order, employ a unique mechanism of discontinuous minus-strand RNA synthesis to generate subgenome-length templates for the synthesis of a nested set of sg mRNAs. Non-structural protein 1 (nsp1) of the arterivirus equine arteritis virus (EAV), a multifunctional regulator of viral RNA synthesis and virion biogenesis, was previously implicated in controlling the balance between genome replication and sg mRNA synthesis. Here, we employed reverse and forward genetics to gain insight into the multiple regulatory roles of nsp1. Our analysis revealed that the relative abundance of viral mRNAs is tightly controlled by an intricate network of interactions involving all nsp1 subdomains. Distinct nsp1 mutations affected the quantitative balance among viral mRNA species, and our data implicate nsp1 in controlling the accumulation of full-length and subgenome-length minus-strand templates for viral mRNA synthesis. The moderate differential changes in viral mRNA abundance of nsp1 mutants resulted in similarly altered viral protein levels, but progeny virus yields were greatly reduced. Pseudorevertant analysis provided compelling genetic evidence that balanced EAV mRNA accumulation is critical for efficient virus production. This first report on protein-mediated, mRNA-specific control of nidovirus RNA synthesis reveals the existence of an integral control mechanism to fine-tune replication, sg mRNA synthesis, and virus production, and establishes a major role for nsp1 in coordinating the arterivirus replicative cycle.

Vyšlo v časopise: Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs. PLoS Pathog 6(2): e32767. doi:10.1371/journal.ppat.1000772
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1000772

Souhrn

Zdroje

1. MillerWA

KoevG

2000 Synthesis of subgenomic RNAs by positive-strand RNA viruses. Virology 273 1 8

2. PerlmanS

NetlandJ

2009 Coronaviruses post-SARS: update on replication and pathogenesis. Nat Rev Microbiol 7 439 450

3. GorbalenyaAE

EnjuanesL

ZiebuhrJ

SnijderEJ

2006 Nidovirales: evolving the largest RNA virus genome. Virus Res 117 17 37

4. MolenkampR

van TolH

RozierBCD

van der MeerY

SpaanWJM

SnijderEJ

2000 The arterivirus replicase is the only viral protein required for genome replication and subgenomic mRNA transcription. J Gen Virol 81 2491 2496

5. SawickiSG

SawickiDL

1995 Coronaviruses use discontinuous extension for synthesis of subgenome-length negative strands. Adv Exp Med Biol 380 499 506

6. PasternakAO

van den BornE

SpaanWJM

SnijderEJ

2001 Sequence requirements for RNA strand transfer during nidovirus discontinuous subgenomic RNA synthesis. EMBO J 20 7220 7228

7. van MarleG

DobbeJC

GultyaevAP

LuytjesW

SpaanWJM

SnijderEJ

1999 Arterivirus discontinuous mRNA transcription is guided by base pairing between sense and antisense transcription-regulating sequences. Proc Natl Acad Sci U S A 96 12056 12061

8. ZunigaS

SolaI

AlonsoS

EnjuanesL

2004 Sequence motifs involved in the regulation of discontinuous coronavirus subgenomic RNA synthesis. J Virol 78 980 994

9. PasternakAO

SpaanWJ

SnijderEJ

2006 Nidovirus transcription: how to make sense…? J Gen Virol 87 1403 1421

10. SawickiSG

SawickiDL

SiddellSG

2007 A contemporary view of coronavirus transcription. J Virol 81 20 29

11. van den BornE

PosthumaCC

GultyaevAP

SnijderEJ

2005 Discontinuous subgenomic RNA synthesis in arteriviruses is guided by an RNA hairpin structure located in the genomic leader region. J Virol 79 6312 6324

12. den BoonJA

SpaanWJM

SnijderEJ

1995 Equine arteritis virus subgenomic RNA transcription: UV inactivation and translation inhibition studies. Virology 213 364 372

13. TijmsMA

van DintenLC

GorbalenyaAE

SnijderEJ

2001 A zinc finger-containing papain-like protease couples subgenomic mRNA synthesis to genome translation in a positive-stranded RNA virus. Proc Natl Acad Sci U S A 98 1889 1894

14. TijmsMA

NedialkovaDD

Zevenhoven-DobbeJC

GorbalenyaAE

SnijderEJ

2007 Arterivirus subgenomic mRNA synthesis and virion biogenesis depend on the multifunctional nsp1 autoprotease. J Virol 81 10496 10505

15. den BoonJA

FaabergKS

MeulenbergJJM

WassenaarALM

PlagemannPGW

GorbalenyaAE

SnijderEJ

1995 Processing and evolution of the N-terminal region of the arterivirus replicase ORF1a protein - identification of two papainlike cysteine proteases. J Virol 69 4500 4505

16. SunY

XueF

GuoY

MaM

HaoN

ZhangXC

LouZ

LiX

RaoZ

2009 Crystal structure of porcine reproductive and respiratory syndrome virus (PRRSV) leader protease nsp1α. J Virol 83 10931 40

17. PosthumaCC

PedersenKW

LuZ

JoostenRG

RoosN

Zevenhoven-DobbeJC

SnijderEJ

2008 Formation of the arterivirus replication/transcription complex: a key role for nonstructural protein 3 in the remodeling of intracellular membranes. J Virol 82 4480 4491

18. van AkenD

Zevenhoven-DobbeJ

GorbalenyaAE

SnijderEJ

2006 Proteolytic maturation of replicase polyprotein pp1a by the nsp4 main proteinase is essential for equine arteritis virus replication and includes internal cleavage of nsp7. J Gen Virol 87 3473 3482

19. van DintenLC

RensenS

GorbalenyaAE

SnijderEJ

1999 Proteolytic processing of the open reading frame 1b-encoded part of arterivirus replicase is mediated by nsp4 serine protease and is essential for virus replication. J Virol 73 2027 2037

20. CunninghamBC

WellsJA

1989 High-resolution epitope mapping of hGH-receptor interactions by alanine-scanning mutagenesis. Science 244 1081 1085

21. WertmanKF

DrubinDG

BotsteinD

1992 Systematic mutational analysis of the yeast ACT1 gene. Genetics 132 337 350

22. BordoD

ArgosP

1991 Suggestions for “safe” residue substitutions in site-directed mutagenesis. J Mol Biol 217 721 729

23. BaricRS

YountB

2000 Subgenomic negative-strand RNA function during mouse hepatitis virus infection. J Virol 74 4039 4046

24. SawickiD

WangT

SawickiS

2001 The RNA structures engaged in replication and transcription of the A59 strain of mouse hepatitis virus. J Gen Virol 82 385 396

25. van MarleG

van DintenLC

SpaanWJM

LuytjesW

SnijderEJ

1999 Characterization of an equine arteritis virus replicase mutant defective in subgenomic mRNA synthesis. J Virol 73 5274 5281

26. WieringaR

de VriesAA

van derMJ

GodekeGJ

OnderwaterJJ

vanTH

KoertenHK

MommaasAM

SnijderEJ

RottierPJ

2004 Structural protein requirements in equine arteritis virus assembly. J Virol 78 13019 13027

27. SnijderEJ

DobbeJC

SpaanWJM

2003 Heterodimerization of the two major envelope proteins is essential for arterivirus infectivity. J Virol 77 97 104

28. de VriesAA

PostSM

RaamsmanMJ

HorzinekMC

RottierPJ

1995 The two major envelope proteins of equine arteritis virus associate into disulfide-linked heterodimers. J Virol 69 4668 4674

29. WieringaR

de VriesAA

RottierPJ

2003 Formation of disulfide-linked complexes between the three minor envelope glycoproteins (GP2b, GP3, and GP4) of equine arteritis virus. J Virol 77 6216 6226

30. SnijderEJ

WassenaarALM

SpaanWJM

1992 The 5′end of the equine arteritis virus replicase gene encodes a papain-like cysteine protease. J Virol 66 7040 7048

31. JonesCT

MurrayCL

EastmanDK

TasselloJ

RiceCM

2007 Hepatitis C virus p7 and NS2 proteins are essential for production of infectious virus. J Virol 81 8374 8383

32. PengCW

NapuliAJ

DoljaVV

2003 Leader proteinase of beet yellows virus functions in long-distance transport. J Virol 77 2843 2849

33. TijmsMA

van der MeerY

SnijderEJ

2002 Nuclear localization of non-structural protein 1 and nucleocapsid protein of equine arteritis virus. J Genl Virol 83 795 800

34. BrierleyI

DigardP

InglisSC

1989 Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell 57 537 547

35. WassenaarALM

SpaanWJM

GorbalenyaAE

SnijderEJ

1997 Alternative proteolytic processing of the arterivirus replicase ORF1a polyprotein: evidence that NSP2 acts as a cofactor for the NSP4 serine protease. J Virol 71 9313 9322

36. PasternakAO

SpaanWJM

SnijderEJ

2004 Regulation of relative abundance of arterivirus subgenomic mRNAs. J Virol 78 8102 8113

37. van HemertMJ

de WildeAH

GorbalenyaAE

SnijderEJ

2008 The in vitro RNA synthesizing activity of the isolated arterivirus replication/transcription complex is dependent on a host factor. J Biol Chem 283 16525 16536

38. BeerensN

SeliskoB

RicagnoS

ImbertI

van derZL

SnijderEJ

CanardB

2007 De novo initiation of RNA synthesis by the arterivirus RNA-dependent RNA polymerase. J Virol 81 8384 8395

39. KroeseMV

Zevenhoven-DobbeJC

Bos-de RuijterJN

PeetersBP

MeulenbergJJ

CornelissenLA

SnijderEJ

2008 The nsp1alpha and nsp1 papain-like autoproteinases are essential for porcine reproductive and respiratory syndrome virus RNA synthesis. J Gen Virol 89 494 499

40. MinskaiaE

HertzigT

GorbalenyaAE

CampanacciV

CambillauC

CanardB

ZiebuhrJ

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

41. EckerleLD

LuX

SperrySM

ChoiL

DenisonMR

2007 High fidelity of murine hepatitis virus replication is decreased in nsp14 exoribonuclease mutants. J Virol 81 12135 12144

42. SnijderEJ

BredenbeekPJ

DobbeJC

ThielV

ZiebuhrJ

PoonLLM

GuanY

RozanovM

SpaanWJM

GorbalenyaAE

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

43. ChenY

CaiH

PanJ

XiangN

TienP

AholaT

GuoD

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

44. WuB

WhiteKA

2007 Uncoupling RNA virus replication from transcription via the polymerase: functional and evolutionary insights. EMBO J 26 5120 5130

45. van DintenLC

den BoonJA

WassenaarALM

SpaanWJM

SnijderEJ

1997 An infectious arterivirus cDNA clone: Identification of a replicase point mutation that abolishes discontinuous mRNA transcription. Proc Natl Acad Sci U S A 94 991 996

46. LandtO

GrunertHP

HahnU

1990 A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96 125 128

47. van den BornE

GultyaevAP

SnijderEJ

2004 Secondary structure and function of the 5′-proximal region of the equine arteritis virus RNA genome. RNA 10 424 437

48. van der MeerY

van TolH

LockerJK

SnijderEJ

1998 ORF1a-encoded replicase subunits are involved in the membrane association of the arterivirus replication complex. J Virol 72 6689 6698

49. PedersenKW

van der MeerY

RoosN

SnijderEJ

1999 Open reading frame 1a-encoded subunits of the arterivirus replicase induce endoplasmic reticulum-derived double-membrane vesicles which carry the viral replication complex. J Virol 73 2016 2026

50. MacLachlanNJ

BalasuriyaUB

HedgesJF

SchweidlerTM

McCollumWH

TimoneyPJ

HullingerPJ

PattonJF

1998 Serologic response of horses to the structural proteins of equine arteritis virus. J Vet Diagn Invest 10 229 236

51. NovakJE

KirkegaardK

1991 Improved method for detecting poliovirus negative strands used to demonstrate specificity of positive-strand encapsidation and the ratio of positive to negative strands in infected cells. J Virol 65 3384 3387

52. de VriesAA

ChirnsideED

HorzinekMC

RottierPJ

1992 Structural proteins of equine arteritis virus. J Virol 66 6294 6303

53. VersteegGA

SlobodskayaO

SpaanWJ

2006 Transcriptional profiling of acute cytopathic murine hepatitis virus infection in fibroblast-like cells. J Gen Virol 87 1961 1975

54. ScheltingaSA

TempletonKE

BeersmaMF

ClaasEC

2005 Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR. J Clin Virol 33 306 311

55. den BoonJA

KleijnenMF

SpaanWJM

SnijderEJ

1996 Equine arteritis virus subgenomic mRNA synthesis: analysis of leader-body junctions and replicative-form RNAs. J Virol 70 4291 4298