Arterivirus Nsp1 Modulates the Accumulation of Minus-Strand Templates to Control the Relative Abundance of Viral mRNAs
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
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.
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
Štítky
Hygiena a epidemiológia Infekčné lekárstvo LaboratóriumČlánok vyšiel v časopise
PLOS Pathogens
2010 Číslo 2
- Očkování proti virové hemoragické horečce Ebola experimentální vakcínou rVSVDG-ZEBOV-GP
- Parazitičtí červi v terapii Crohnovy choroby a dalších zánětlivých autoimunitních onemocnění
- Koronavirus hýbe světem: Víte jak se chránit a jak postupovat v případě podezření?
Najčítanejšie v tomto čísle
- Caspase-1 Activation via Rho GTPases: A Common Theme in Mucosal Infections?
- Kaposi's Sarcoma Associated Herpes Virus (KSHV) Induced COX-2: A Key Factor in Latency, Inflammation, Angiogenesis, Cell Survival and Invasion
- IL-1β Processing in Host Defense: Beyond the Inflammasomes
- Reverse Genetics in Predicts ARF Cycling Is Essential for Drug Resistance and Virulence