Abstract:
UNLABELLED:Coronaviruses (CoVs) are unique in encoding a 3'→5' exoribonuclease within nonstructural protein 14 (nsp14-ExoN) that is required for high-fidelity replication, likely via proofreading. nsp14 associates with the CoV RNA-dependent RNA polymerase (nsp12-RdRp), and nsp14-ExoN activity is enhanced by binding nsp10, a small nonenzymatic protein. However, it is not known whether nsp10 functions in the regulation of CoV replication fidelity. To test this, we engineered single and double alanine substitution mutations into the genome of murine hepatitis virus (MHV-A59) containing ExoN activity [ExoN(+)] at positions within nsp10 known to disrupt the nsp10-nsp14 interaction in vitro. We show that an nsp10 mutant, R80A/E82A-ExoN(+), was five to ten times more sensitive to treatment with the RNA mutagen 5-fluorouracil (5-FU) than wild-type (WT)-ExoN(+), suggestive of decreased replication fidelity. This decreased-fidelity phenotype was confirmed using two additional nucleoside analogs, 5-azacytidine and ribavirin. R80A/E82A-ExoN(+) reached a peak titer similar to and demonstrated RNA synthesis kinetics comparable to those seen with WT-ExoN(+). No change in 5-FU sensitivity was observed for R80A/E82A-ExoN(-) relative to MHV-ExoN(-), indicating that the decreased-fidelity phenotype of R80A/E82A-ExoN(-) is linked to the presence of ExoN activity. Our results demonstrate that nsp10 is important for CoV replication fidelity and support the hypothesis that nsp10 functions to regulate nsp14-ExoN activity during virus replication. IMPORTANCE:The adaptive capacity of CoVs, as well as all other RNA viruses, is partially attributed to the presence of extensive population genetic diversity. However, decreased fidelity is detrimental to CoV replication and virulence; mutant CoVs with decreased replication fidelity are attenuated and more sensitive to inhibition by RNA mutagens. Thus, identifying the viral protein determinants of CoV fidelity is important for understanding CoV replication, pathogenesis, and virulence. In this report, we show that nsp10, a small, nonenzymatic viral protein, contributes to CoV replication fidelity. Our data support the hypothesis that CoVs have evolved multiple proteins, in addition to nsp14-ExoN, that are responsible for maintaining the integrity of the largest known RNA genomes.
journal_name
J Viroljournal_title
Journal of virologyauthors
Smith EC,Case JB,Blanc H,Isakov O,Shomron N,Vignuzzi M,Denison MRdoi
10.1128/JVI.00110-15subject
Has Abstractpub_date
2015-06-01 00:00:00pages
6418-26issue
12eissn
0022-538Xissn
1098-5514pii
JVI.00110-15journal_volume
89pub_type
杂志文章abstract::Hepatitis E virus replicons containing the neomycin resistance gene expressed from open reading frames (ORFs) 2 and 3 were transfected into Huh-7 cells, and stable cell lines containing functional replicons were selected by constant exposure to G418 sulfate. Northern blot analyses detected full-length replicon RNA and...
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doi:10.1128/JVI.72.8.6637-6645.1998
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pub_type: 杂志文章
doi:10.1128/JVI.69.5.2946-2953.1995
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pub_type: 杂志文章
doi:10.1128/JVI.75.13.6086-6094.2001
更新日期:2001-07-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01815-09
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pub_type: 杂志文章
doi:10.1128/JVI.00505-06
更新日期:2006-09-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.67.3.1350-1356.1993
更新日期:1993-03-01 00:00:00
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pub_type: 杂志文章
doi:10.1128/jvi.76.5.2131-2140.2002
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pub_type: 杂志文章
doi:10.1128/JVI.43.1.348-351.1982
更新日期:1982-07-01 00:00:00
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pub_type: 杂志文章
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journal_title:Journal of virology
pub_type: 杂志文章
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pub_type: 杂志文章
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更新日期:2004-05-01 00:00:00
abstract:UNLABELLED:The safety and efficacy of the live-attenuated Japanese encephalitis virus (JEV) SA14-14-2 vaccine are attributed to mutations that accumulated in the viral genome during its derivation. However, little is known about the contribution that is made by most of these mutations to virulence attenuation and vacci...
journal_title:Journal of virology
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更新日期:2015-06-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.67.6.3534-3543.1993
更新日期:1993-06-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.13.5.1148-1150.1974
更新日期:1974-05-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.42.2.730-733.1982
更新日期:1982-05-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.69.3.1564-1574.1995
更新日期:1995-03-01 00:00:00
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journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.01522-06
更新日期:2007-02-01 00:00:00
abstract::In this study we report the complete sequence and genome organization of the serotype I feline coronavirus (FCoV) strain Black. Furthermore, a reverse genetic system was established for this FCoV strain by cloning a full-length cDNA copy into vaccinia virus. This clone served as basis for the generation of recombinant...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.02339-07
更新日期:2008-02-01 00:00:00
abstract:UNLABELLED:Recent experiments suggest that some glycoprotein (GP)-specific monoclonal antibodies (MAbs) can protect experimental animals against the filovirus Ebola virus (EBOV). There is a need for isolation of MAbs capable of neutralizing multiple filoviruses. Antibody neutralization assays for filoviruses frequently...
journal_title:Journal of virology
pub_type: 杂志文章
doi:10.1128/JVI.00101-16
更新日期:2016-03-28 00:00:00