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Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis.

ABSTRACT: Genomes of positive (+)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5'-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5' untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic mRNA7 (sgmRNA7). The natural DI RNA is structurally a fusion of the two ends of the BCoV genome that results in a single open reading frame between a partial nsp1-coding region and the entire N gene. (i) In the first examination, mutation analyses of a recently discovered long-range RNA-RNA base-paired structure between the 5' untranslated region and the partial nsp1-coding region showed that it, possibly in concert with adjacent stem-loops, is a cis-acting replication signal in the (+) strand. We postulate that the higher-order structure promotes (+)-strand synthesis. (ii) In the second examination, analyses of multiple frame shifts, truncations, and point mutations within the partial nsp1-coding region showed that synthesis of a PEFP core amino acid sequence within a group A lineage betacoronavirus-conserved NH2-proximal WAPEFPWM domain is required in cis for DI RNA replication. We postulate that the nascent protein, as part of an RNA-associated translating complex, acts to direct the DI RNA to a critical site, enabling RNA replication. We suggest that these results have implications for viral genome replication and explain, in part, why coronavirus sgmRNAs fail to replicate.cis-Acting RNA and protein structures that regulate (+)-strand RNA virus genome synthesis are potential sites for blocking virus replication. Here we describe two: a previously suspected 5'-proximal long-range higher-order RNA structure and a novel nascent NH2-terminal protein component of nsp1 that are common among betacoronaviruses of group A lineage.

SUBMITTER: Su YP

PROVIDER: S-EPMC4136265 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis.

Su Yu-Pin YP Fan Yi-Hsin YH Brian David A DA

Journal of virology 20140528 16

<h4>Unlabelled</h4>Genomes of positive (+)-strand RNA viruses use cis-acting signals to direct both translation and replication. Here we examine two 5'-proximal cis-replication signals of different character in a defective interfering (DI) RNA of the bovine coronavirus (BCoV) that map within a 322-nucleotide (nt) sequence (136 nt from the genomic 5' untranslated region and 186 nt from the nonstructural protein 1 [nsp1]-coding region) not found in the otherwise-identical nonreplicating subgenomic ...[more]

PMID: 24872586

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Project description:UnlabelledCoronaviruses (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.ImportanceThe 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.

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Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis.

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Dependence of coronavirus RNA replication on an NH2-terminal partial nonstructural protein 1 in cis.

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