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Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3'-Terminal Adenylyltransferase Activity.

ABSTRACT: Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs. Using partially double-stranded RNAs, very efficient TATase activity was observed if the opposite (template) strand contained a short 5' oligo(U) sequence, while very little (if any) activity was detected for substrates with other homopolymeric or heteropolymeric sequences in the 5' overhang. The oligo(U)-assisted/templated TATase activity on partial-duplex RNAs was confirmed for two other coronavirus nsp8 proteins, suggesting that the activity is conserved among coronaviruses. Replacement of a conserved Lys residue with Ala abolished the in vitro RNA-binding and TATase activities of nsp8 and caused a nonviable phenotype when the corresponding mutation was introduced into the HCoV-229E genome, confirming that these activities are mediated by nsp8 and critical for viral replication. In additional experiments, we obtained evidence that nsp8 has a pronounced specificity for adenylate and is unable to incorporate guanylate into RNA products, which strongly argues against the previously proposed template-dependent RNA polymerase activity of this protein. Given the presence of an oligo(U) stretch at the 5' end of coronavirus minus-strand RNAs, it is tempting to speculate (but remains to be confirmed) that the nsp8-mediated TATase activity is involved in the 3' polyadenylation of viral plus-strand RNAs.IMPORTANCE Previously, coronavirus nsp8 proteins were suggested to have template-dependent RNA polymerase activities resembling those of RNA primases or even canonical RNA-dependent RNA polymerases, while more recent studies have suggested an essential cofactor function of nsp8 (plus nsp7) for nsp12-mediated RNA-dependent RNA polymerase activity. In an effort to reconcile conflicting data from earlier studies, the study revisits coronavirus nsp8-associated activities using additional controls and proteins. The data obtained for three coronavirus nsp8 proteins provide evidence that the proteins share metal ion-dependent RNA 3' polyadenylation activities that are greatly stimulated by a short oligo(U) stretch in the template strand. In contrast, nsp8 was found to be unable to select and incorporate appropriate (matching) nucleotides to produce cRNA products from heteropolymeric and other homooligomeric templates. While confirming the critical role of nsp8 in coronavirus replication, the study amends the list of activities mediated by coronavirus nsp8 proteins in the absence of other proteins.

SUBMITTER: Tvarogova J

PROVIDER: S-EPMC6613758 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3'-Terminal Adenylyltransferase Activity.

Tvarogová Jana J Madhugiri Ramakanth R Bylapudi Ganesh G Ferguson Lyndsey J LJ Karl Nadja N Ziebuhr John J

Journal of virology 20190529 12

Coronavirus nonstructural protein 8 (nsp8) has been suggested to have diverse activities, including noncanonical template-dependent polymerase activities. Here, we characterized a recombinant form of the human coronavirus 229E (HCoV-229E) nsp8 and found that the protein has metal ion-dependent RNA 3'-terminal adenylyltransferase (TATase) activity, while other nucleotides were not (or very inefficiently) transferred to the 3' ends of single-stranded and (fully) double-stranded acceptor RNAs. Usin ...[more]

PMID: 30918070

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Project description: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.

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Dataset Information

Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3'-Terminal Adenylyltransferase Activity.

Publications

Identification and Characterization of a Human Coronavirus 229E Nonstructural Protein 8-Associated RNA 3'-Terminal Adenylyltransferase Activity.

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