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

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 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. 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(