Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein

Abstract

The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.

Nidovirales: Evolving the largest RNA virus genome

“…The first candidate to be added to the constellation is another proteinase. Besides the conserved M pro (3CL pro ), arteri-, coronaand toroviruses encode so-called accessory proteinases (one to four, depending on the virus) that autocatalytically process the large N-proximal regions of the replicase polyproteins in which they reside ((Baker et al, 1989;Snijder et al, 1992) (reviewed by Ziebuhr et al, 2000); see also Draker et al, 2006). They belong to the same superfamily of papain-like (PL pro ) cysteine proteinases despite often having little sequence resemblance and very different sizes (den Boon et al, 1995;Gorbalenya et al, 1991) (Fig.…”

RNA structure analysis of alphacoronavirus terminal genome regions

“…Over the past years, viral components of the coronavirus RTC have been characterized in considerable detail, providing a wealth of functional and structural information (reviewed in Imbert et al, 2010;Masters, 2006;Ulferts et al, 2010;Ulferts and Ziebuhr, 2011;Ziebuhr, 2008). A large number of virally encoded enzymes, including protease, ADP-ribose-1 -phosphatase, NTPase, 5 -to-3 helicase, RNA 5 -triphosphatase, RNA polymerase, guanosine-N7 and ribose 2 -O methyltransferases, 3 -to-5 exoribonuclease and uridylatespecific endoribonuclease, have been identified (Baker et al, 1989;Bhardwaj et al, 2004;Chen et al, 2009Chen et al, , 2011Decroly et al, 2008Decroly et al, , 2011Eckerle et al, 2010;Ivanov et al, 2004;Kanjanahaluethai and Baker, 2000;Minskaia et al, 2006;Putics et al, 2005;Saikatendu et al, 2005;Seybert et al, 2000;te Velthuis et al, 2010;Ziebuhr et al, 1995). In some cases, these activities could be linked to specific steps of viral RNA synthesis and/or RNA processing or were shown to interfere with cellular functions (reviewed in Masters and Perlman, 2013;Ziebuhr, 2008).…”

Comparative in vivo analysis of the nsp15 endoribonuclease of murine, porcine and severe acute respiratory syndrome coronaviruses

“…Upon infection, the viral genomic RNA, which is capped at the 5 -end and polyadenylated at the 3 -end, serves as a mRNA for translation of the two overlapping open reading frames (ORF) (gene 1a/1b) at the 5 two-third of the genome via the ribosomal frameshifting translation mechanism (Bredenbeek et al, 1990;Brierley et al, 1987Brierley et al, , 1989Lee et al, 1991). The resultant protein product, polyprotein 1a/b, is then proteolytically cleaved by virus-encoded proteases into 16 nonstructural proteins, termed nsp 1-16, many of which have enzymatic activities, such as papain-like proteases (nsp3), 3C-like protease (nsp5), RNA-dependent RNA polymerase (RdRp, nsp12), helicase (nsp13), exoribonuclease and methyltransferase (nsp14), endoribonuclease (nsp15), 2-O-ribosyl-transferase (nsp16) (Baker et al, 1989;Bost et al, 2001;Denison et al, 1992Denison et al, , 1998Harcourt et al, 2004;Snijder et al, 2003;Thiel et al, 2001;Ziebuhr, 2005). These nsps are believed to form replication/transcription complexes along with putative cellular factors that catalyze the synthesis of genomic RNA (replication) and subgenomic RNAs (transcription).…”