Intracellular processing of the N-terminal ORF 1a proteins of the coronavirus MHV-A59 requires multiple proteolytic events

Abstract

Several polypeptide products of MHV-A59 ORF 1a were characterized in MHV-A59 infected DBT cells, using antisera directed against fusion proteins encoded in the first 6.5 kb of ORF1a. These included the previously identified N-terminal ORF 1a product, p28, as well as 290-, 240-, and 50-kDa polypeptides. P28 was always detected as a discrete band without larger precursors, suggesting rapid cleavage of p28 immediately after its synthesis. Once p28 was cleaved there was little degradation of the protein over a 2-hr period. The intracellular cleavage of p28 was not inhibited by the protease inhibitor leupeptin, in contrast to results obtained during in vitro translation of genome RNA (Denison and Perlman, 1986). These data suggest that different protease activities may be responsible for the cleavage of p28 in vitro and in vivo. The 290-kDa protein was an intermediate cleavage product derived from a precursor of greater than 400 kDa. The 290-kDa product was subsequently cleaved into secondary products of 50 and 240 kDa. The intracellular cleavage of the 290-kDa polypeptide was inhibited by leupeptin at concentrations which did not inhibit the early cleavage of p28 or the cleavage of the 290-kDa product from its larger polyprotein precursor. In the presence of zinc chloride, a product of greater than 320 kDa was detected, which appears to incorporate p28 at its amino terminus. This suggests that at least two protease activities may be necessary for processing of ORF1a proteins, one of which cleaves p28 and is sensitive to zinc chloride but resistant to leupeptin, and the other which cleaves the 290-kDa precursor and is sensitive to both inhibitors. Both the 290- and 240-kDa proteins should contain sequences predicted to encode two papain-like protease activities.

Toroviruses: replication, evolution and comparison with other members of the coronavirus-like superfamily

“…As described for other viral replicases, the CVL replicases are proteolytically cleaved into smaller active units. A number of cleavage products has recently been detected in coronavirus- (Brierley et al, 1990;Denison et al, 1991Denison et al, , 1992 and arterivirus-infected (Snijder etal., 1992; E. J. Snijder, A. L. M. Wassenaar & W. J. M. Spaan, unpublished) cells. The proteases involved in replicase protein processing are encoded by the ORF la sequence.…”

Localization of mouse hepatitis virus open reading frame 1A derived proteins

“…Most of the polyclonal antibodies used in this study (UP1, UP102, 600A, 81043) have been described previously (Denison et al, 1992Hughes et al, 1993). These antisera were raised in rabbits using prokaryotic/viral fusion proteins as immunogens.…”

“…Biochemical studies using antibodies that recognize discrete portions of the protein(s) encoded by gene 1 have been useful tools to elucidate the proteolytic cleavages which process the gene 1 primary translation product (Denison et al, 1991(Denison et al, , 1992Hughes et al, 1995;Lu et al, 1995). To date, these studies have been most successful for ORF1a.…”

“…An N-terminal 28 kDa protein, p28, is cotranslationally cleaved from the primary translation product by the ®rst papain-like protease encoded in ORF1a Perlman, 1986, 1987;Baker et al, 1989;Denison et al, 1992). Subsequently, a 65 kDa protein (p65) is cleaved from a large precursor protein and additional discrete polypeptides approximately 290 kDa (p290), 240 kDa (p240), 50 kDa (p50), and p29 kDa (3CLpro) molecular mass are generated by proteases (Denison et al, 1992Hughes et al, 1993;Pin Ä o  n et al, 1997;Lu and Denison, 1997). The presumed relationships of these cleavage products to each other and their positions on the MHV genome are shown in Figure 1.…”

Internal Ribosome Entry in the Coding Region of Murine Hepatitis Virus mRNA 5

“…The unique region of MHV mRNA 1 contains two large ORFs that constitute the coronavirus RNA polymerase locus. Expression of viral proteins from this locus involves both ribosomal frameshifting and autoproteolytic processing (Baker et al, 1989;Bredenbeek et al, 1990;Denison et al, 1991Denison et al, , 1992. MHV mRNA 5 also contains two ORFs in its unique region, designated ORF 5a and ORF 5b.…”