A 100-KILODALTON POLYPEPTIDE ENCODED BY OPEN READING FRAME (ORF) 1B OF THE CORONAVIRUS INFECTIOUS-BRONCHITIS VIRUS IS PROCESSED BY ORF 1A PRODUCTS

The genome-length mRNA (mRNA 1) of the coronavirus infectious bronchit is virus (IBV) contains two large open reading frames (ORFs), 1a and 1 b, with the potential to encode polypeptides of 441 and 300 kDa, respe ctively. The downstream ORF, ORF 1b, is expressed by a ribosomal frame shifting mechanism. In an effort to detect viral polypeptides encoded by ORF 1b in virus-infected cells, immunoprecipitations were carried o ut with a panel of region-specific antisera. A polypeptide of approxim ately 100 kDa was precipitated from IBV-infected, but not mock-infecte d Vero cells by one of these antisera (V58). Antiserum V58 was raised against a bacterially expressed fusion protein containing polypeptide sequences encoded by ORF 1b nucleotides 14492 to 15520; it recognizes specifically the corresponding in vitro-synthesized target protein. A polypeptide comigrating with the 100,000-molecular-weight protein (100 K protein) identified in infected cells was also detected when the IBV sequence from nucleotides 8693 to 16980 was expressed in Vero cells b y using a vaccinia virus-T7 expression system. Deletion analysis revea led that the sequence encoding the C terminus of the 100K polypeptide lies close to nucleotide 15120; it may therefore be generated by prote olysis at a potential QS cleavage site encoded by nucleotides 15129 to 15135. In contrast, expression of IBV sequences from nucleotides 1075 2 to 16980 generated two polypeptides of approximately 62 and 235 kDa, which represent the ORF 1a stop product and the 1a-1b fused product g enerated by a frameshifting mechanism, respectively, but no processed products were observed. Since the putative picornavirus 3C-like protei nase domain is located in ORF 1a between nucleotides 8937 and 9357, th is observation suggests that deletion of the picornavirus 3C-like prot einase domain and surrounding regions abolishes processing of the 1b p olyprotein. In addition, the in vitro translation and in vivo transfec tion studies also indicate that the ORF 1a region between nucleotides 8763 and 10720 contains elements that down-regulate the expression of ORF 1b.