PROCESSING OF THE CORONAVIRUS MHV-JHM POLYMERASE POLYPROTEIN - IDENTIFICATION OF PRECURSORS AND PROTEOLYTIC PRODUCTS SPANNING 400 KILODALTONS OF ORF1A

The replicase of mouse hepatitis virus strain JHM (MHV-JHM) is encoded by two overlapping open reading frames, ORF1a and ORF1b, which are tr anslated to produce a 750-kDa precursor polyprotein. The polyprotein i s proposed to be processed by viral proteinases to generate the functi onal replicase complex. To date, only the MHV-JHM amino-terminal prote ins p28 and p72, which is processed to p65, have been identified. To f urther elucidate the biogenesis of the MHV-JHM replicase, we cloned an d expressed five regions of ORF1a in bacteria and prepared rabbit anti sera to each region. Using the immune sera to immunoprecipitate radiol abeled proteins from MHV-JHM infected cells, we determined that the MH V-JHM ORF1a is initially processed to generate p28, p72, p250, and p15 0. Pulse-chase analysis revealed that these intermediates are further processed to generate p65, p210, p40, p27, the MHV 3C-like proteinase, and p15. A putative replicase complex consisting of p250, p210, p40, p150, and a large protein (>300 kDa) coprecipitate from infected cells disrupted with NP-40, indicating that these proteins are closely asso ciated even after initial proteolytic processing. Immunofluorescence s tudies revealed punctate labeling of ORF1a proteins in the perinuclear region of infected cells, consistent with a membrane-association of t he replicase complex. Furthermore, in vitro transcription/translation studies of the MHV-JHM 3Cpro and flanking hydrophobic domains confirm that 3C protease activity is significantly enhanced in the presence of canine microsomal membranes. Overall, our results demonstrate that th e MHV-JHM ORF1a polyprotein: (1) is processed into more than 10 protei n intermediates and products, (2) requires membranes for efficient bio genesis, and (3) is detected in discrete membranous regions in the cyt oplasm of infected cells. (C) 1998 Academic Press.