Identification of mouse hepatitis virus papain-like proteinase 2 activity

Mouse hepatitis virus (MHV) is a 31-kb positive-strand RNA virus that is re plicated in the cytoplasm of infected cells by a viral RNA-dependent RNA po lymerase, termed the replicase, The replicase is encoded in the 5'-most 22 kb of the genomic RNA, which is translated to produce a polyprotein of >800 kDa, The replicase polyprotein is extensively processed by viral and perha ps cellular proteinases to give rise to a functional replicase complex. To date, two of the MHV replicase-encoded proteinases, papain-like proteinase 1 (PLP1) and the poliovirus 3C-like proteinase (3CLpro), have been shown to process the replicase polyprotein. In this report, we describe the cloning , expression, and activity of the third MHV proteinase domain, PLP2, We sho w that PLP2 cleaves a substrate encoding the first predicted membrane-spann ing domain (MP1) of the replicase polyprotein. Cleavage of MP1 and release of a 150-kDa intermediate, p150, are likely to be important for embedding t he replicase complex in cellular membranes. Using an antiserum (anti-D11) d irected against the C terminus of the MP1 domain, we verified that p150 enc ompasses the MP1 domain and identified a 44-kDa protein (p44) as a processe d product of p150. Pulse-chase experiments showed that p150 is rapidly gene rated in MHV-infected cells and that p44 is processed from the p150 precurs or. Protease inhibitor studies revealed that unlike 3CLpro activity, PLP2 a ctivity is not sensitive to cysteine protease inhibitor E64d, Furthermore, coexpression studies using the PLP2 domain and a substrate encoding the MP1 cleavage site showed that PLP2 acts efficiently in trans. Site-directed mu tagenesis studies confirmed the identification of cysteine 1715 as a cataly tic residue of PLP2, This study is the first to report enzymatic activity o f the PLP2 domain and to demonstrate that three distinct viral proteinase a ctivities process the MHV replicase polyprotein.