Identification and characterization of mouse hepatitis virus papain-like proteinase 2 activity

Mouse hepatitis virus (MHV) is a 31.2-kb positive-stranded RNA virus that replicates in the cytoplasm of infected cells. The enzyme that replicates the viral RNA is the viral RNA-dependent RNA polymerase, termed the replicase. The replicase is encoded in the 5′-most 22 kb of the viral genomic RNA. Thus, the incoming viral RNA is translated to produce a replicase polyprotein of >800 kDa. The replicase polyprotein is then extensively processed by viral proteinases to give rise to a functional replicase complex. Sequence analysis of the replicase gene led to the prediction of three proteinase domains, two papain-like proteinases (PLP1 and PLP2) and a poliovirus 3C-like proteinase (3CLpro). Prior to the studies presented in this dissertation, only two of the MHV replicase-encoded proteinases, PLP1 and 3CLpro, were shown to process the replicase polyprotein. There were no reports of an enzymatic activity for the predicted PLP2 domain.; In this study, I described the cloning, expression and activity of the MHV PLP2 domain. I showed that PLP2 cleaves the replicase polyprotein at a site located just upstream of the predicted membrane-associated protein domain 1 (MP1). Cleavage of this site releases a 150-kDa intermediate, p150, that contains two predicted membrane associated proteins (MP1 and MP2) and the 3CLpro domain. The MP1 and MP2 proteins are proposed to be important for embedding the replicase complex into cellular membranes. By performing co-expression studies using the PLP2 domain and substrate encoding the MP1 cleavage site, I showed that PLP2 can also act efficiently in trans. Site-directed mutagenesis studies confirmed the identification of cysteine residue 1715 as a catalytic residue and also demonstrated that four cysteine residues predicted to form a zinc-binding domain are required for PLP2 activity.; To identify the cleavage site recognized by PLP2, I generated a number of constructs expressing the substrate cleavage site and performed site-directed mutagenesis analysis. I demonstrated that PLP2 recognizes glycine residues 2839 and 2840 as the critical determinants for cleavage of the precursor polyprotein. Analysis of the amino acid sequences downstream of the cleavage site predicted that the MP1 contains two possible N-linked glycosylation sites and four putative transmembrane domains. I showed that the p150 intermediate and the MP1 product are sensitive to treatments with tunicamycin and endoglycosidase H, indicating that they are glycoproteins. Mutagenesis and expression studies confirmed that the predicted asparagine residues 3016 and 3077 are glycosylated. Biochemical fractionation experiments revealed that MP1 acts as an integral membrane protein and is resistant to extraction by detergent Triton X-114. Thus, the MHV replicase product MP1 is an integral membrane glycoprotein that may act as a scaffolding component for the membrane-associated viral replicase complex.; Overall, this study is the first to report enzymatic activity of the PLP2 domain, to characterize the site recognized by PLP2, and to demonstrate that three distinct viral proteinase activities are required to generate the coronavirus replicase complex.