Cleavage between replicase proteins p28 and p65 of mouse hepatitis virus is not required for virus replication.

Two families of large positive-strand RNA viruses, the arteriviruses and the coronaviruses, belong to the order Nidovirales. Despite differences in the genome sizes of the arteriviruses (13 to 16 kb) and coronaviruses (27 to 32 kb), these virus families have several important features in common, including a polycistronic genome, an array of conserved protein domains, and a discontinuous RNA transcription strategy. Another important conserved feature of nidovirus replication is the expression of viral replicase proteins from large polyprotein precursors. These replicase polyproteins are cleaved co- and posttranslationally by virus-encoded proteinases to yield up to 15 mature replicase protein products. Along with putative cellular factors, the mature replicase proteins and precursors are thought to mediate all stages of viral RNA synthesis on membrane-bound viral replication complexes in the cytoplasm. Previous studies have demonstrated roles for arterivirus replicase proteins in the formation and function of viral replication complexes (17, 21, 22, 26). However, the functions of the majority of coronavirus replicase proteins during viral replication have not been determined. The coronavirus replicase gene (gene 1) is composed of two overlapping open reading frames (ORF1a and ORF1b) that are translated by host cell ribosomes to produce the replicase polyproteins. Translation of ORF1a yields a polyprotein of nearly 490 kDa, while a −1 ribosomal frameshift allows translation of an ORF1a-ORF1b fusion polyprotein that is approximately 800 kDa. Cleavage of the coronavirus replicase polyproteins is mediated by one or two papain-like cysteine proteinases (PLP1 and PLP2) and a picornavirus 3C-like cysteine proteinase (3CLpro) to yield 12 to 16 mature replicase protein products. 3CLpro cleavage sites have been identified at conserved regions in all coronaviruses, including the newly discovered coronavirus associated with severe acute respiratory syndrome (SARS-CoV) (23, 26). In contrast, the conservation and activity of PLP1 and PLP2 domains vary among the three known coronavirus serogroups (groups 1, 2, and 3). The group 1 and 2 coronaviruses express both PLP1 and PLP2 activities and have in common the feature that PLP1 cleaves the first two amino-terminal products from the polyproteins (26). Specifically, viruses in group 1, such as human coronavirus 229E, liberate an amino-terminal 9-kDa protein and an 87-kDa protein after PLP1-mediated processing (12, 27). For group 2 coronaviruses, including bovine coronavirus and mouse hepatitis virus (MHV), PLP1 has been shown in vitro to cleave an amino-terminal 28-kDa protein and a 65-kDa protein, referred to as p28 and p65, respectively (Fig. ​(Fig.1)1) (1, 2, 7, 8, 10, 14). In contrast, SARS-CoV, as well as the avian coronaviruses (group 3), such as infectious bronchitis virus (IBV), only express active PLP2, which is responsible for cleavage of amino-terminal proteins (23, 26). These observations suggest that PLP1-mediated cleavages may not be essential for coronavirus replication or that the amino-terminal proteins liberated by PLP1s have group-specific functions during replication. FIG. 1. A59 genome organization and replicase antibodies. The A59 genome is 32 kb long. The replicase gene (gene 1, 22 kb) is shown, with replicase protein domains in overlapping ORF1a and ORF1b. Hatched boxes indicate proteinases: PLP1 domain (papain-like proteinase ... MHV has been extensively used as a model for studies of coronavirus replicase protein expression and processing. The determinants of MHV PLP1-mediated cleavages have been studied in detail by using in vitro expression and processing assays (2, 3, 10, 14). PLP1 has been shown to proteolytically process the first two cleavage sites in the MHV replicase polyprotein: between p28 and p65 at 247G↓V248 (referred to as cleavage site 1, or CS1, in this report) and between p65 and p210 at 832A↓G833 (CS2) (3, 10, 14). Until recently, the lack of a reverse genetics system has hindered the characterization of these cleavage sites in the context of MHV infection. Several lines of evidence indicate that continuous translation and processing of the MHV replicase polyprotein are required for virus replication (15, 18, 19). However, the requirements for processing at specific cleavage sites in the polyprotein, such as between p28 and p65, have not been shown. Biochemical experiments determined that both p28 and p65 are associated with intracellular membranes (4, 11, 20), and confocal immunofluorescence microscopy data have shown that both p28 and p65 localize to viral replication complexes in punctate perinuclear foci (20; S. M. Brockway, X. T. Lu, T. R. Peters, T. S. Dermody, and M. R. Denison, submitted for publication). These results suggest that p28 and p65 may play a role in replication complex formation or RNA synthesis. In the present study, we used a reverse genetics approach to verify the critical CS1 residues necessary for cleavage and to determine if cleavage of p28 from p65 is required for MHV replication. By using the MHV strain A59 infectious clone, viruses were generated that had either predicted cleaving or noncleaving mutations in CS1. Viruses with predicted noncleaving CS1 mutations were viable but were unable to process p28 and p65, instead expressing an uncleaved p93 precursor protein. Although noncleaving CS1 mutants were able to replicate, these viruses had diminished peak titers, smaller plaques, and decreased RNA synthesis compared with wild-type virus and cleaving CS1 mutant viruses. Despite the defect in viral replication, the mutant viruses did not demonstrate phenotypic reversion or changes at introduced mutations after repeated passage in culture. This study demonstrates that while cleavage of p28 from p65 is not required for viral replication, alterations at the first cleavage site alter virus growth, protein processing, and RNA synthesis.