Positive-strand RNA viruses induce modifications of cytoplasmic membranes to form replication complexes. For coronaviruses, replicase nonstructural protein 4 (nsp4) has been proposed to function in the formation and organization of replication complexes. Murine hepatitis virus (MHV) nsp4 is glycosylated at residues Asn176 (N176) and N237 during plasmid expression of nsp4 in cells. To test if MHV nsp4 residues N176 and N237 are glycosylated during virus replication and to determine the effects of N176 and N237 on nsp4 function and MHV replication, alanine substitutions of nsp4 N176, N237, or both were engineered into the MHV-A59 genome. The N176A, N237A, and N176A/N237A mutant viruses were viable, and N176 and N237 were glycosylated during infection of wild-type (wt) and mutant viruses. The nsp4 glycosylation mutants exhibited impaired virus growth and RNA synthesis, with the N237A and N176A/N237A mutant viruses demonstrating more profound defects in virus growth and RNA synthesis. Electron microscopic analysis of ultrastructure from infected cells demonstrated that the nsp4 mutants had aberrant morphology of virus-induced double-membrane vesicles (DMVs) compared to those infected with wt virus. The degree of altered DMV morphology directly correlated with the extent of impairment in viral RNA synthesis and virus growth of the nsp4 mutant viruses. The results indicate that nsp4 plays a critical role in the organization and stability of DMVs. The results also support the conclusion that the structure of DMVs is essential for efficient RNA synthesis and optimal replication of coronaviruses.

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Partial or complete deletion of several coronavirus nonstructural proteins (nsps), including open reading frame 1a (ORF1a)-encoded nsp2, results in viable mutant proteins with specific replication defects. It is not known whether expression of nsps from alternate locations in the genome can complement replication defects. In this report, we show that the murine hepatitis virus nsp2 sequence was tolerated in ORF1b with an in-frame insertion between nsp13 and nsp14 and in place of ORF4. Alternate encoding or duplication of the nsp2 gene sequence resulted in differences in nsp2 expression, processing, and localization, was neutral or detrimental to replication, and did not complement an ORF1a ⌬nsp2 replication defect. The results suggest that wild-type genomic organization and expression of nsps are required for optimal replication.Coronaviruses are positive-sense RNA viruses that translate the first open reading frames (ORFs; ORF1a and ORF1b) of their 30-kb genome RNA into polyproteins that are co-and posttranslationally processed into intermediate and mature nonstructural proteins (nsps; nsps 1 to 16). The nsps interact on cytoplasmic membranes at sites of viral RNA synthesis, referred to as replication complexes (4,5,10,17,22,25). Translation of ORF1b, which encodes several proteins confirmed or predicted to be essential for viral RNA synthesis, requires a ribosomal frameshift event at the end of ORF1a that occurs at 10 to 40% efficiency in vitro (2, 9, 13-15, 18, 21, 23).The murine hepatitis virus (MHV) nsp2 is a 65-kDa protein that has minimal sequence identity or similarity among different coronavirus groups and has no known or predicted functions. We have shown for MHV and severe acute respiratory syndrome coronavirus (SARS-CoV) that in-frame deletion of nsp2 (⌬nsp2) yields viable mutant viruses (12). However, both MHV and SARS-CoV ⌬nsp2 mutants exhibit a 90% reduction in peak titer and a 50% reduction in viral RNA synthesis. To determine if expression of nsp2 from nonnative sites could complement the defect in MHV ⌬nsp2 replication, we engineered the nsp2 coding sequence at alternate sites in the genome both in the absence and in the presence of the wild-type ORF1a nsp2 sequence. The results indicate that nsp2 can be encoded and expressed alone from ORF4, as a sequence duplication in ORF1a and ORF1b or in ORF1a and ORF4, but not near the end of ORF1a or alone in ORF1b. Duplication or expression of the nsp2 sequence from ORF4 was detrimental to replication compared to that of the wild type, indicating that the native context of nsp2 expression, and possibly a single copy of the sequence, may be necessary for optimal function in replication. Results also indicate that the addition of amino acids at the N and C termini of natively expressed nsp2 has no effect on peak viral growth.nsp2 can be encoded in replication-competent mutant viruses in ORF4 and between nsp13 and nsp14 in ORF1b. MHV nonessential ORFs have been shown to tolerate foreign gene insertion (3,8,19). In order to test the effects of nsp2 expressi...

Coronavirus nonstructural proteins 1 to 3 are processed by one or two papain-like proteases (PLP1 and PLP2) at specific cleavage sites (CS1 to -3). Murine hepatitis virus (MHV) PLP2 and orthologs recognize and cleave at a position following a p4-Leu-X-Gly-Gly-p1 tetrapeptide, but it is unknown whether these residues are sufficient to result in processing by PLP2 at sites normally cleaved by PLP1. We demonstrate that exchange of CS1 and/or CS2 with the CS3 p4-p1 amino acids in engineered MHV mutants switches specificity from PLP1 to PLP2 at CS2, but not at CS1, and results in altered protein processing and virus replication. Thus, the p4-p1 residues are necessary for PLP2 processing but require a specific protein or cleavage site context for optimal PLP recognition and cleavage.Coronaviruses are positive-strand RNA viruses that translate their first open reading frames (ORF1a and ORF1b) into polyproteins that are processed by viral proteases into intermediate and mature nonstructural proteins (nsp1 to -16) (Fig. 1A) (4,7,17,20). nsp1, -2, and -3 are liberated at cleavage sites (CSs) between nsp1-2 (CS1), nsp2-3 (CS2), and nsp3-4 (CS3) by one or two papain-like protease (PLP) activities encoded within nsp3 (1,2,12,13,15) (Fig. 1B). Murine hepatitis virus (MHV) and human coronavirus 229E (HCoV-229E) use two PLPs (PLP1 and PLP2) to process at CS1 to -3, while severe acute respiratory syndrome coronavirus (SARS-CoV) and avian infectious bronchitis virus (IBV) use a single PLP each (PLpro and PLP2, respectively) (10,20,25,26). The factors determining the evolution and use of one versus two PLPs by different coronaviruses for processing of nsp1, -2, and -3 are unknown. Mutations at MHV CSs or within PLP1 alter replication and protein processing in surprising ways (8, 13). Loss of processing at MHV CS1 and CS2 by CS deletion or mutation results in changes in the timing and extent of virus replication. Inactivation of MHV PLP1 is more detrimental for virus replication than deletion of CS1 and CS2 or than inactivation of PLP1 combined with the CS deletions, even though not all of the mutant viruses process at CS1 or CS2 or display similar protein processing phenotypes. In contrast to MHV results, the HCoV-229E PLP1 and PLP2 have both been shown to process at CS1 and CS2, albeit at different efficiencies (Fig. 1B) (24). Finally, the single SARS-CoV PLP2 homolog (PLpro) mediates efficient processing at CS1 to -3, each of which has an upstream position 4-Leu-X-Gly-Gly-position 1 (p4-LXGG-p1) amino acid motif implicated in PLpro processing (10,16,18). MHV possesses a p4-LXGG-p1 sequence only at CS3 and is cleaved by PLP2. These results suggest that p4-LXGG-p1 may be the critical determinant of recognition by PLP2/PLpro, but this hypothesis has not been tested in studies of replicating virus. Thus, it remains unknown whether the differences in PLP/CS recognition and processing are determined by the proximal p4-p1 residues (22).In this study, we used MHV as a model to test whether PLP/CS specificities could be switched by an e...