Murine Coronaviruses Encoding nsp2 at Different Genomic Loci Have Altered Replication, Protein Expression, and Localization

Abstract

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...

Emerging coronaviruses: Genome structure, replication, and pathogenesis

“…[65][66][67] Plasma and antibodies obtained from the convalescent patients have been proposed for use in treatment. 68 In addition, various vaccine strategies, such as using inactivated viruses, live-attenuated viruses, viral vector-based Cellular mRNA degradation, inhibiting IFN signaling 15,16 nsp2 Unknown 17,18 nsp3 PLP, polypeptides cleaving, blocking host innate immune response, promoting cytokine expression 19,20 nsp4 DMV formation 21,22 nsp5 3CL pro , M pro , polypeptides cleaving, inhibiting IFN signaling [23][24][25] nsp6…”

Biogenesis and Dynamics of the Coronavirus Replicative Structures

“…The function of nsp2 is not yet known, although this protein was shown not to be essential for virus replication [58,59]. The reader is referred to several excellent reviews on this topic for more detailed insights [10,60,61].…”

Recombinant Live Vaccines to Protect Against the Severe Acute Respiratory Syndrome Coronavirus

“…Deletion of nsp2 in MHV and SARS-CoV viruses caused 0.5-1 log10 reductions in peak titers in single-cycle growth assays, as well as a reduction in viral RNA synthesis and growth [117,119]. These findings indicate that nsp2 is not essential for virus replication and that its deletion may lead to viruses with an attenuated phenotype.…”

“…Most of the experimental information on the influence of coronavirus replicase protein modification in attenuation has been obtained changing nsp1 and nsp2 [114][115][116][117][118][119]. In the case of SARS-CoV, it has been shown that nsp1 significantly inhibited IFN-dependent signaling by decreasing the phosphorylation levels of STAT1 while having little effect on those of STAT2, JAK1, and TYK2 [115].…”