Abstract
The coronavirus replicase-transcriptase complex is an assembly of viral and cellular proteins that mediate the synthesis of genome and subgenome-sized mRNAs in the virus-infected cell. Here, we report a genetic and functional analysis of 19 temperature-sensitive (ts) mutants of Murine hepatitis virus MHV-A59 that are unable to synthesize viral RNA when the infection is initiated and maintained at the non-permissive temperature. Both classical and biochemical complementation analysis leads us to predict that the majority of MHV-A59 ORF1a replicase gene products (non-structural proteins nsp1–nsp11) form a single complementation group (cistron1) while the replicase gene products encoded in ORF1b (non-structural proteins nsp12–nsp16) are able to function in trans and comprise at least three, and possibly five, further complementation groups (cistrons II–VI). Also, we have identified mutations in the non-structural proteins nsp 4, nsp5, nsp10, nsp12, nsp14, and nsp16 that are responsible for the ts phenotype of eight MHV-A59 mutants, which allows us to conclude that these proteins are essential for the assembly of a functional replicase-transcriptase complex. Finally, our analysis of viral RNA synthesis in ts mutant virus-infected cells allows us to discriminate three phenotypes with regard to the inability of specific mutants to synthesize viral RNA at the non-permissive temperature. Mutant LA ts6 appeared to be defective in continuing negative-strand synthesis, mutant Alb ts16 appeared to form negative strands but these were not utilized for positive-strand RNA synthesis, and mutant Alb ts22 was defective in the elongation of both positive- and negative-strand RNA. On the basis of these results, we propose a model that describes a pathway for viral RNA synthesis in MHV-A59-infected cells. Further biochemical analysis of these mutants should allow us to identify intermediates in this pathway and elucidate the precise function(s) of the viral replicase proteins involved.
Highlights
Coronaviruses are positive-strand, enveloped RNA viruses that infect vertebrates and are associated mainly with respiratory and enteric disease
The virus produced at 37 8C by Alb ts22, Wuts18, Wuts36, and Wuts38 was ts, i.e., the efficiency of plating (EOP) was less than 10À4
Our conclusion must mean that a large proportion of nsp1– nsp11 proteins function as a polyprotein, if only initially or transiently, or they associate as a cis-acting complex before they are proteolytically processed
Summary
Coronaviruses are positive-strand, enveloped RNA viruses that infect vertebrates and are associated mainly with respiratory and enteric disease. They have long been recognized as important pathogens of livestock and companion animals, and they are a common cause of respiratory tract infections in humans [1,2,3]. The genomic RNA encodes the structural proteins of the virus, non-structural proteins involved in viral RNA synthesis (the nsp or replicase proteins), and proteins that are non-essential for replication in cell culture but appear to confer a selective advantage in vivo (accessory proteins) [1].
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