Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins

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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. Coronaviruses infect both humans and animals and are associated mainly with respiratory and enteric diseases. The recent outbreak of SARS emphasizes the need to develop new strategies to control these infections. This paper focuses on the proteins involved in the replication of the coronavirus genome and the production of viral mRNAs in the host cell. These so-called replicase-transcriptase proteins are likely to make good targets for the development of anti-coronaviral drugs. The approach used here is to analyze conditional, temperature-sensitive mutants of Murine hepatitis virus that are normal at 33 °C (the permissive temperature) but are unable to replicate and transcribe viral RNAs at 39.5 °C (the restrictive temperature). By identifying the genetic changes responsible for these temperature-sensitive mutations and by analyzing the precise nature of the defect in RNA synthesis at the restrictive temperature, the authors are able to propose a model that describes a pathway for viral RNA synthesis in the infected cell. Further analysis of these mutants should allow the elucidation of the precise function(s) of the viral proteins involved.

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