Functional and Genetic Analysis of Coronavirus Replicase-Transcriptase Proteins

PLoS Pathogens

Stuart G Siddell¹  Helen Stokes¹  Dorothea L Sawicki²  Stanley G Sawicki²  Diane Younker²  Volker Thiel³  Yvonne Meyer³ 

[1] Department of Cellular and Molecular Medicine, University of Bristol, Bristol, United Kingdom;Department of Medical Microbiology and Immunology, Medical University of Ohio, Toledo, Ohio, United States of America;Institute of Virology, University of Würzburg, Würzburg, Germany

关键词: RNA synthesis;    Phenotypes;    Proteases;    Viral replication;    RNA structure;    Messenger RNA;    RNA viruses;    RNA annealing;   

DOI  :  10.1371/journal.ppat.0010039

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY