Translation of MHV virion RNA in a cell-free system produces a large polyprotein that is rapidly cleaved into smaller proteins, a process inhibited by leupeptin and ZnCl2.
MHV virion RNA is translated into a large polyprotein that is subsequently cleaved into smaller functional proteins, contrasting with the minimal proteolytic processing of other MHV proteins.
The first event after infection with mouse hepatitis virus strain A59 (MHV-A59) is presumed to be the synthesis of an RNA-dependent RNA polymerase from the input genomic RNA. The synthesis and processing of this putative polymerase protein was studied in a cell-free translation system utilizing 60S RNA from MHV-A59 virions. The polypeptide products of this reaction included two major species of 220 and 28 kilodaltons. Kinetics experiments indicated that both p220 and p28 appeared after 60 min of incubation and that protein p28 was synthesized initially as the N-terminal portion of a larger precursor protein. When the cell-free translation products were labeled with N-formyl35Smethionyl-tRNAi, p28 was the predominant radioactive product, confirming its N-terminal location within a precursor protein. Translation in the presence of the protease inhibitors leupeptin and ZnCl2 resulted in the disappearance of p28 and p220 and the appearance of a new protein, p250. This product, which approached the maximal size predicted for a protein synthesized from genomic RNA, was not routinely detected in the absence of inhibitors even under conditions which optimized the translation reaction for elongation of proteins. Subsequent chelation of ZnCl2 resulted in the partial cleavage of the precursor protein and the reappearance of p28. One-dimensional peptide mapping with Staphylococcus aureus V-8 protease confirmed the precursor-product relationship of p250 and p28. The results show that MHV virion RNA, like many other viral RNAs, is translated into a large polyprotein, which is cleaved soon after synthesis into smaller, presumably functional proteins. This is in marked contrast to the synthesis of other MHV proteins, in which minimal proteolytic processing occurs.
Denison et al. (Wed,) conducted a other in Mouse hepatitis virus (MHV-A59) infection. Protease inhibitors (leupeptin and ZnCl2) vs. Absence of inhibitors was evaluated on Polypeptide products of cell-free translation. Translation of MHV virion RNA in a cell-free system produces a large polyprotein that is rapidly cleaved into smaller proteins, a process inhibited by leupeptin and ZnCl2.