Identification of a domain required for autoproteolytic cleavage of murine coronavirus gene A polyprotein.
The 5'-most gene of the murine coronavirus genome, gene A, is presumed to encode viral RNA-dependent RNA polymerase. It has previously been shown that the N-terminal portion of this gene product is cleaved into a protein of 28 kilodaltons (p28). To further understand the mechanism of synthesis of the p28 protein, cDNA clones representing the 5'-most 5.3 kilobases of murine coronavirus mouse hepatitis virus strain JHM were sequenced and subcloned into pT7 vectors from which RNAs were transcribed and translated in vitro. The sequence was found to encode a single long open reading frame continuing from near the 5' terminus of the genome. Although p28 is encoded from the first 1 kilobase at the 5' end of the genome, translation of in vitro-transcribed RNAs indicated that this protein was not detected unless the product of the entire 5.3-kilobase region was synthesized. Translation of RNAs of 3.9 kilobases or smaller yielded proteins which contained the p28 sequence, but p28 was not cleaved. This suggests that the sequence in the region between 3.9 and 5.3 kilobases from the 5' end of the genomic RNA is essential for proteolytic cleavage and contains autoproteolytic activity. The p28 protein could not be cleaved from the smaller primary translation products of gene A, even in the presence of the larger autocleaving protein. Cleavage of the p28 protein was inhibited by addition of the protease inhibitor ZnCl2. This study thus identified a protein domain essential for autoproteolytic cleavage of the gene A polyprotein.
Baker
S C
SC
Department of Microbiology, School of Medicine, University of Southern California, Los Angeles 90033.
Shieh
C K
CK
Soe
L H
LH
Chang
M F
MF
Vannier
D M
DM
Lai
M M
MM
D000818:Animals; D001483:Base Sequence; D004279:DNA, Viral / Q000032:analysis; D012321:DNA-Directed RNA Polymerases / Q000032:analysis / Q000235:genetics; D005814:Genes, Viral; D051379:Mice; D008969:Molecular Sequence Data; D006517:Murine hepatitis virus / Q000235:genetics*; D010447:Peptide Hydrolases / Q000032:analysis*; D014176:Protein Biosynthesis; D014764:Viral Proteins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism
D004279:DNA, Viral; D014764:Viral Proteins; D012321:DNA-Directed RNA Polymerases; D010447:Peptide Hydrolases