Molecular cloning of the gene encoding the putative polymerase of mouse hepatitis coronavirus, strain A59

Abstract

Complementary DNA (cDNA) libraries were constructed representing the genome RNA of the coronavirus mouse hepatitis virus, strain A59 (MHV-A59). From these libraries clones were selected to form a linear map across the entire gene A, the putative viral polymerase gene. This gene is approximately 23 kb in length, considerably larger than earlier estimates. Sequence analysis of the 5' terminal region of the genome indicates the presence of the 66-nucleotide leader that is found on all mRNAs. Secondary structure analysis of the 5' terminal region suggests that transcription of leader terminates in the region of nucleotide 66. The sequence of the first 2000 nucleotides is very similar to that reported for the closely related JHM strain of MHV and potentially encodes p28, a basic protein thought to be a component of the viral polymerase (L. Soe, C. K. Shieh, S. Baker, M. F. Chang, and M. M. C. Lai, 1987, J. Virol., 61, 3968-3976). Gene A contains two of the consensus sequences found in intergenic regions. One is adjacent to the 5' leader sequence and the other is upstream from the initiation codon for translation of gene B.

Internal Ribosome Entry in the Coding Region of Murine Hepatitis Virus mRNA 5

“…Mouse hepatitis virus (MHV), a member of the Coronaviridae, has a positive-strand RNA genome of about 31300 nucleotides (Pachuk et al, 1989;Lee et al, 1991 ;Bonilla et al, 1994). In the infected cell, the expression of viral proteins is mediated by the genomic-length RNA, also known as mRNA 1, together with seven subgenomic mRNAs.…”

“…These mRNAs appear to be functionally monocistronic. However, mRNA 1 and the subgenomic mRNA 5 are exceptions (Skinner et al, 1984;Budzilowicz & Weiss, 1987;Pachuk et al, 1989). The unique region of MHV mRNA 1 contains two large ORFs that constitute the coronavirus RNA polymerase locus.…”

Proteolytic cleavage of the murine coronavirus surface glycoprotein is not required for fusion activity

“…The murine coronaviruses are generically called murine hepatitis virus (MHV), although different MHV strains have quite different organ tropisms and cause a variety of different diseases (Wege et al, 1982;Barthold, 1986). The MHV genome is a positive-strand RNA of about 32 kb (Pachuk et al, 1989;Lee et al, 1991) which encodes four major structural proteins: the nucleocapsid protein N (50000 to 60000 Mr), the membrane protein M (23000 to 26000Mr), the haemagglutinin-esterase protein HE (65000 Mr) and the surface protein S (180000 Mr) (Siddell, 1982).…”

Subgenomic Negative-Strand RNA Function during Mouse Hepatitis Virus Infection

“…Importantly, following treatment, previously transcribed negative strands remain in transcriptionally active complexes even in the absence of new negative-strand synthesis. These findings indicate that the subgenomic-length negative strands are the principal templates of positive-strand synthesis during MHV infection.Mouse hepatitis virus (MHV), a coronavirus in the Nidovirales order, contains a ϳ32-kb linear, single-stranded, positivepolarity RNA genome (8,21,28). Upon entry into the cell, the viral genome is transcribed into seven to eight subgenomic mRNAs ranging in size from ϳ1.0 to 32.0 kb (21, 32).…”