Date of Award
1984
Degree Type
Dissertation
Degree Name
Doctor of Philosophy
Abstract
L-2 cells infected with murine coronavirus (MHV) strains MHV-3 and JHM shut off host cell protein synthesis and synthesized polypeptides with apparent molecular weights 180,000 (p180), 56,000 (p56), 24,000, (p24) and 22,000 (p22) after a 15 minute pulse with {lcub}('35)S{rcub}-methionine. During a 2 hour chase period performed late in infection (5.5 h PI) a fifth polypeptide of molecular weight 50,000 (p50) was detected. The corresponding p56 and p50 polypeptides of the two viruses were enriched in the basic amino acid arginine. Pulse-chase and peptide mapping experiments confirmed a precursor-product relationship between the p56 and p50 proteins. MHV-3 virions, however, showed no trace of the p50 protein. Moreover, of the total five detected polypeptides only three, p22, p56 and p180 were labeled in MHV-3-infected cells during a short two minute pulse with {lcub}('35)S{rcub}-methionine and, are therefore, presumed to be immediate translation products. Pulse-chase and peptide mapping experiments confirmed that p24 is derived from p22 by post-translational modification. Further analyses with the glycosylation inhibitor tunicamycin provided strong evidence that conversion of p22 to p24 apparently does not involve N-linked glycosylation.;Analysis of {lcub}('35)S{rcub}-methionine-labeled viral proteins by SDS polyacrylamide gel electrophoresis on five different murine hepatitis strains: A59, JHM, MHV-1, MHV-3, and MHV-S revealed that the proteins of each strain of MHV have very similar molecular weights. The strong degree of interstrain homology was confirmed by comparative HPLC peptide mapping of the viral nucleocapsid proteins.;Concurrent with the study of MHV polypeptides, intracellular viral-specific RNAs were also examined by using {lcub}('32)P{rcub}-complementary DNA prepared against the A59 nucleocapsid-encoding mRNA in a Northern Transfer procedure as well as by in vitro labeling of MHV-infected cultures in the presence of actinomycin D and subsequent analyses in DMSO-urea agarose gels. The results demonstrate that MHV-infected cells generate six overlapping, polyadenylated, subgenomic RNAs all of which share common sequences and thus provide evidence for a nested set arrangement of coronavirus mRNAs. Hence coronavirus replication differs from that of other well characterized single-stranded, plus-sense RNA animal viruses such as that of the picornaviruses and alphaviruses.
Recommended Citation
Cheley, Stephen Benet, "Murine Hepatitis Virus (mhv) Replication: A Molecular Study" (1984). Digitized Theses. 1366.
https://ir.lib.uwo.ca/digitizedtheses/1366