Coronavirus replicational strategy
Date Issued
December 1, 1980
Author(s)
Dennis, Douglas Edward
Advisor(s)
David A. Brian
Additional Advisor(s)
Austin Brown
W. Stuart Riggsby
Robert Bryant
Abstract
An enzymatic activity which incorporates (-3H) UMP into acid-precipitable material in the presence of endogenous template was found in the cytoplasm of porcine cells infected with the transmissible gastroenteritis virus of swine. This activity was not found in uninfected control cells, nor was it found in purified virus. The activity was associated with rapidly-sedimenting, particulate structures, suggesting that the enzyme is bound to a membrane-containing replication complex. The membrane association was confirmed by demonstrating detergent-dependent changes in sedimentation behavior. The enzymatic activity required the presence of all three ribonucleoside triphosphates in addition to (-3H)UTP and it was not inhibited by actinomycin D. The heated product was digested by ribonuclease but not by deoxyribonuclease. Mg++ was required for enzymatic activity and its optimal concentration was determined to be approximately 5 mM. These results support the classification of this coronavirus as a positive-stranded RNA virus. In an effort to determine the coronaviral specificity of the in vitro polymerase products, we examine the molecular weight, strandedness, (i.e., whether the RNA is single or double stranded), and polyadenylation of the in vitro polymerase products and compared these to the same properties for intracellular virus-specific RNA. Cells pulse-labeled with (-3H) uridine between 4 and 6 h postinfection, a time when the rate of viral RNA synthesis is maximal, contained five major species of single-stranded, polyadenylated RNA with apparent molecular weights of 6.8-, 3.15-, 1.40-, 0.94-, and 0.66 x 106. Seven minor species of single-stranded, polyadenylated RNA with apparent molecular weights of 6.2-, 1.55-, 1.05-, 0.64-, 0.39-, and 0.24 x 106 were also found. The 6.8 x 106 molecular weight species was indistinguishable from virion genomic RNA by denaturing gel electrophoresis. Intracellular virus-specified species were closely associated with cytoplasmic membrane structures and are probably part of a membrane-associated replication complex. When the membrane-associated complex containing the viral RNA polymerase and endogenous template were used for the in vitro synthesis of RNA, three species of single-stranded RNA were seen, of which the two smaller species appeared to be nonpolyadenylated. These species electrophoretically comigrated with three of the species made in vivo and they had molecular weights of approximately 3.15-, 0.94-, and 0.66 x 106. This coronavirus, therefore, apparently replicates by the transcription and subsequent translation of multiple subgenomic mRNA molecules and the mechanism for regulating the length of at least some of the subgenomic transcripts resides within the replication complex.
Degree
Doctor of Philosophy
Major
Microbiology
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Thesis80b.D358.pdf
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