Graduate Thesis Or Dissertation
 

Host cell nuclear involvement in vaccinia virus replication

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  • It was previously shown that vaccinia virus is unable to complete its replicative cycle in cells that have been subjected to cytochalasin-B mediated enucleation prior to infection. Likewise, in the presence of the drug α-amanitin, a potent inhibitor of host RNA polymerase II but not of viral transcription, the replication of vaccinia virus is inhibited by approximately 95%. These findings led to the conclusion that active participation of the host cell transcriptional apparatus is required for the production of infectious vaccinia virus progeny. In order to identify the viral gene(s) that interacts with the host cell transcriptional apparatus during viral replication, I isolated and characterized a vaccinia virus mutant (α-27) capable of replicating in the presence of the drug α-amanitin. A biochemical analysis of the replication of α-27 versus wild-type vaccinia virus in the presence and absence of the drug revealed no differences with respect to DNA synthesis or viral protein synthesis. However, a marked difference was observed in the ability of the two viruses to direct the proteolytic processing of the two major core precursor polypeptides, P94 and P65, in the presence of the drug. The processing reaction was completely blocked by α-amanitin in wild-type vaccinia virus-infected cells, but proceeded normally in α-27-infected cells. In an attempt to map the mutation responsible for α-amanitin resistance, an α-amanitin-resistant temperature sensitive vaccinia XT virus mutant (α[superscript r]ts7), in which both phenotypes were the result of one or two very closely linked mutations, was isolated. Marker rescue experiments using the cloned Hindlll DNA fragments from wild-type vaccinia virus, mapped the a-amanitin-resistant mutation to the 1.5Kb Hindlll-N fragment. As a preliminary step toward determining the nature and number of the gene(s) encoded within the Hindlll-N fragment, I sequenced this region of the genome using the Sanger dideoxynucleotide chain termination method. The sequencing data showed that there are two open reading frames in this area of the genome. One of the open reading frames is translated into a 20K polypeptide while the second one is translated into a 48K polypeptide.
  • Note: Figures in original document are black and white photocopies. Best scan available.
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file details VillarrealElciraC1987.pdf 2017-11-06 公开 下载