Determination of mutation rates of RNA viruses and characterization of the receptor-destroying enzyme of bovine coronavirus.
Item
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Title
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Determination of mutation rates of RNA viruses and characterization of the receptor-destroying enzyme of bovine coronavirus.
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Identifier
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AAI9009755
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identifier
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9009755
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Creator
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Leider, Jason Mark.
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Contributor
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Adviser: Peter Palese
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Date
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1989
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, Microbiology | Biology, Molecular
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Abstract
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The extreme variability of some RNA-containing animal viruses is an important factor which limits the effectiveness of vaccine development against viral infection. For this thesis, two studies were performed which led to the determination of the mutation rates of three RNA viruses. First, the mutation rates of influenza A/WSN/33 virus and the Mahoney strain of poliovirus type 1 were measured. The rate of mutation for the nonstructural (NS) gene of influenza A virus and for the VP1 gene in poliovirus type 1 was assayed by direct sequence analysis. Each gene was repeatedly sequenced in over 100 viral clones which were descended from a single virion in one plaque generation. We obtained values of 1.5 {dollar}\times{dollar} 10{dollar}\sp{lcub}-5{rcub}{dollar} and less than 2.1 {dollar}\times{dollar} 10{dollar}\sp{lcub}-6{rcub}{dollar} mutations per nucleotide per infectious cycle for the mutation rates of influenza A virus and poliovirus, respectively.;In our second study, the newly developed technology of denaturing gradient gel analysis was applied in order to determine the mutation rate of the retrovirus Rous sarcoma virus (RSV). Progeny descended from a single virion were collected after one replication cycle, and seven regions of the genome were analyzed for mutations by denaturing gradient gel electrophoresis. In all, 65,250 nucleotides were screened, yielding nine mutations, and the RSV mutation rate was calculated as 1.4 {dollar}\times{dollar} 10{dollar}\sp{lcub}-4{rcub}{dollar} mutations per nucleotide per replication cycle. These results indicate that RSV is an extremely mutable virus.;Extensive virus variability, as described in these first two studies, is in stark contrast to the need of a virus to preserve a conserved pocket at its receptor-binding site and the active sites of any enzymes it may contain (such as polymerases and receptor-destroying enzymes). In our third project, we described the activity of the receptor-destroying enzyme of bovine coronavirus (BCV), and the effect on viral replication following its inhibition. We identified the E3 protein of BCV as possessing serine esterase activity. Furthermore, treatment of BCV with the serine esterase inhibitor diisopropylfluorophosphate dramatically reduced its infectivity in a plaque assay. It is assumed that the esterase activity of BCV is required in an early step of virus replication, possibly during virus entry or uncoating.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
