Role of the potyvirus P1 protein in polyprotein processing and viral infectivity /
Tobacco etch polyvirus (TEV) is a positive strand RNA virus
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| Format: | Thesis Book |
| Language: | English |
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[Place of publication not identified] :
[publisher not identified] ;
1995.
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| Subjects: | |
| Online Access: | http://proxy.library.tamu.edu/login?url=http://proquest.umi.com/pqdweb?did=742145691&sid=1&Fmt=2&clientId=2945&RQT=309&VName=PQD |
| Summary: | Tobacco etch polyvirus (TEV) is a positive strand RNA virus and has a genome encoding a large polyprotein that is proteolytically processed by three viral proteinases. In this study, the PI protein, located near the N-terminus of the viral polyprotein, was identified as one of the TEV proteinases, which autocatalytically cleaves a site between itself and the helper component proteinase (HC-Pro). Mutational analysis was conducted to identify sequences within the PI protein required for proteolysis. A C-terminal proteolytic domain was identified containing three amino acid residues, His2l4, ASP223, and Ser256, that are strictly conserved among potyviruses and resemble the active site catalytic triad of chymotrypsin-like proteinases. A factor present in plant cell extracts was also required for separation of PI and HC-Pro. Mutations debilitating PI autoproteolysis inhibited virus infectivity in plants, indicating that either PI proteinase activity or separation of P 1 and HC-Pro was required. These data also suggested that additional activities provided by PI and/or HC-Pro, may require separation of these two proteins. To determine if PI proteinase activity was required and if PI was a multi-functional protein, a variety of mutations were introduced into the PI coding region of the TEV genome. Elimination of the N-terminal 157 amino acid residues had little effect on virus accumulation in tobacco protoplasts, however, deletion of the entire PI protein severely reduced virus accumulation in protoplasts. Accumulation of PI- defective virus was enhanced in transgenic cells expressing PI/HCPro polyproteins. Only mutations within the C-terminal proteolytic domain that were inhibitory to proteolysis were inhibitory to virus accumulation in plants. Suffogate cleavage sites recognized by the TEV NIa proteinase were inserted into mutant TEV genomes at positions resulting in NIa-mediated proteolysis between PI and HC-Pro, and were able to restore infectivity to these PI-processing defective viruses. Only mutations eliminating or altering sequences within the C-terminal half of PI were inhibitory to virus accumulation in isolated cells or whole plants indicating that the proteolytic domain of PI may contribute to virus replication beyond its role in proteolysis. |
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| Item Description: | Vita. "Major Subject: Microbiology". In title, numerals are used. |
| Physical Description: | x, 124 leaves : illustrations ; 28 cm. Issued also on microfiche from University Microfilms Inc. |
| Bibliography: | Includes bibliographical references. |