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|Title:||Studies on Rous sarcoma virus reverse transcriptase biogenesis|
|Advisor:||Ghosh, Hara P.|
|Abstract:||<p>The purpose of this thesis was to investigate the biosynthesis and function of reverse transcriptase in relation to the assembly and maturation of RNA tumor viruses. The assembly and maturation of RNA tumor viruses were examined by biochemical characterization of a conditional lethal mutant of Rous sarcoma virus (RSV). The functional aspects of reverse transcriptase was investigated by recombinant DNA methods. A temperature-sensitive coordinate mutant tsLA83 of Prague B (PR-B) strain of RSV at the nonpermissive temperature (41℃) produced noninfectious virus particles (NI-LA83) which contained only 3% of the reverse transcriptase activity present in infectious virions. Analyses of [³⁵S] methionine labeled NI-LA83 showed the presence of all of the viral proteins except revere transcriptase. Pulse-chase analyses of the virus specified proteins in cells infected with LA83 or PR-B showed that the gag and glycoprotein precursors, Pr76ᵍᵃᵍ and gPr92ᵉⁿᵛ, respectively, were processed at both 35℃ and 41℃. The reverse transcriptase precursor, Pr180ᵍᵃᵍ⁻ᵖᵒˡ, however, was not processed in La83-infected cells at 41℃. In contrast, cells infected with LA83 or PR-B at 35℃ as well as with PR-B at 41℃ showed normal cleavage of Pr180ᵍᵃᵍ⁻ᵖᵒˡ. A shiftdown of LA83-infected cells at 41℃ to the permissive temperature, 35℃, resulted in the normal processing of Pr180ᵍᵃᵍ⁻ᵖᵒˡ and production of infectious virus containing reverse transcriptase. Electron microscopic analysis showed that at 41℃ cells infected with LA83 showed a large number of budding structures but fewer released particles. A shiftdown from 41 to 35℃ resulted in an increase of virus particles with a concomitant decrease in budding structures suggesting that the processing of reverse transcriptase precursor is related to virion assembly. The mechanism of synthesis of reverse transcriptase was investigated. Immunoprecipitation experiments have revealed possible intermediates in Pr180ᵍᵃᵍ⁻ᵖᵒˡ processing. These are 130,000- and 150,000-dialton intracellular proteins (Pr130ᵍᵃᵍ⁻ᵖᵒˡ and Pr150ᵍᵃᵍ⁻ᵖᵒˡ) that contain antigenic determinants of reverse transcriptase and gag proteins. Pr130ᵍᵃᵍ⁻ᵖᵒˡ contains antigenic determinants of reverse-transcriptase and p15, Pr150ᵍᵃᵍ⁻ᵖᵒˡ contains all the gag-proteins, but not the complete sequence of reverse transcriptase. Immuno-complexes of Pr180ᵍᵃᵍ⁻ᵖᵒˡ molecules with anti-reverse transcriptase antiserum were tested for in-vitro cleavage in the presence of detergent-disrupted virus (a source of p15, the viral protease). The results identified two possible cleavage intermediates (Pr130 and Pr70). A precursor-product relationship has been demonstrated between Pr180ᵍᵃᵍ⁻ᵖᵒˡ and Pr130ᵍᵃᵍ⁻ᵖᵒˡ, although a direct precursor product relationship could not be demonstrated between Pr180ᵍᵃᵍ⁻ᵖᵒˡ and Pr150ᵍᵃᵍ⁻ᵖᵒˡ. A possible biosynthetic scheme of reverse transcriptase was proposed. In an attempt to establish the structural and functional relationships of the reverse transcriptase molecule, site directed mutagenesis on molecularly cloned RSV proviral DNA was undertaken: Expression of the reverse transcriptase gene in both bacteria and avian cell cultures was studied. E. coli harboring the recombinant plasmids containing proviral DNAs from different strains of RSV synthesized a protein of 65,000 molecular weight (p65) which is immunoprecipitated with anti-reverse transcriptase antiserum. The RNA dependent-DNA polymerase activities from E.coli containing the recombinant plasmids were about 30-fold higher than the background activity present in E.coli containing pBR322. The RNA dependent-DNA polymerase activity was specifically neutralized by the antibody against the reverse transcriptase. A possible translational initiation site was suggested to be an internal methionine residue, which is located at residue number 295 or 297 from the amino-terminus of the mature reverse transcriptase molecule. The amino-terminal one-third of the reverse-transcriptase molecule thus appears to be dispensable for the polymerase activity. The presence of a promoter-like sequence in the reverse transcriptase gene which is involved in the transcription of the polymerase gene for synthesis of reverse transcriptase activity in E.coli has also been suggested. Biological activity of the site directed mutagenized viral DNAs were tested by transfection into chick embryofibroblast cells. Deletion near the 5-end region of the pol gene resulted in inefficient production of virus due to possible functional defect in the truncated reverse transcriptase molecules of inefficient processing of the precursor molecules. However, the cells were transformed. Deletion near the 3-end region of the pol gene resulted in inefficient production of virus and the cells were not transformed. These results suggest that the pp32 DNA binding protein, which is encoded from the 3-end region of the pol gene, or the carboxy terminal moiety of the β subunit, or both, are required in the life cycle of avian retroviruses.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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