Date of Award
Doctor of Philosophy (PhD)
Laura K. Moen
Mark S. Elliot
Patricia A. Pleban
Howard S. White
Human T-cell leukemia virus 1 (HTLV-1) is a type C human retrovirus which has been the causative agent of Adult T-cell leukemia. Replication of the retrovirus; requires a reverse transcriptase which converts the retroviral RNA into DNA which is later incorporated into the host's genome. Very little is known about the reverse transcriptase of HTLV-1. Researchers have attempted to purify HTLV-1 RT by isolating the enzyme from human cell lines. Because large amounts of protein could not be produced by this isolation method, the reverse transcriptase cannot be fully characterized. In this research, a recombinant protein expressed in E. coli was purified, which has an apparent molecular weight consistent with that from virion-derived reverse transcriptase. This recombinant protein was purified to approximately 90% homogeneity by a two-step chromatographic process on phosphocellulose and sepharose CL-6B.
The reverse transcriptase of HTLV-1 is synthesized as a Gag-Pro-Pol precursor protein and undergoes proteolytic processing during maturation. By using sequence comparisons from a number of retroviral pol genes as well as information about the location of the ribosomal frameshift, the location of the putative coding sequence for the enzyme has been identified. This information and PCR amplification was used to construct a clone, which spans a region of the pro-poljunction of HTLV-1, for overexpression in E. coli. The Pro-Pol protein was processed in vitro with a recombinant HTLV-1 protease to mimic the proteolytic processing of the Gag-Pro-Pol precursor in the virion. SDS-PAGE analysis of the cleavage sample revealed a 5.5 kDa fragment. This processed fragment of the Pro-Pol protein was further treated with trypsin and analyzed by capillary LC-MS and MS/MS. Analysis of the fragment by MS/MS revealed the N-terminal cleavage at Leu157-Pro158 of the pro ORF. These results confirm the authentic amino acid sequence of the reverse transcriptase of HTLV-1 RT. The data reported here provides a basis for further investigation of the functional and structural aspects of protein-nucleic interaction of the enzyme.
Agbuya, Pinky G..
"Characterization of Human T-Cell Leukemia Virus 1 Reverse Transcriptase"
(2000). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/qbyf-zb02