CLONING AND EXPRESSION OF A HUMAN T-LYMPHOTROPIC VIRUS TYPE-1 PROTEINWITH REVERSE-TRANSCRIPTASE ACTIVITY

Unlike most other characterized retroviruses, there is little publishe d information on the biochemical properties of human T-lymphotropic vi rus type 1 (HTLV-1) reverse transcriptase (RT). Specifically, no repor ts of a cloned functional RT enzyme have been published. Since the RT enzyme is an essential component of the virus, our objective was to cl one, express, and purify a functional RT enzyme from HTLV-1. Our appro ach was to clone and express a protein of approximately 60 to 65 kDa t hat we hypothesized would correspond to the RT region encoded by the p ol reading frame. The predicted region encoding the RT enzyme comprise d nucleotides 2617 to 4312 of the HTLV-1 MT-2 isolate. A putative RT g ene was obtained by PCR and was ligated into various prokaryotic expre ssion vectors. A novel cloning approach allowed us to generate a stabl e clone in the prokaryotic expression vector pGEX-4T-1 and produce a r ecombinant protein of approximately 60 to 65 kDa. The partially purifi ed protein displays RT activity in both amplification RT (AMP-RT) assa ys and traditional RT assays. This is the first report of a cloned pro tein from HTLV-1 which displays RT activity and is the first step in t he characterization of HTLV-1 RT.