Cloning and expression of a human T-lymphotropic virus type 1 protein with reverse transcriptase activity.
Unlike most other characterized retroviruses, there is little published information on the biochemical properties of human T-lymphotropic virus type 1 (HTLV-1) reverse transcriptase (RT). Specifically, no reports of a cloned functional RT enzyme have been published. Since the RT enzyme is an essential component of the virus, our objective was to clone, express, and purify a functional RT enzyme from HTLV-1. Our approach was to clone and express a protein of approximately 60 to 65 kDa that we hypothesized would correspond to the RT region encoded by the pol reading frame. The predicted region encoding the RT enzyme comprised nucleotides 2617 to 4312 of the HTLV-1 MT-2 isolate. A putative RT gene was obtained by PCR and was ligated into various prokaryotic expression vectors. A novel cloning approach allowed us to generate a stable clone in the prokaryotic expression vector pGEX-4T-1 and produce a recombinant protein of approximately 60 to 65 kDa. The partially purified protein displays RT activity in both amplification RT (AMP-RT) assays and traditional RT assays. This is the first report of a cloned protein from HTLV-1 which displays RT activity and is the first step in the characterization of HTLV-1 RT.
Owen
S M
SM
HIV and Retrovirus Diseases Branch, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333, USA.
Lal
R B
RB
Ikeda
R A
RA
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D005814:Genes, Viral; D015368:Human T-lymphotropic virus 1 / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D010957:Plasmids; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D014764:Viral Proteins; D012194:RNA-Directed DNA Polymerase