Proteolytic processing of the human T-cell lymphotropic virus 1 reverse transcriptase: Identification of the N-terminal cleavage site by mass spectrometry

Human T-cell lymphotropic virus 1 (HTLV-1) is a type C human retrovirus, wh ich is the causative agent of Adult T-cell Leukemia and other diseases. The reverse transcriptase of HTLV-1 (E.C. 2.7.7.49) is synthesized as part of a Gag-Pro-Pol precursor protein, and the mature Gag, Pro, and Pol proteins, including the reverse transcriptase, are created by proteolytic processing catalyzed by the viral protease. The location of the proteolytic cleavage site, which creates the N-terminus of mature HTLV-1 reverse transcriptase, has not been previously identified. By using sequence comparisons of severa l retroviral polymerases, as well as information about the location of the ribosomal frameshift, we tentatively identified this N-terminal processing site. PCR amplification was used to construct a clone, which spans a region of the pro-pol junction of HTLV-1, to produce a recombinant Pro-Pol protei n spanning the locations of those cleavage sites proposed by others as well as the one identified by our sequence alignment. Cleavage of the recombina nt Pro-Pol protein by HTLV-1 protease generated a 5.5-kDa fragment. Analysi s of this fragment by capillary LC-MS and MS/MS revealed the N-terminal cle avage site to be between Leu(147)-Pro(148) of the pro ORF. This is the firs t physical identification of the authentic amino acid sequence of the rever se transcriptase of HTLV-1. The data reported here provides a basis for fur ther investigation of the function and structural aspects of protein-nuclei c interaction. (C) 2001 Academic Press.