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, which 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 several 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 protein spanning the locations of those cleavage sites proposed by others as well as the one identified by our sequence alignment. Cleavage of the recombinant Pro--Pol protein by HTLV-1 protease generated a 5.5-kDa fragment. Analysis of this fragment by capillary LC-MS and MS/MS revealed the N-terminal cleavage site to be between Leu(147)--Pro(148) of the pro ORF. This is the first physical identification of the authentic amino acid sequence of the reverse transcriptase of HTLV-1. The data reported here provides a basis for further investigation of the function and structural aspects of protein-nucleic interaction.
Agbuya
P G
PG
Department of Chemistry and Biochemistry, Old Dominion University, Norfolk, Virginia, USA.
Sherman
N E
NE
Moen
L K
LK
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D015368:Human T-lymphotropic virus 1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014357:Trypsin
D017931:DNA Primers; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D014357:Trypsin