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Comparative studies of recombinant reverse transcriptases from HIV-2 and SIV.
Somers D; Rud E; Clarke B; Kirby I; Stammers DK; Wellcome Research
December 30, 1992
Int Conf AIDS. 1992 Jul 19-24;8(3):38 (abstract no. PuA 6166). Unique

OBJECTIVE: Production of HIV-2 and SIV RTs for comparative biochemical and structural analysis. METHODS: The coding sequences for HIV-2 and SIV RTs were obtained by PCR from genomic clones pROD35#2 and SIVmac32H(pj5) respectively. The 3' PCR primer incorporated sequences to mutate the C-termini to GLU-GLU-PHE. This sequence is recognised by a Mab (YL1/2) which was used in immobilised form to purify these RTs expressed in E.coli. Purified RTs were characterised both kinetically as well as by reaction with antibodies (YL1/2 and an antipeptide antibody to an SIV C-terminal peptide). RESULTS AND CONCLUSIONS: Both HIV-2 and SIV RTs were purified in a single step using the antibody column, and were mainly observed as 68K polypeptides with small amounts of 55K. A C-terminal fragment of apparent Mr 40K was seen for HIV-2 but not SIV RT. The reaction of the 55K form with the SIV peptide antibody indicated the cleavage site for the protease in both HIV-2 and SIV RTs could be further towards the C-terminus than in HIV-1 RT.

Cloning, Molecular Comparative Study HIV-2/*ENZYMOLOGY/GENETICS Molecular Weight Polymerase Chain Reaction Recombinant Proteins/ISOLATION & PURIF/METABOLISM RNA-Directed DNA Polymerase/GENETICS/ISOLATION & PURIF/ *METABOLISM SIV/*ENZYMOLOGY/GENETICS ABSTRACT