IDENTIFICATION OF THE NUCLEOTIDE-BINDING SITE OF HIV-1 REVERSE-TRANSCRIPTASE USING DTTP AS A PHOTOAFFINITY LABEL

We have utilized UV-induced cross-linking of [methyl-H-3]dTTP to ident ify the nucleotide binding site on heterodimeric HIV-1 reverse transcr iptase (RT). RT was derivatized by irradiating a solution containing [ methyl-H-3]dTTP and purified recombinant RT for 10 min. The UV-induced cross-linking reaction between dTTP and RT is linear with time of UV exposure up to 10 min, and it has been determined previously that dTTP cross-linking is half-maximal at 90 muM [Cheng, N., Painter, G. R., & Furmann, P.A. (1991) Biochem. Biophys. Res. Commun. 174, 785-789]. Un der these reaction conditions, only the 66-kDa subunit of the 66-kDa/5 1-kDa RT heterodimer was labeled with dTTP. The [methyl-H-3]dTTP-label ed RT was fragmented with trypsin and endoproteinase Asp-N, and peptid es were purified on reversed phase HPLC. The peptide covalently linked to [methyl-H-3]dTTP was subjected to amino acid sequence analysis. Th e sequencing data localized the nucleotide binding site of RT to Lys-7 3 in the vicinity of several mutation sites linked to antiviral drug r esistance. Since most effective anti-AIDS compounds are inhibitors of RT, information about its dNTP binding site may make it possible to un derstand the basis for the antiviral activity of nucleoside analogs su ch as AZT, ddI, and ddC. This information may also be useful for a mor e rationally based design of anti-HIV agents.