Mutational analysis of Phe160 within the "palm" subdomain of human immunodeficiency virus type 1 reverse transcriptase

The highly conserved Phe160 residue is located in the "palm" subdomain of h uman immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT), and makes contact with Tyr115, a residue which is involved in deoxynucleoside t riphosphate (dNTP) binding and fidelity of DNA synthesis. Five mutant RTs h aving Tyr, Trp, lie, Ala or Gin instead of Phe160 were obtained by site-dir ected mutagenesis. F160Y and F160W retained substantial DNA polymerase acti vity, whereas the catalytic efficiency of nucleotide incorporation of mutan ts F160I, F160A and F160Q was less than 10% that of the wild-type RT, using poly(rA).oligo(dT)(20) as the template-primer. The low catalytic efficienc y of mutants F160I, F160A and F160Q was due to their lower affinity for the dNTP substrate. F160Y displayed similar kinetic parameters as the wild-typ e RT in nucleotide insertion assays carried out with heteropolymeric DNA/DN A template-primers. However, nucleotide affinity was two- to sixfold reduce d in the case of mutant F160W. Fidelity assays revealed similar misinsertio n and mispair extension ratios for the three enzymes, although F160W showed a slightly higher accuracy of DNA synthesis, particularly in the presence of high concentrations of dNTP. When introduced in an infectious proviral c lone, mutations F160I, F160A and F160Q rendered non-viable virus. The impor tance of Phe160 for polymerase function and viral replication could be medi ated by its interaction with Tyr115, as suggested by the analysis of the av ailable crystal structures of HIV-1 RT. (C) 1999 Academic Press.