MUTATING THE PRIMER GRIP OF P66 HIV-1 REVERSE-TRANSCRIPTASE IMPLICATES TRYPTOPHAN-229 IN TEMPLATE-PRIMER UTILIZATION

''BcgI cassette'' mutagenesis was used to prepare variants of p66 huma n immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu(224) and Trp(229). Mutant polypept ides were reconstituted in vitro with wild type p51 to generate the '' selectively mutated'' heterodimer series p66(224A)/p51-p66(229A)/p51. Purified enzymes were characterized with respect to dimerization, DNA polymerase, RNase H, and tRNA(Lys-3) binding. The combined analyses in dicate that while alteration of p66 residues Glu(224)-Leu(228) has min imal consequences, the DNA polymerase activities of mutant p66(229A)/p 51 are impaired. DNase I footprinting illustrates that this mutant doe s not form a stable replication complex with a model template-primer. In vivo studies indicate that the equivalent mutation eliminates viral infectivity, suggesting a contribution of Trp(229) toward architectur e of the p66 primer grip.