Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates Tryptophan-229 in template-primer utilization

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Jacques, Pamela S. ; Wöhrl, Birgitta M. ; Ottmann, Michele ; Darlix, Jean-Luc ; Le Grice, Stuart F.J.:
Mutating the "primer grip" of p66 HIV-1 reverse transcriptase implicates Tryptophan-229 in template-primer utilization.
In: The Journal of Biological Chemistry. Bd. 269 (1994) Heft 42 . - S. 26472-26478.
ISSN 1083-351X

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Abstract

"BcgI cassette" mutagenesis was used to prepare variants of p66 human immunodeficiency virus (HIV)-1 reverse transcriptase with amino acid substitutions between residues Glu224 and Trp229. Mutant polypeptides 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 indicate that while alteration of p66 residues Glu224-Leu228 has minimal consequences, the DNA polymerase activities of mutant p66(229A)/p51 are impaired. DNase I footprinting illustrates that this mutant does 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 Trp229 toward architecture of the p66 primer grip.