ALANINE-SCANNING MUTATIONS IN THE PRIMER GRIP OF P66 HTV-1 REVERSE-TRANSCRIPTASE RESULT IN SELECTIVE LOSS OF RNA PRIMING ACTIVITY

Alanine-scanning mutants of the primer grip region of human immunodefi ciency virus type 1 reverse transcriptase were tested for their abilit y to extend RNA and DNA versions of the polypurine tract primer, and a n oligonucleotide representing the 18-nucleotide sequence at the 3' en d of tRNA(Lys3). A majority of the mutant enzymes were either complete ly or severely deficient in RNA priming activity, but, with only one e xception, were able to efficiently extend DNA versions of the same pri mers, The mutant enzymes were able to bind to RNA primers, indicating that the defect in RNA priming was not simply a loss of binding activi ty, Mutations at positions 229, 233, and 235 dramatically reduced the amount of specific RNase H cleavage at the 3' terminus of the polypuri ne tract, which is required for primer removal, An alanine substitutio n at position 232 led to loss of cleavage specificity, although total activity was close to the wild-type level, Taken together, these resul ts demonstrate for the first time that there are residues in human imm unodeficiency virus type 1 reverse transcriptase which are specificall y involved in protein-nucleic acid interactions with RNA primers.