The K65R mutation confers increased DNA polymerase processivity to HIV-1 reverse transcriptase.
The K65R mutation in HIV-1 reverse transcriptase (RT) is associated with viral cross-resistance to 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, and 2',3'-dideoxy-3'-thiacytidine. We have found that in vitro DNA synthesis by K65R RT is significantly more processive than that of wild type (wt) RT. Depending on the template/primer (T/P) used, the total incorporation of nucleotides under single processive cycle conditions was 20-50% higher with K65R RT than with wt RT. With heteropolymeric T/P, the total incorporation of dNMP by K65R and wt RT was similar under continuous DNA synthesis reaction conditions. However, under single processive cycle conditions, the rate of full-length polymerization product synthesis by K65R RT was about 2-fold higher than that by wt RT. We also found a decreased rate of T/P dissociation during K65R RT DNA synthesis, which is consistent with the increased processivity of the enzyme. We postulate that the increased processivity of the K65R RT may be a compensatory response to the decreased affinity of this mutant for certain dNTP substrates, allowing normal viral replication kinetics.
Arion
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Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote Ste-Catherine Road, Montreal, Quebec, H3T 1E2 Canada.
Borkow
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Gu
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Wainberg
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Parniak
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MA
D004247:DNA / Q000096:biosynthesis; D004351:Drug Resistance; D054303:HIV Reverse Transcriptase; D017354:Point Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013698:Templates, Genetic
D011994:Recombinant Proteins; D004247:DNA; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase