Background:  Mutations in the polymerase domain of HIV-1 reverse transcriptase (RT) that confer non-nucleoside RT inhibitor (NNRTI) resistance reduce ribonuclease H (RNase H) activity. Little is know, however about the effects on RNase H activity of nucleoside RT inhibitor (NRTI) resistance mutations.

Methods:  Purified RT heterodimer was prepared by expressing cloned p66 and p51RT subunits of wild type (18A) and zidovudine (AZT)-resistant (18C) isolates. The 184V mutation for lamivudine (3TC) resistance was introduced into the 18A and 18C RT by site-directed mutagenesis. In addition, thymidine analog resistance mutations (TAM:  41L, 67N, 70R, 210W, 215Y, 219Q) and the 184V mutation were introduced into p51 and p66 subunits of HIV-1 RT from Hxb2. The DNA 3’-end-directed and RNA 5’-end-directed RNase H cleavage activity of purified recombinant RTs was characterized using RNA·DNA hybrid substrates.

Results:  Introduction of 184V into the 18A RT substantially reduced the overall rate of both DNA 3’-end-directed and RNA 5’-end-directed RNase H cleavage relative to the wild type (18A) enzyme. However, 184V appeared selectively to enhance the secondary cleavage step. The 18C RT (41L/67N/70R/215Y/219Q) showed a moderate reduction in RNase H activity compared to 18A. RNase H activity of the 18C-184V RT was similar to 18C. Likewise, introduction of 184V into an Hxb2 RT substantially reduced 3’-end- and 5’-end-directed RNase H cleavage relative to WT Hxb2 RT. However, introduction of TAM-1 pattern mutations (41L, 210W, 215Y) did not substantially alter the 5’-end-directed cleavage rate.

Conclusions:  The 184V mutation had a marked effect on RNase H function, reducing rates and modifying kinetics during both DNA 3’-end- and RNA 5’-end directed cleavages. The effect of TAM on RNase H activity depended on the specific combination of mutations and the genetic backbone of the RT. These findings provide evidence that NRTI-resistance mutations, like NNRTI-resistance mutations, can alter RNase H function.