Effects of the K65R and K65R/M184V reverse transcriptase mutations in subtype C HIV on enzyme function and drug resistance.

BACKGROUND: We investigated the effects of mutations K65R and K65R plus M184V on enzymatic function and mechanisms of drug resistance in subtype C reverse transcriptase (RT). METHODS: Recombinant subtype C HIV-1 RTs containing K65R or K65R+M184V were purified from Escherichia coli. Enzyme activities and tenofovir (TFV) incorporation efficiency by wild-type (WT) and mutant RTs of both subtypes were determined in cell-free assays. Efficiency of (-) ssDNA synthesis and initiation by subtype C RTs was measured using gel-based assays with HIV-1 PBS RNA template and tRNA3Lys as primer. Single-cycle processivity was assayed under variable dNTP concentrations. Steady-state analysis was performed to measure the relative inhibitory capacity (ki/km) of TFV-disphosphate (TFV-DP). ATP-dependent excision and rescue of TFV-or ZDV-terminated DNA synthesis was monitored in time-course experiments. RESULTS: The efficiency of tRNA-primed (-)ssDNA synthesis by subtype C RTs was: WT > K65R > K65R+M184V RT. At low dNTP concentration, K65R RT exhibited lower activity in single-cycle processivity assays while the K65R+M184V mutant showed diminished processivity independent of dNTP concentration. ATP-mediated excision of TFV-or ZDV-terminated primer was decreased for K65R and for K65R+M184V RT compared to WT RT. K65R and K65R+M184V displayed 9.8-and 5-fold increases in IC50 for TFV-DP compared to WT RT. The Ki/Km of TFV was increased by 4.1-and 7.2-fold, respectively, for K65R and K65R+M184V compared to WT RT. CONCLUSION: The diminished initiation efficiency of K65R-containing RTs at low dNTP concentrations have been confirmed for subtype C as well as subtype B. Despite decreased excision, this decreased binding/incorporation results in diminished susceptibility of K65R and K65R+M184 RT to TFV-DP.

Kinetics of inhibition of HIV type 1 reverse transcriptase-bearing NRTI-associated mutations by apricitabine triphosphate.

We wished to investigate the effects of various mutations in HIV-1 reverse transcriptase (RT) on biochemical inhibition by the active form of a novel nucleoside termed apricitabine. Accordingly, we studied the efficiency of chain-termination mediated by apricitabine triphosphate (TP) in cell-free assays that used either recombinant wild-type or mutated RTs. We also performed steady-state-kinetics and primer-unblocking assays. Subtype C RTs were also analysed. The results showed that the K65R mutation in RT caused reductions in the efficiency of chain-termination of apricitabine-TP by increasing its Ki. However, K65R did not affect rates of primer unblocking for apricitabine-TP. No significant differences were found between subtype C and subtype B RTs with regard to any of the parameters studied. Other mutations such as M184V, L74V and K103N had no effect on the efficiency of chain termination by apricitabine-TP. Thus, the mechanism of reduced susceptibility to apricitabine of viruses containing K65R in RT seems to be mediated exclusively through a reduction in binding or incorporation of apricitabine-TP. Unlike some other nucleoside analogues, increased excision of incorporated apricitabine does not seem to be a cause of resistance to apricitabine.

Arginine methylation of the HIV-1 nucleocapsid protein results in its diminished function.

OBJECTIVE: The HIV-1 nucleocapsid protein (NC) is involved in transfer RNA3 annealing to the primer binding site of viral genomic RNA by means of two basic regions that are similar to the N-terminal portion of the arginine-rich motif (ARM) of Tat. As Tat is known to be asymmetrically arginine dimethylated by protein arginine methyltransferase 6 (PRMT6) in its ARM, we investigated whether NC could also act as a substrate for this enzyme. METHODS: Arginine methylation of NC was demonstrated in vitro and in vivo, and sites of methylation were determined by mutational analysis. The impact of the arginine methylation of NC was measured in RNA annealing and reverse transcription initiation assays. An arginine methyltransferase inhibitor (AMI)3.4 was tested for its effects on viral infectivity and replication in vivo. RESULTS: NC is a substrate for PRMT6 both in vitro and in vivo. NC possesses arginine dimethylation sites in each of its two basic regions at positions R10 and R32, and methylated NC was less able than wild-type to promote RNA annealing and participate in the initiation of reverse transcription. Exposure of HIV-1-infected MT2 and primary cord blood mononuclear cells to AMI3.4 led to increased viral replication, whereas viral infectivity was not significantly affected in multinuclear-activation galactosidase indicator assays. CONCLUSION: NC is an in-vivo target of PRMT6, and arginine methylation of NC reduces RNA annealing and the initiation of reverse transcription. These findings may lead to ways of driving HIV-infected cells out of latency with drugs that inhibit PRMT6.

Diminished efficiency of HIV-1 reverse transcriptase containing the K65R and M184V drug resistance mutations.

OBJECTIVES: To determine the underlying biochemical mechanisms responsible for the diminished viral replicative capacity associated with K65R/M184V-containing viruses. METHODS: We studied the efficiency of (-)ssDNA synthesis by recombinant wild-type and mutated HIV-1 reverse transcriptases in cell-free assays. In addition, we determined susceptibility levels to nucleoside analog reverse transcriptase inhibitors (NRTIs) both in cell-free and cell culture assays. RESULTS: We observed that the K65R/M184V mutations in reverse transcriptase caused reductions in the efficiency of initiation of (-)ssDNA synthesis by increasing pausing at positions +3 and +5 as well as diminished RNA usage. These findings were confirmed in cell culture data using MT-4 cells and cord blood mononuclear cells. CONCLUSIONS: The simultaneous presence of K65R and M184V in reverse transcriptase has a negative impact with regard to the efficiency of initiation of (-)ssDNA synthesis and RNA usage, that exceeds the effect of either mutation on its own. These mechanisms, among others, are responsible for the diminished viral replicative capacity observed in tissue culture when K65R/M184V-containing viruses are studied.

Impaired rescue of chain-terminated DNA synthesis associated with the L74V mutation in human immunodeficiency virus type 1 reverse transcriptase.

The L74V and M184V mutations in the reverse transcriptase (RT) gene of human immunodeficiency virus type 1 (HIV-1) are frequently associated with resistance to the nucleoside reverse transcriptase inhibitors abacavir, didanosine, and lamivudine. Yet viruses containing any of these mutations often display hypersusceptibility to zidovudine (ZDV). Two distinct mechanisms have been described to explain HIV-1 drug resistance. One of these involves diminished rates of incorporation of the nucleotide analogue by mutated RT, while the other mechanism involves increased rates of phosphorolytic excision of the drug-terminated primer. To understand the biochemical mechanisms responsible for the hypersensitization of L74V-containing viruses to ZDV, we studied the efficiency of excision of ZDV-monophosphate (ZDV-MP)-terminated primers by recombinant wild-type and mutated HIV-1 RTs in cell-free assays. We observed that the L74V mutation in RT caused reductions in ATP-dependent removal of ZDV-MP from newly synthesized viral DNA. In addition, we determined that the L74V and M184V mutations did not affect the ratio between the populations of RT-DNA/DNA complexes found at pre- and posttranslocational stages; however, they might have affected proper alignment between incorporated chain terminator and pyrophosphate donor, substrate orientation, affinity for ATP, and/or primer-template substrate. Finally, we confirmed previous findings that L74V-containing viruses display diminished replication capacity and that this is associated with reduced levels of synthesis of early reverse-transcribed viral DNA molecules.