Enhanced impairment of chain elongation by inhibitors of HIV reverse transcriptase in cell-free reactions yielding longer DNA products

Abstract

We have studied the relationship between the length of HIV-1 reverse transcriptase (RT)-mediated nucleotide polymerization and inhibitors of these reactions in cell-free RT assays performed in the presence of either of two dideoxynucleoside triphosphates (ddNTPs), i.e. AZTTP or 3TCTP, or nevirapine, a non-nucleoside RT inhibitor. These reactions employed a heterologous RNA template and three DNA oligonucleotide primers, i.e. pAR, dPR and PA, that yielded distinct full-length products of 65, 192 and 376 nt, respectively, in the absence of inhibitor. We now show that the extent of inhibition of RT activity was greatest with use of the PA primer, which normally yielded the longest reaction product, and that lesser degrees of inhibition were noted in the reactions that generated shorter products. For example, at a concentration of 5 microM AZTTP, the extent of inhibition was 75% with the PA primer but only 40% and <10% when reactions were primed by the dPR and pAR primers, respectively. Similar results were obtained when either a mutated form of HIV RT (i.e. M184V), associated with resistance to 3TC, was tested in the presence of 3TCTP or when RT derived from Moloney murine leukemia virus (M-MuLV) was tested in the presence of AZTTP.

Mechanism of Inhibition of HIV-1 Reverse Transcriptase by 4′-Ethynyl-2-fluoro-2′-deoxyadenosine Triphosphate, a Translocation-defective Reverse Transcriptase Inhibitor

“…Hence, we used a shorter template (T d31 /P d18 (Table 1)), which allowed unambiguous identification of the stopping sites. As expected, the inhibitory potential of EFdA (and other NRTIs) appears lower in these shorter T/P (IC 50 for suppression of full primer extension was 104 nM) in which there are fewer opportunities for incorporation (24). This substrate allows incorporation of dA, ddA, or EFdA at positions 1, 6, and 10 ( Fig.…”

Molecular Impact of the M184V Mutation in Human Immunodeficiency Virus Type 1 Reverse Transcriptase

“…A 200-fold increased level of resistance to 3TC-TP has also been seen in an endogenous RT assay (49,50). Although the level of resistance to 3TC-TP in cell-free assays is much lower than the 1,000-fold increase in IC 50 s measured in tissue culture, the biochemical data clearly indicate diminished rates of incorporation of 3TC monophosphate (3TC-MP).…”

“…(i) M184V is the only single mutation known to encode as much as 1,000-fold increased resistance to an NRTI, i.e., 3TC, as well as low-level resistance to other drugs. The increase in the 50% inhibitory concentration (IC 50 ) of 3TC can approach 1,000-fold in tissue culture evaluations. (ii) The mutation is observed very rapidly both in cell culture and in patients receiving treatment with 3TC, but not in those receiving ddI or zalcitabine (ddC) (6,32,58,63).…”

A Cell-Based Strategy To Assess Intrinsic Inhibition Efficiencies of HIV-1 Reverse Transcriptase Inhibitors

“…ϪKX ϭ H. A similar approach was previously used to assess the drug concentration-based impacts of other RTIs (AZT, 3TC, and NVP) on vDNA product accumulation (18,22). Best-fit values for TFV, FTC, and EFV are shown in Table 1.…”

“…Comparison of k pol /K d between the natural dNTP and ddNTP analog defines discrimination efficiency (selectivity) of RT (13,14). While informative, these kinetic parameters of inhibition determined under cell-free conditions can vary widely depending on the type (RNA or DNA), nature (homopolymeric or heteropolymeric), sequence, and length of T/P complex used (12,(15)(16)(17)(18)(19). Moreover, such conditions that allow polymerization from multiple start sites, monitor complete and incomplete DNA products, and/or use variable ratios of T/P to RT concentrations all do not emulate conditions found in the natural cellular milieu.…”