Nucleoside Analog Reverse Transcriptase Inhibitors
Increasing biochemical evidence strongly suggests that the excision of incorporated chain-terminating nucleotides is an important mechanism in HIV resistance to AZT. Earlier reports showed that reverse transcriptase (RT) enzymes with mutations associated with resistance to AZT facilitated rescue of DNA synthesis in the presence of pyrophosphate (PPi) or ATP, alternatively. Now, a new study by Meyer et al (Abstract 14) confirms recently published data, showing that ATP- and PPi-dependent removal of chain-terminators is reduced in RT enzymes that confer resistance to foscarnet -- an analogue of PPi. The data suggest a common binding site for PPi and ATP, respectively [1].

Meyer et al also studied the effects of several combinations of amino acid substitutions associated with resistance to AZT (Abstract 15) [2]. The strongest effect in regard to ATP-dependent primer unblocking was observed with four mutations at positions 67, 70, 215Y and 219. Increased rates of pyrophosphorolysis were not observed in their assay system that distinguishes between primer unblocking, conducted with HIV-1 RT, and rescue of DNA synthesis, conducted with the Klenow fragment of E. coli DNA polymerase I. In contrast, increased rates of pyrophosphorolysis were seen with the same mutant enzyme, when both reactions were performed with HIV-1 RT (Selmi et al, Abstract 17), in agreement with previously published papers [3]. Selmi at al further developed an approach to specifically target mutant enzymes that recruit primer unblocking reactions as possible mechanisms for drug resistance. The authors showed that nucleotides bearing a boranophosphate group on the monophosphate of thymidine analogues decreased rates of pyrophosphorolysis by several relevant mutant enzymes.

Lennerstrand et al (Abstract 16) reported that relatively high-level resistance to stavudine (d4T) was conferred by the 69 mutation in an AZT-resistant background, as revealed by in vitro drug susceptibility screening and cell-free assays using recombinant mutant enzymes [4]. The data suggest an important role for primer unblocking reactions, while mutation 75T and the multidrug resistant mutation 151M may contribute to low-level d4T resistance by causing decreased binding of d4T-triphosphates. Low-level resistance to abacavir (ABC), conferred by the M184V mutation, involves improved substrate discrimination, as seen with 3TC (Abstract 41) [5]. However, the rates of incorporation of carbovir-TP, the physiologically relevant metabolite of ABC, was still extremely high with either wild-type RT, mutant enzyme containing the M184V mutation, RT containing AZT-resistance conferring mutations, or a combination of M184V and AZT-resistance mutations. In good agreement with these results, Jaccard et al (Abstract 61) reported that isolates from AZT and 3TC experienced patients remained largely susceptible to ABC [6]. Although these data confirm that additional mutations, e.g. the multi-drug resistant Q151M mutant, may be required to confer higher levels of resistance to ABC, the drug is capable of maintaining the pressure on HIV-1 variants containing the M184V mutation (Abstract 21) [7].

Nonnucleoside Reverse Transcriptase Inhibitors
A novel NNRTI resistance-conferring mutation, i.e. M230L, that was identified in NNRTI-experienced patients, has been associated with a dose-dependent stimulation of HIV replication (Abstracts 29, 30) [8,9]. This mutation, in combination with other well-known mutations associated with resistance to NNRTI, can dramatically decrease susceptibilities to nevirapine, efavirenz and delavirdine (up to 800-fold). An HIV variant containing both the M230L and K103N mutations showed a 50-100% stimulation of replication, compared to replication levels observed in absence of drug. The M230L mutation alone caused lower levels of stimulation and drug resistance. In contrast, other NNRTI resistance mutations, e.g. the P236L mutation associated with delavirdine therapy, showed diminished replication levels (Abstract 35) [10]. Cell-free assays, conducted with recombinant RT containing the P236L mutation showed a slightly diminished formation of the primer for plus-strand DNA synthesis (Abstract 31) [11]. Maximum levels of drug-resistance combined with high levels of viral fitness may also help to explain the emergence of the G138L mutation under TSAO drug pressure.

Protease Inhibitors
Kempf et al and Molla et al (Abstracts 38, 39) analyzed HIV isolates from patients failing single or multiple PI therapy, with particular focus on drug resistance to ABT-378 [12,13]. Correlations between phenotypic and genotypic resistance suggest that multiple mutations may be required to achieve clinically relevant resistance. Pre-existing mutations may provide a platform for the evolution of resistance to ABT-378. To elucidate the genetic nature of tipranavir resistance, Kemp et al performed site-directed mutagenesis and in vitro drug selection studies (Abstract 40) [14]. However, the genetic changes responsible for relevant clinical resistance has not yet been identified and appear to be more complex than previously expected. Notably in this context, Peters at al (Abstract 51) suggested that regions outside the RT or protease genes may contribute to HIV-resistance. In this respect, the authors reported duplication in a conserved region of p6^Gag that is selected under drug pressure [15].