Phenotypic susceptibility to bevirimat in isolates from HIV-1-infected patients without prior exposure to bevirimat.

Bevirimat (BVM) is the first of a new class of anti-HIV drugs with a novel mode of action known as maturation inhibitors. BVM inhibits the last cleavage of the Gag polyprotein by HIV-1 protease, leading to the accumulation of the p25 capsid-small peptide 1 (SP1) intermediate and resulting in noninfectious HIV-1 virions. Early clinical studies of BVM showed that over 50% of the patients treated with BVM did not respond to treatment. We investigated the impact of prior antiretroviral (ARV) treatment and/or natural genetic diversity on BVM susceptibility by conducting in vitro phenotypic analyses of viruses made from patient samples. We generated 31 recombinant viruses containing the entire gag and protease genes from 31 plasma samples from HIV-1-infected patients with (n = 21) or without (n = 10) prior ARV experience. We found that 58% of the patient isolates tested had a >10-fold reduced susceptibility to BVM, regardless of the patient's ARV experience or the level of isolate resistance to protease inhibitors. Analysis of mutants with site-directed mutations confirmed the role of the V370A SP1 polymorphism (SP1-V7A) in resistance to BVM. Furthermore, we demonstrated for the first time that a capsid polymorphism, V362I (CA protein-V230I), is also a major mutation conferring resistance to BVM. In contrast, none of the previously defined resistance-conferring mutations in Gag selected in vitro (H358Y, L363M, L363F, A364V, A366V, or A366T) were found to occur among the viruses that we analyzed. Our results should be helpful in the design of diagnostics for prediction of the potential benefit of BVM treatment in HIV-1-infected patients.

The A62V and S68G mutations in HIV-1 reverse transcriptase partially restore the replication defect associated with the K65R mutation.

BACKGROUND: The K65R mutation in human immunodeficiency virus type 1 reverse transcriptase can be selected by abacavir, didanosine, tenofovir, and stavudine in vivo resulting in reduced susceptibility to these drugs and decreased viral replication capacity. In clinical isolates, K65R is frequently accompanied by the A62V and S68G reverse transcriptase mutations. METHODS: The role of A62V and S68G in combination with K65R was investigated using phenotypic, viral growth competition, pre-steady-state kinetic, and excision analyses. RESULTS: Addition of A62V and S68G to K65R caused no significant change in human immunodeficiency virus type 1 resistance to abacavir, didanosine, tenofovir, or stavudine but partially restored the replication defect of virus containing K65R. The triple mutant K65R+A62V+S68G still showed some replication defect compared with wild-type virus. Pre-steady-state kinetic analysis demonstrated that K65R resulted in a decreased rate of incorporation (kpol) for all natural dNTPs, which were partially restored to wild-type levels by addition of A62V and S68G. When added to K65R and S68G, the A62V mutation seemed to restore adenosine triphosphate-mediated excision of tenofovir to wild-type levels. CONCLUSIONS: A62V and S68G serve as partial compensatory mutations for the K65R mutation in reverse transcriptase by improving the viral replication capacity, which is likely due to increased incorporation efficiency of the natural substrates.

Effects of the translocation status of human immunodeficiency virus type 1 reverse transcriptase on the efficiency of excision of tenofovir.

The ATP-dependent phosphorolytic excision of nucleoside analogue reverse transcriptase inhibitors can diminish their inhibitory effects on human immunodeficiency virus replication. Previous studies have shown that excision can occur only when the reverse transcriptase complex exists in its pretranslocational state. Binding of the next complementary nucleotide causes the formation of a stable dead-end complex in the posttranslocational state, which blocks the excision reaction. To provide mechanistic insight into the excision of the acyclic phosphonate nucleotide analog tenofovir, we compared the efficiencies of the reaction in response to changes in the translocation status of the enzyme. We found that rates of excision of tenofovir with wild-type reverse transcriptase can be as high as those seen with 3'-azido-3'-deoxythymidine monophosphate (AZT-MP). Thymidine-associated mutations, which confer >100-fold and 3-fold decreased susceptibility to AZT and tenofovir, respectively, caused substantial increases in the efficiency of excision of both inhibitors. However, in contrast to the case for AZT-MP, the removal of tenofovir was highly sensitive to dead-end complex formation. Site-specific footprinting experiments revealed that complexes with AZT-terminated primers exist predominantly pretranslocation. In contrast, complexes with tenofovir-terminated primers are seen in both configurations. Low concentrations of the next nucleotide are sufficient to trap the complex posttranslocation despite the flexible, acyclic character of the compound. Thus, the relatively high rate of excision of tenofovir is partially neutralized by the facile switch to the posttranslocational state and by dead-end complex formation, which provides a degree of protection from excision in the cellular environment.

The balance between NRTI discrimination and excision drives the susceptibility of HIV-1 RT mutants K65R, M184V and K65r+M184V.

The HIV-1 reverse transcriptase (RT) resistance mutations K65R and M184V occur individually and in combination, and can contribute to decreased treatment responses in patients. In order to understand how these mutations interact with one another to confer drug resistance, the susceptibilities and underlying resistance mechanisms of these mutants to nucleoside RT inhibitors (NRTIs) were determined. Virus carrying K65R have reduced susceptibility to most NRTIs, but retain full susceptibility to zidovudine (AZT). M184V mutants have reduced susceptibility to lamivudine (3TC), emtricitabine (FTC) and didanosine (ddl), and contribute to reduced susceptibility to abacavir; however, they remain fully susceptible to tenofovir (TFV), AZT and stavudine (d4T). In cell culture, the K65R+M184V virus showed slightly increased susceptibility to TFV, AZT and d4T compared with K65R alone, but showed further decreases in susceptibility to 3TC, FTC, ddl and abacavir. There are two major biochemical mechanisms of resistance: altered NRTI binding/incorporation and altered NRTI excision after incorporation. For most NRTIs, the primary mechanism of resistance by K65R, M184V and K65R+M184V mutant RTs is to disrupt the NRTI-binding/incorporation steps. In the case of AZT, however, decreased binding/incorporation by K65R and K65R+M184V was counteracted by decreased AZT excision resulting in wild-type susceptibility. For TFV, decreased excision by K65R and K65R+M184V may partially counteract the K65R-driven decrease in incorporation relative to wild-type resulting in only low levels of TFV resistance. The K65R-mediated effect on decreasing NRTI excision was stronger than for M184V. These studies show that both mechanisms of resistance (binding/incorporation and excision) must be considered when defining resistance mechanisms.

In vitro combination studies of tenofovir and other nucleoside analogues with ribavirin against HIV-1.

In patients coinfected and treated for both HIV-1 and hepatitis C virus (HCV), administration of ribavirin (RBV) may result in altered intracellular drug levels of nucleoside reverse transcriptase inhibitors through inhibition of inosine 5'-monophosphate dehydrogenase. Drug interactions between tenofovir and RBV were studied in vitro in order to provide insights into the safety of co-administration of tenofovir disoproxil fumarate (DF) and RBV in HCV/HIV-1-coinfected patients. In accordance with previous in vitro studies, strongly increased anti-HIV activity was observed when RBV was combined with didanosine (ddl). In contrast, low-level anti-HIV antagonism was observed when RBV was combined with either tenofovir or abacavir. Significantly stronger anti-HIV antagonism was observed when RBV was combined with either zidovudine, stavudine, emtricitabine or lamivudine. Thus, although tenofovir and ddl are both adenosine analogues, their in vitro interactions with RBV are markedly different. These results suggest a low potential for increased toxicity upon co-administration of tenofovir DF with RBV in patients.

Molecular mechanisms of tenofovir resistance conferred by human immunodeficiency virus type 1 reverse transcriptase containing a diserine insertion after residue 69 and multiple thymidine analog-associated mutations.

Two amino acids inserted between residues 69 and 70 of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) are rare mutations that may develop in viruses containing multiple thymidine analog (zidovudine [AZT], stavudine)-associated mutations and that confer high-level resistance to all currently approved chain-terminating nucleoside and nucleotide RT inhibitors (NRTIs). The two known mechanisms of resistance to NRTIs are decreased incorporation and increased excision. The mechanism used by RT insertion mutants has not been described for tenofovir (TFV), a recently approved agent in this class. A patient-derived HIV-1 strain (strain FS-SSS) that contained an insertion mutation in a background of additional resistance mutations M41L, L74V, L210W, and T215Y was obtained. A second virus (strain FS) was derived from FS-SSS. In strain FS the insertion and T69S were reverted but the other resistance mutations were retained. The FS virus showed strong resistance to AZT but low-level changes in susceptibilities to other NRTIs and TFV. The FS-SSS virus showed reduced susceptibilities to all NRTIs including TFV. Steady-state kinetics demonstrated that the relative binding or incorporation of TFV was slightly decreased for FS-SSS RT compared to those for wild-type RT. However, significant ATP-mediated excision of TFV was detected for both mutant RT enzymes and followed the order FS-SSS RT > FS RT > wild-type RT. The presence of physiological concentrations of the +1 nucleotide inhibited TFV excision by the wild-type RT and slightly inhibited excision by the FS RT, whereas the level of excision by the FS-SSS RT remained high. Computer modeling suggests that the increased mobility of the beta3-beta4 loop may contribute to the high-level and broad NRTI resistance caused by the T69 insertion mutation.

Molecular mechanisms of resistance to human immunodeficiency virus type 1 with reverse transcriptase mutations K65R and K65R+M184V and their effects on enzyme function and viral replication capacity.

Human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) resistance mutations K65R and M184V result in changes in susceptibility to several nucleoside and nucleotide RT inhibitors. K65R-containing viruses showed decreases in susceptibility to tenofovir, didanosine (ddI), abacavir, and (-)-beta-D-dioxolane guanosine (DXG; the active metabolite of amdoxovir) but appeared to be fully susceptible to zidovudine and stavudine in vitro. Viruses containing the K65R and M184V mutations showed further decreases in susceptibility to ddI and abacavir but increased susceptibility to tenofovir compared to the susceptibilities of viruses with the K65R mutation. Enzymatic and viral replication analyses were undertaken to elucidate the mechanisms of altered drug susceptibilities and potential fitness defects for the K65R and K65R+M184V mutants. The relative inhibitory capacities (K(i)/K(m)) of the active metabolites of tenofovir, ddI, and DXG were increased for the RT containing the K65R mutation compared to that for the wild-type RT, but the relative inhibitory capacity of abacavir was only minimally increased. For the mutant viruses with the K65R and M184V mutations, the increase in tenofovir susceptibility compared to that of the mutants with K65R correlated with a decrease in the tenofovir inhibitory capacity that was mediated primarily by an increased K(m) of dATP. The decrease in susceptibility to ddI by mutants with the K65R and M184V mutations correlated with an increase in the inhibitory capacity mediated by an increased K(i). ATP-mediated removal of carbovir as well as small increases in the inhibitory capacity of carbovir appear to contribute to the resistance of mutants with the K65R mutation and the mutants with the K65R and M184V mutations to abacavir. Finally, both the HIV-1 K65R mutant and, more notably, the HIV-1 K65R+M184V double mutant showed reduced replication capacities and reduced RT processivities in vitro, consistent with a potential fitness defect in vivo and the low prevalence of the K65R mutation among isolates from antiretroviral agent-experienced patients.

ATP-dependent removal of nucleoside reverse transcriptase inhibitors by human immunodeficiency virus type 1 reverse transcriptase.

Removal of nucleoside chain terminator inhibitors mediated by human immunodeficiency virus (HIV) reverse transcriptase (RT) using ATP as an acceptor molecule has been proposed as a novel mechanism of HIV resistance. Recombinant wild-type and mutant HIV type 1 (HIV-1) RT enzymes with thymidine analog resistance mutations D67N, K70R, and T215Y were analyzed for their ability to remove eight nucleoside reverse transcriptase inhibitors in the presence of physiological concentrations of ATP. The order for the rate of removal of the eight inhibitors by the mutant RT enzyme was zidovudine (AZT) > stavudine (d4T) >> zalcitabine (ddC) > abacavir > amdoxovir (DAPD) > lamivudine (3TC) > didanosine (ddI) > tenofovir. Thymidine analogs AZT and d4T were the most significantly removed by the mutant enzyme, suggesting that removal of these inhibitors by the ATP-dependent removal mechanism contributes to the AZT and d4T resistance observed in patients with HIV expressing thymidine analog resistance mutations. ATP-dependent removal of tenofovir was 22- to 35-fold less efficient than removal of d4T and AZT, respectively. The addition of ATP and the next complementary deoxynucleoside triphosphate caused a reduction of ATP-mediated removal of d4T, ddC, and DAPD, while AZT and abacavir removal was unaffected. The reduction of d4T, ddC, and DAPD removal in the presence of the deoxynucleoside triphosphate could explain the minor changes in susceptibility to these drugs observed in conventional in vitro phenotypic assays using cells that have higher deoxynucleoside triphosphate pools. The minimal removal of abacavir, ddC, DAPD, 3TC, ddI, and tenofovir is consistent with the minor changes in susceptibility to these drugs observed for HIV mutants with thymidine analog resistance mutations.