The effects of RNase H inhibitors and nevirapine on the susceptibility of HIV-1 to AZT and 3TC.
ABSTRACT: It was recently proposed that HIV RT mutations that decrease RNase H activity increase zidovudine (AZT) resistance by delaying the degradation of the RNA template, allowing more time for AZTMP excision from the 3' end of the viral DNA. This predicts that suboptimal concentrations of an RNase H Inhibitor (RNHI), which would decrease RNaseH activity, would decrease AZT susceptibility. Conversely, a suboptimal concentration of a nonnucleoside RT inhibitor (NNRTI) would decrease polymerase activity and increase AZT susceptibility. We determined the effect of several RNHIs and an NNRTI (nevirapine) on AZT and lamivudine (3TC) susceptibility with vectors that replicate using WT or AZT resistant RTs. Susceptibility to 3TC, which is not readily excised, did not change significantly. Nevirapine, and most RNHIs tested, had only small effects on the susceptibility of either HIV vector to AZT and 3TC. One RNHI, F0444-0019, increased the IC(50) for AZT for either vector by ~5-fold, which may be a concern.
Project description:Single-dose nevirapine (sdNVP)-which prevents mother-to-child transmission of HIV-selects non-nucleoside reverse-transcriptase inhibitor (NNRTI) resistance mutations in the majority of women and HIV-infected infants receiving it. This open-label, randomised trial examined the efficacy of short-course zidovudine (AZT) and lamivudine (3TC) with sdNVP in reducing NNRTI resistance in mothers, and as a secondary objective, in infants, in a setting where sdNVP was standard-of-care.sdNVP alone, administered at the onset of labour and to the infant, was compared to sdNVP with AZT plus 3TC, given as combivir (CBV) for 4 (NVP/CBV4) or 7 (NVP/CBV7) days, initiated simultaneously with sdNVP in labour; their newborns received the same regimens. Women were randomised 1ratio1ratio1. HIV-1 resistance was assessed by population sequencing at: baseline, 2, and 6 wk after birth. An unplanned interim analysis resulted in early stopping of the sdNVP arm. 406 pregnant women were randomised and took study medication (sdNVP 74, NVP/CBV4 164, and NVP/CBV7 168). HIV-1 resistance mutations emerged in 59.2%, 11.7%, and 7.3% of women in the sdNVP, NVP/CBV4, and NVP/CBV7 arms by 6 wk postpartum; differences between NVP-only and both NVP/CBV arms were significant (p<0.0001), but the difference between NVP/CBV4 and NVP/CBV7 was not (p = 0.27). Estimated efficacy comparing combined CBV arms with sdNVP was 85.6%. Similar resistance reductions were seen in infants who were HIV-infected by their 6-wk visit.A short course of AZT plus 3TC, supplementing maternal and infant sdNVP, reduces emergent NNRTI resistance mutations in both mothers and their infants. However, this trial was not powered to detect small differences between the CBV arms.www.ClinicalTrials.govNCT 00144183.
Project description:We previously shown that mutations in the connection (CN) subdomain of human immunodeficiency virus type 1 (HIV-1) subtype B reverse transcriptase (RT) increase 3'-azido-3'-deoxythymidine (AZT) resistance in the context of thymidine analog mutations (TAMs) by affecting the balance between polymerization and RNase H activity. To determine whether this balance affects drug resistance in other HIV-1 subtypes, recombinant subtype CRF01_AE was analyzed. Interestingly, CRF01_AE containing TAMs exhibited 64-fold higher AZT resistance relative to wild-type B, whereas AZT resistance of subtype B containing the same TAMs was 13-fold higher, which in turn correlated with higher levels of AZT-monophosphate (AZTMP) excision on both RNA and DNA templates. The high level of AZT resistance exhibited by CRF01_AE was primarily associated with the T400 residue in wild-type subtype AE CN subdomain. An A400T substitution in subtype B enhanced AZT resistance, increased AZTMP excision on both RNA and DNA templates, and reduced RNase H cleavage. Replacing the T400 residue in CRF01_AE with alanine restored AZT sensitivity and reduced AZTMP excision on both RNA and DNA templates, suggesting that the T400 residue increases AZT resistance in CRF01_AE at least in part by directly increasing the efficiency of AZTMP excision. These results show for the first time that CRF01_AE exhibits higher levels of AZT resistance in the presence of TAMs and that this resistance is primarily associated with T400. Our results also show that mixing the RT polymerase, CN, and RNase H domains from different subtypes can underestimate AZT resistance levels, and they emphasize the need to develop subtype-specific genotypic and phenotypic assays to provide more accurate estimates of clinical drug resistance.
Project description:We previously proposed that mutations in the connection subdomain (cn) of HIV-1 reverse transcriptase increase AZT resistance by altering the balance between nucleotide excision and template RNA degradation. To test the predictions of this model, we analyzed the effects of previously identified cn mutations in combination with thymidine analog mutations (D67N, K70R, T215Y, and K219Q) on in vitro RNase H activity and AZT monophosphate (AZTMP) excision. We found that cn mutations G335C/D, N348I, A360I/V, V365I, and A376S decreased primary and secondary RNase H cleavages. The patient-derived cns increased ATP- and PPi-mediated AZTMP excision on an RNA template compared with a DNA template. One of 5 cns caused an increase in ATP-mediated AZTMP excision on a DNA template, whereas three cns showed a higher ratio of ATP- to PPi-mediated excision, indicating that some cn mutations also affect excision on a DNA substrate. Overall, the results strongly support the model that cn mutations increase AZT resistance by reducing template RNA degradation, thereby providing additional time for RT to excise AZTMP.
Project description:N348I in HIV-1 reverse transcriptase (RT) confers resistance to zidovudine (AZT) and nevirapine. Biochemical studies demonstrated that N348I indirectly increases AZT resistance by decreasing the frequency of secondary ribonuclease H (RNase H) cleavages that reduce the RNA/DNA duplex length of the template/primer (T/P) and diminish the efficiency of AZT-monophosphate (MP) excision. By contrast, there is some discrepancy in the literature in regard to the mechanisms associated with nevirapine resistance: one study suggested that it is due to decreased inhibitor binding while others suggest that it may be related to the decreased RNase H cleavage phenotype. From a structural perspective, N348 in both subunits of RT resides distal to the enzyme's active sites, to the T/P binding tract and to the nevirapine-binding pocket. As such, the structural mechanisms associated with the resistance phenotypes are not known.Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the ?14-?15 loop in the p51 subunit of RT and the RNA template. Substitution of the asparagine at codon 348 in the p51 subunit with either isoleucine or leucine abrogated the observed protein-RNA interaction, thus, providing a possible explanation for the decreased RNase H phenotype. By contrast, alanine or glutamine substitutions exerted no effect. To validate this model, we introduced the N348I, N348L, N348A and N348Q mutations into RT and purified enzymes that contained subunit-specific mutations. N348I and N348L significantly decreased the frequency of secondary RNase H cleavages and increased the enzyme's ability to excise AZT-MP. As predicted by the modelling, this phenotype was due to the mutation in the p51 subunit of RT. By contrast, the N348A and N348Q RTs exhibited RNase H cleavage profiles and AZT-MP excision activities similar to the wild-type enzyme. All N348 mutant RTs exhibited decreased nevirapine susceptibility, although the N348I and N348L mutations conferred higher fold resistance values compared to N348A and N348Q. Nevirapine resistance was also largely due to the mutation present in the p51 subunit of RT.This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.
Project description:BACKGROUND: The precise efficacy of nucleoside analogue reverse-transcriptase inhibitors (NRTIs) in preventing and inhibiting virus replication remains unknown in RT-SHIV infected Chinese-origin rhesus macaques (Ch RM). FINDINGS: Ch RM were inoculated intravenously with 200 TCID50 RT-SHIV and treated by gavage with NRTIs (20 mg AZT and 10 mg 3TC twice per day) for four consecutive weeks beginning at one hour, on day 217 or 297 post inoculation, respectively. Treatment with AZT/3TC inhibited transiently RT-SHIV replication during chronic infection, but did not significantly affect peripheral blood CD4+ T cells in macaques. Treatment with AZT/3TC at 1 hour post infection prevented RT-SHIV infection in two out of four animals during the 120-day observation period. CONCLUSIONS: Therefore, the Ch RM model with RT-SHIV infection can be used to evaluate the efficacy of new NRTIs.
Project description:Drug resistance-associated mutations in HIV-1 reverse transcriptase (RT) can affect the balance between polymerase and ribonuclease H (RNase H) activities of the enzyme. We have recently demonstrated that the N348I mutation in the connection domain causes selective dissociation from RNase H-competent complexes, whereas the functional integrity of the polymerase-competent complex remains largely unaffected. N348I has been associated with resistance to the non-nucleoside RT inhibitor (NNRTI), nevirapine; however, a possible mechanism that links changes in RNase H activity to changes in NNRTI susceptibility remains to be established. To address this problem, we consider recent findings suggesting that NNRTIs may affect the orientation of RT on its nucleic acid substrate and increase RNase H activity. Here we demonstrate that RNase H-mediated primer removal is indeed more efficient in the presence of NNRTIs; however, the N348I mutant enzyme is able to counteract this effect. Efavirenz, a tight binding inhibitor, restricts the influence of the mutation. These findings provide strong evidence to suggest that N348I can thwart the inhibitory effects of nevirapine during initiation of (+)-strand DNA synthesis, which provides a novel mechanism for resistance. The data are in agreement with clinical data, which demonstrate a stronger effect of N348I on susceptibility to nevirapine as compared with efavirenz.
Project description:Antiretroviral drug resistance following pMTCT strategies remains a significant problem. With rapid advancements in next generation sequencing technologies, there is more focus on HIV drug-resistant variants of low frequency, or the so-called minority variants. In South Africa, AZT monotherapy for pMTCT, similar to World Health Organization option A, has been used since 2008. In 2010, a single dose of co-formulated TDF/FTC was included in the strategy for prevention of resistance conferred by single-dose nevirapine (sd NVP). The study was conducted in KwaZulu-Natal, South Africa, among pMTCT participants who received AZT monotherapy from 14 weeks of gestation, intrapartum AZT and sd NVP, and postpartum sd TDF/FTC. Twenty-six specimens collected at 6 weeks post-delivery were successfully sequenced using 454 ultra-deep sequencing. Non-nucleoside reverse transcriptase inhibitor (NNRTI) resistance was detected in 17 of 26 (65%) patients, 2 (7%) had Thymidine analogue mutations, and 3 (11%) had K65R. Of the 17 patients with NNRTI resistance, 11 (65%) had high-level NNRTI resistance, whereas 6 (35%) had intermediate NNRTI resistance. The levels of NNRTI resistance are much higher than would be expected, given the inclusion of antepartum AZT and postpartum TDF/FTC. This high level of NNRTI resistance could impact future NNRTI-containing treatment for a large proportion of pMTCT-exposed women. The detection of Thymidine analogue mutations highlights the need to understand the clinical impact of these on AZT-containing antiretroviral treatment in women exposed to AZT monotherapy.
Project description:We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.
Project description:INTRODUCTION: Tanzanian guidelines for prevention of mother-to-child-transmission of HIV (PMTCT) recommend an antiretroviral combination regimen involving zidovudine (AZT) during pregnancy, single-dosed nevirapine at labor onset, AZT plus Lamivudine (3TC) during delivery, and AZT/3TC for 1-4 weeks postpartum. As drug toxicities are a relevant concern, we assessed hematological alterations in AZT-exposed women and their infants. METHODS AND MATERIALS: A cohort of HIV-positive women, either with AZT intake (n?=?82, group 1) or without AZT intake (n?=?62, group 2) for PMTCT during pregnancy, was established at Kyela District Hospital, Tanzania. The cohort also included the infants of group 1 with an in-utero AZT exposure ?4 weeks, receiving AZT for 1 week postpartum (n?=?41), and infants of group 2 without in-utero AZT exposure, receiving a prolonged 4-week AZT tail (n?=?58). Complete blood counts were evaluated during pregnancy, birth, weeks 4-6 and 12. RESULTS: For women of group 1 with antenatal AZT intake, we found a statistically significant decrease in hemoglobin level, red blood cells, white blood cells, granulocytes, as well as an increase in red cell distribution width and platelet count. At delivery, the median red blood cell count was significantly lower and the median platelet count was significantly higher in women of group 1 compared to group 2. At birth, infants from group 1 showed a lower median hemoglobin level and granulocyte count and a higher frequency of anemia and granulocytopenia. At 4-6 weeks postpartum, the mean neutrophil granulocyte count was significantly lower and neutropenia was significantly more frequent in infants of group 2. CONCLUSIONS: AZT exposure during pregnancy as well as after birth resulted in significant hematological alterations for women and their newborns, although these changes were mostly mild and transient in nature. Research involving larger cohorts is needed to further analyze the impact of AZT-containing regimens on maternal and infant health.