Subunit-specific mutational analysis of residue N348 in HIV-1 reverse transcriptase

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>Using a novel modelled structure of RT in complex with an RNA/DNA T/P, we identified a putative interaction between the β14-β15 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.</p> <p>Conclusions</p> <p>This study demonstrates that N348I-mediated AZT and nevirapine resistance is due to the mutation in the p51 subunit of RT.</p

The RNA binding protein HuR does not interact directly with HIV-1 reverse transcriptase and does not affect reverse transcription in vitro

<p>Abstract</p> <p>Background</p> <p>Lemay <it>et al </it>recently reported that the RNA binding protein HuR directly interacts with the ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) and influences the efficiency of viral reverse transcription (Lemay <it>et al</it>., 2008, Retrovirology 5:47). HuR is a member of the embryonic lethal abnormal vision protein family and contains 3 RNA recognition motifs (RRMs) that bind AU-rich elements (AREs). To define the structural determinants of the HuR-RT interaction and to elucidate the mechanism(s) by which HuR influences HIV-1 reverse transcription activity <it>in vitro</it>, we cloned and purified full-length HuR as well as three additional protein constructs that contained the N-terminal and internal RRMs, the internal and C-terminal RRMs, or the C-terminal RRM only.</p> <p>Results</p> <p>All four HuR proteins were purified and characterized by biophysical methods. They are well structured and exist as monomers in solution. No direct protein-protein interaction between HuR and HIV-1 RT was detected using NMR titrations with ¹⁵N labeled HuR variants or the ¹⁵N labeled RNase H domain of HIV-1 RT. Furthermore, HuR did not significantly affect the kinetics of HIV-1 reverse transcription <it>in vitro</it>, even on RNA templates that contain AREs.</p> <p>Conclusions</p> <p>Our results suggest that HuR does not impact HIV-1 replication through a direct protein-protein interaction with the viral RT.</p

Zidovudine (AZT) Monotherapy Selects for the A360V Mutation in the Connection Domain of HIV-1 Reverse Transcriptase

Background: We previously demonstrated in vitro that zidovudine (AZT) selects for A371V in the connection domain and Q509L in ribonuclease H (RNase H) domain of HIV-1 reverse transcriptase (RT) which, together with the thymidine analog mutations D67N, K70R and T215F, confer greater than 100-fold AZT resistance. The goal of the current study was to determine whether AZT monotherapy in HIV-1 infected patients also selects the A371V, Q509L or other mutations in the C-terminal domains of HIV-1 RT. Methodology/Principal Findings: Full-length RT sequences in plasma obtained pre- and post-therapy were compared in 23 participants who received AZT monotherapy from the AIDS Clinical Trials Group study 175. Five of the 23 participants reached a primary study endpoint. Mutations significantly associated with AZT monotherapy included K70R (p = 0.003) and T215Y (p = 0.013) in the polymerase domain of HIV-1 RT, and A360V (p = 0.041) in the connection domain of HIV-1 RT. HIV-1 drug susceptibility assays demonstrated that A360V, either alone or in combination with thymidine analog mutations, decreased AZT susceptibility in recombinant viruses containing participant-derived full-length RT sequences or site-directed mutant RT. Biochemical studies revealed that A360V enhances the AZT-monophosphate excision activity of purified RT by significantly decreasing the frequency of secondary RNase H cleavage events that reduce the RNA/DNA duplex length and promote template/primer dissociation. Conclusions: The A360V mutation in the connection domain of RT was selected in HIV-infected individuals that received AZT monotherapy and contributed to AZT resistance. © 2012 Brehm et al

N348I in the Connection Domain of HIV-1 Reverse Transcriptase Confers Zidovudine and Nevirapine Resistance

Analyzing HIV sequences from a Canadian cohort, Gilda Tachedjian and colleagues identify a common mutation in a little-studied domain of reverse transcriptase that confers resistance to two drug classes

Evidence for biphasic uncoating during HIV-1 infection from a novel imaging assay

BACKGROUND: Uncoating of the HIV-1 core plays a critical role during early post-fusion stages of infection but is poorly understood. Microscopy-based assays are unable to easily distinguish between intact and partially uncoated viral cores. RESULTS: In this study, we used 5-ethynyl uridine (EU) to label viral-associated RNA during HIV production. At early time points after infection with EU-labeled virions, the viral-associated RNA was stained with an EU-specific dye and was detected by confocal microscopy together with viral proteins. We observed that detection of the viral-associated RNA was specific for EU-labeled virions, was detected only after viral fusion with target cells, and occurred after an initial opening of the core. In vitro staining of cores showed that the opening of the core allowed the small molecule dye, but not RNase A or antibodies, inside. Also, staining of the viral-associated RNA, which is co-localized with nucleocapsid, decays over time after viral infection. The decay rate of RNA staining is dependent on capsid (CA) stability, which was altered by CA mutations or a small molecule inducer of HIV-1 uncoating. While the staining of EU-labeled RNA was not affected by inhibition of reverse transcription, the kinetics of core opening of different CA mutants correlated with initiation of reverse transcription. Analysis of the E45A CA mutant suggests that initial core opening is independent of complete capsid disassembly. CONCLUSIONS: Taken together, our results establish a novel RNA accessibility-based assay that detects an early event in HIV-1 uncoating and can be used to further define this process

The Time is Gone!? Pink Floyds "Time" (1973)

This paper provides a hermeneutic approach on Pink Floyds popular song "Time" from 1973s album Dark Side Of The Moon. The primary aim of this examination is the analysis of the conceptual connection of music and lyrics in this song. Moreover, derivation of semantic meaning potential is used to form an intersubjectively verifiable song interpretation. All in all, the main results of this analysis suggest a close connection of the songs musical parameters, i.e. form, harmony, melody, rhythm as well as sound, and its lyrical theme: (1) first the musique concrète in the songs introduction and the subsequent instrumental passage establish evident semantic relations to the following lyrical theme of fleeting time, (2) a devils circle of transitoriness of life is presented in the songs lyrics and is musically mirrored by periodic formal and harmonic repetitions, (3) the ambivalence of the bridges negative lyrics in combination with the sweet melodic thirds in the vocals suggests a melancholic atmosphere, (4) the lyrics description of a human life from birth to death is reflected in the formal structure and the harmony of »Time« as well as in the conceptual arrangement of Dark Side Of The Moons first LP-side from »Speak To Me« to »The Great Gig In The Sky«

Potent Nonnucleoside Reverse Transcriptase Inhibitors Target HIV-1 Gag-Pol

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) target HIV-1 reverse transcriptase (RT) by binding to a pocket in RT that is close to, but distinct, from the DNA polymerase active site and prevent the synthesis of viral cDNA. NNRTIs, in particular, those that are potent inhibitors of RT polymerase activity, can also act as chemical enhancers of the enzyme's inter-subunit interactions. However, the consequences of this chemical enhancement effect on HIV-1 replication are not understood. Here, we show that the potent NNRTIs efavirenz, TMC120, and TMC125, but not nevirapine or delavirdine, inhibit the late stages of HIV-1 replication. These potent NNRTIs enhanced the intracellular processing of Gag and Gag-Pol polyproteins, and this was associated with a decrease in viral particle production from HIV-1-transfected cells. The increased polyprotein processing is consistent with premature activation of the HIV-1 protease by NNRTI-enhanced Gag-Pol multimerization through the embedded RT sequence. These findings support the view that Gag-Pol multimerization is an important step in viral assembly and demonstrate that regulation of Gag-Pol/Gag-Pol interactions is a novel target for small molecule inhibitors of HIV-1 production. Furthermore, these drugs can serve as useful probes to further understand processes involved in HIV-1 particle assembly and maturation

Discovery of a small molecule agonist of phosphatidylinositol 3-kinase p110α that reactivates latent HIV-1

Combination antiretroviral therapy (cART) can effectively suppress HIV-1 replication, but the latent viral reservoir in resting memory CD4+ T cells is impervious to cART and represents a major barrier to curing HIV-1 infection. Reactivation of latent HIV-1 represents a possible strategy for elimination of this reservoir. In this study we describe the discovery of 1,2,9,10-tetramethoxy-7H-dibenzo[de,g]quinolin-7-one (57704) which reactivates latent HIV-1 in several cell-line models of latency (J89GFP, U1 and ACH-2). 57704 also increased HIV-1 expression in 3 of 4 CD8+-depleted blood mononuclear cell preparations isolated from HIV-1-infected individuals on suppressive cART. In contrast, vorinostat increased HIV-1 expression in only 1 of the 4 donors tested. Importantly, 57704 does not induce global T cell activation. Mechanistic studies revealed that 57704 reactivates latent HIV-1 via the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway. 57704 was found to be an agonist of PI3K with specificity to the p110a isoform, but not the p110β, δ or γ isoforms. Taken together, our work suggests that 57704 could serve as a scaffold for the development of more potent activators of latent HIV-1. Furthermore, it highlights the involvement of the PI3K/Akt pathway in the maintenance of HIV-1 latency. © 2014 Doyon et al

Disparities in the Burden of HIV/AIDS in Canada

Background We aimed to characterize changes in patterns of new HIV diagnoses, HIV-related mortality, and HAART use in Canada from 1995 to 2008. Methods Data on new HIV diagnoses were obtained from Health Canada, HIV-related mortality statistics were obtained from Statistics Canada, and information on the number of people on HAART was obtained from the single antiretroviral distribution site in British Columbia (BC), and the Intercontinental Marketing Services Health for Ontario and Quebec. Trends of new HIV-positive tests were assessed using Spearman rank correlations and the association between the number of individuals on HAART and new HIV diagnoses were estimated using generalized estimating equations (GEE). Results A total of 34,502 new HIV diagnoses were observed. Rates of death in BC are higher than those in Ontario and Quebec with the rate being 2.03 versus 1.06 and 1.21 per 100,000 population, respectively. The number of HIV infected individuals on HAART increased from 5,091 in 1996 to 20,481 in 2008 in the three provinces (4 fold increase). BC was the only province with a statistically significant decrease (trend test p&lt;0.0001) in the rate of new HIV diagnoses from 18.05 to 7.94 new diagnoses per 100,000 population. Our analysis showed that for each 10% increment in HAART coverage the rate of new HIV diagnoses decreased by 8% (95% CI: 2.4%, 13.3%) Interpretation Except for British Columbia, the number of new HIV diagnoses per year has remained relatively stable across Canada over the study period. The decline in the rate of new HIV diagnoses per year may be in part attributed to the greater expansion of HAART coverage in this province