The R262K Substitution Combined with H51Y in HIV-1 Subtype B Integrase Confers Low-Level Resistance against Dolutegravir

ABSTRACTClinical studies have shown that integrase strand transfer inhibitors (INSTIs) can be used effectively against HIV-1 infection. To date, no resistance substitution has been found in INSTI-naive patients treated with the new integrase inhibitor dolutegravir (DTG). In a recent selection study with DTG, using a virus bearing the H51Y substitution in integrase, the emergence of an R to K substitution at position 262 (R262K) was observed. We characterized this double mutant with respect to integrase strand transfer activity and susceptibility to DTG both biochemically and in tissue culture. We showed that the addition of R262K to H51Y decreased recombinant integrase strand transfer activity but improved integrase DNA-binding affinity, compared to wild-type or H51Y-containing enzymes. The defect in strand transfer activity did not translate into a decrease in HIV-1 infectivity. The combination of H51Y and R262K substitutions slightly decreased susceptibility to DTG (fold change = 1.87) in cell-based resistance assays. Although viral replication was not affected and enzyme efficiency was impaired by the addition of R262K to H51Y, there was an overall increase in the level of biochemical drug resistance against DTG. Our findings suggest that the R at position 262 plays an important role in DNA binding.

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Effects of integrase inhibitor-based antiretroviral therapy on brain outcomes according to time since acquisition of HIV-1 infection

Scientific Reports ◽

10.1038/s41598-021-90678-6 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Anna Prats ◽

Ignacio Martínez-Zalacaín ◽

Beatriz Mothe ◽

Eugènia Negredo ◽

Núria Pérez-Álvarez ◽

...

Keyword(s):

Antiretroviral Therapy ◽

Integrase Inhibitor ◽

Strand Transfer ◽

Cognitive Differences ◽

Integrase Strand Transfer Inhibitors ◽

Initiation Of Therapy ◽

Main Component ◽

Living With Hiv ◽

The Impact ◽

Hiv 1

AbstractIntegrase strand transfer inhibitors (INSTI) are a main component of the current antiretroviral regimens recommended for treatment of HIV infection. However, little is known about the impact of INSTI on neurocognition and neuroimaging. We developed a prospective observational trial to evaluate the effects of INSTI-based antiretroviral therapy on comprehensive brain outcomes (cognitive, functional, and imaging) according to the time since HIV-1 acquisition. We recruited men living with HIV who initiated antiretroviral therapy with INSTI < 3 months since the estimated date of HIV-1 acquisition (n = 12) and > 6 months since estimated date of HIV-1 acquisition (n = 15). We also recruited a group of matched seronegative individuals (n = 15). Assessments were performed at baseline (before initiation of therapy in HIV arms) and at weeks 4 and 48. Baseline cognitive functioning was comparable between the arms. At week 48, we did not find cognitive differences between starting therapy with INSTI earlier than 3 months or later than 6 months after acquisition of HIV-1 infection. Functional status was poorer in individuals diagnosed earlier. This effect recovered 48 weeks after initiation of therapy. Regarding brain imaging, we found that men living with HIV initiating antiretroviral therapy later experienced a greater decrease in medial orbitofrontal cortex over time, with expected negative repercussions for decision-making tasks.

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Accumulation of integrase strand transfer inhibitor resistance mutations confers high-level resistance to dolutegravir in non-B subtype HIV-1 strains from patients failing raltegravir in Uganda

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa355 ◽

2020 ◽

Vol 75 (12) ◽

pp. 3525-3533

Author(s):

Emmanuel Ndashimye ◽

Mariano Avino ◽

Abayomi S Olabode ◽

Art F Y Poon ◽

Richard M Gibson ◽

...

Keyword(s):

Prolonged Exposure ◽

Cross Resistance ◽

Strand Transfer ◽

Hiv Integrase ◽

Resistance Mutations ◽

Middle Income ◽

Subtype B ◽

High Level ◽

Hiv 1 ◽

Level Resistance

Abstract Background Increasing first-line treatment failures in low- and middle-income countries (LMICs) have led to increased use of integrase strand transfer inhibitors (INSTIs) such as dolutegravir. However, HIV-1 susceptibility to INSTIs in LMICs, especially with previous raltegravir exposure, is poorly understood due to infrequent reporting of INSTI failures and testing for INSTI drug resistance mutations (DRMs). Methods A total of 51 non-subtype B HIV-1 infected patients failing third-line (raltegravir-based) therapy in Uganda were initially selected for the study. DRMs were detected using Sanger and deep sequencing. HIV integrase genes of 13 patients were cloned and replication capacities (RCs) and phenotypic susceptibilities to dolutegravir, raltegravir and elvitegravir were determined with TZM-bl cells. Spearman’s correlation coefficient was used to determine cross-resistance between INSTIs. Results INSTI DRMs were detected in 47% of patients. HIV integrase-recombinant virus carrying one primary INSTI DRM (N155H or Y143R/S) was susceptible to dolutegravir but highly resistant to raltegravir and elvitegravir (>50-fold change). Two patients, one with E138A/G140A/Q148R/G163R and one with E138K/G140A/S147G/Q148K, displayed the highest reported resistance to raltegravir, elvitegravir and even dolutegravir. The former multi-DRM virus had WT RC whereas the latter had lower RCs than WT. Conclusions In HIV-1 subtype A- and D-infected patients failing raltegravir and harbouring INSTI DRMs, there is high-level resistance to elvitegravir and raltegravir. More routine monitoring of INSTI treatment may be advised in LMICs, considering that multiple INSTI DRMs may have accumulated during prolonged exposure to raltegravir during virological failure, leading to high-level INSTI resistance, including dolutegravir resistance.

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Molecular and genetic characterization of natural variants of HIV-1 Nef gene from North India and its functional implication in down-regulation of MHC-I and CD-4

Current HIV Research ◽

10.2174/1570162x18666200925160755 ◽

2020 ◽

Vol 18 ◽

Author(s):

J. Singh ◽

L. Ronsard ◽

M. Pandey ◽

R. Kapoor ◽

V.G. Ramachandran ◽

...

Keyword(s):

Biological Activities ◽

Sequence Similarity ◽

Eukaryotic Expression ◽

North India ◽

Cell Surface Expression ◽

Functional Domains ◽

Wild Type ◽

Down Regulation ◽

Subtype B ◽

Hiv 1

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of HIV-1 Nef gene shows extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North-India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in eukaryotic expression vector and further characterized with respect to their ability to down regulate cell surface expression of CD4 and MHC-1molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 down regulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising out of amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

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DNA Length and Cationic Cofactor Dependent Strand Transfer Activity of HIV-1 Integrase

2009 3rd International Conference on Bioinformatics and Biomedical Engineering ◽

10.1109/icbbe.2009.5162656 ◽

2009 ◽

Author(s):

Hong-Qiu He ◽

Cun Xin Wang ◽

Wei-Zu Chen

Keyword(s):

Strand Transfer ◽

Transfer Activity ◽

Hiv 1

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Progressive emergence of an S153F plus R263K combination of integrase mutations in the proviral DNA of one individual successfully treated with dolutegravir

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkaa471 ◽

2020 ◽

Author(s):

Hanh T Pham ◽

Brunna M Alves ◽

Sunbin Yoo ◽

Meng A Xiao ◽

Jing Leng ◽

...

Keyword(s):

Drug Resistance ◽

Dna Binding ◽

Drug Resistant ◽

Strand Transfer ◽

Viral Genomes ◽

Proviral Dna ◽

Viral Loads ◽

Subtype B ◽

Transfer Inhibitor ◽

Integrase Strand Transfer Inhibitor

Abstract Objectives The development of HIV drug resistance against the integrase strand transfer inhibitor dolutegravir is rare. We report here the transient detection, by near full-genome ultradeep sequencing, of minority HIV-1 subtype B variants bearing the S153F and R263K integrase substitutions in the proviral DNA from blood cells of one patient who successfully initiated dolutegravir-based ART, over 24 weeks. Our objective was to study the effects of these substitutions. Methods Strand transfer and DNA-binding activities of recombinant integrase proteins were measured in cell-free assays. Cell-based resistance, infectivity and replicative capacities were measured using molecular clones. Structural modelling was performed to understand experimental results. Results R263K emerged first, followed by the addition of S153F at Week 12. By Week 24, both mutations remained present, but at lower prevalence. We confirmed the coexistence of S153F and R263K on single viral genomes. Combining S153F or S153Y with R263K decreased integration and viral replicative capacity and conferred high levels of drug resistance against all integrase inhibitors. Alone, S153Y and S153F did little to infectivity or dolutegravir resistance. We identified altered DNA binding as a mechanism of resistance. The patient remained with undetectable viral loads at all timepoints. Conclusions Drug-resistant minority variants have often been reported under suppressive ART. Our study adds to these observations by unravelling a progression towards higher levels of resistance through a novel pathway despite continuous undetectable viral loads. Poorly replicative HIV drug-resistant minority proviral variants did not compromise viral suppression in one individual treated with dolutegravir.

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Solution structures of the trihelix DNA-binding domains of the wild-type and a phosphomimetic mutant of Arabidopsis GT-1: Mechanism for an increase in DNA-binding affinity through phosphorylation

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.22827 ◽

2010 ◽

Vol 78 (14) ◽

pp. 3033-3047 ◽

Cited By ~ 21

Author(s):

Takashi Nagata ◽

Emi Niyada ◽

Natsuki Fujimoto ◽

Yuuya Nagasaki ◽

Kazuaki Noto ◽

...

Keyword(s):

Dna Binding ◽

Binding Affinity ◽

Wild Type ◽

Solution Structures ◽

Dna Binding Domains ◽

Binding Domains ◽

Dna Binding Affinity

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Detection of novel HIV-1 drug resistance mutations by support vector analysis of deep sequence data and experimental validation

10.1101/804781 ◽

2019 ◽

Author(s):

Mariano Avino ◽

Emmanuel Ndashimye ◽

Daniel J. Lizotte ◽

Abayomi S. Olabode ◽

Richard M. Gibson ◽

...

Keyword(s):

Drug Resistance ◽

Drug Treatment ◽

Treatment Outcomes ◽

Sequence Data ◽

Wild Type Virus ◽

Support Vector ◽

Wild Type ◽

Subtype B ◽

The World ◽

Hiv 1

AbstractThe global HIV-1 pandemic comprises many genetically divergent subtypes. Most of our understanding of drug resistance in HIV-1 derives from subtype B, which predominates in North America and western Europe. However, about 90% of the pandemic represents non-subtype B infections. Here, we use deep sequencing to analyze HIV-1 from infected individuals in Uganda who were either treatment-naïve or who experienced virologic failure on ART without the expected patterns of drug resistance. Our objective was to detect potentially novel associations between mutations in HIV-1 integrase and treatment outcomes in Uganda, where most infections are subtypes A or D. We retrieved a total of 380 archived plasma samples from patients at the Joint Clinical Research Centre (Kampala), of which 328 were integrase inhibitor-naïve and 52 were raltegravir (RAL)-based treatment failures. Next, we developed a bioinformatic pipeline for alignment and variant calling of the deep sequence data obtained from these samples from a MiSeq platform (Illumina). To detect associations between within-patient polymorphisms and treatment outcomes, we used a support vector machine (SVM) for feature selection with multiple imputation to account for partial reads and low quality base calls. Candidate point mutations of interest were experimentally introduced into the HIV-1 subtype B NL4-3 backbone to determine susceptibility to RAL in U87.CD4.CXCR4 cells. Finally, we carried out replication capacity experiments with wild-type and mutant viruses in TZM-bl cells in the presence and absence of RAL. Our analyses not only identified the known major mutation N155H and accessory mutations G163R and V151I, but also novel mutations I203M and I208L as most highly associated with RAL failure. The I203M and I208L mutations resulted in significantly decreased susceptibility to RAL (44.0-fold and 54.9-fold, respectively) compared to wild-type virus (EC50=0.32 nM), and may represent novel pathways of HIV-1 resistance to modern treatments.Author summaryThere are many different types of HIV-1 around the world. Most of the research on how HIV-1 can become resistant to drug treatment has focused on the type (B) that is the most common in high-income countries. However, about 90% of infections around the world are caused by a type other than B. We used next-generation sequencing to analyze samples of HIV-1 from patients in Uganda (mostly infected by types A and D) for whom drug treatment failed to work, and whose infections did not fit the classic pattern of adaptation based on B. Next, we used machine learning to detect mutations in these virus populations that could explain the treatment outcomes. Finally, we experimentally added two candidate mutations identified by our analysis to a laboratory strain of HIV-1 and confirmed that they conferred drug resistance to the virus. Our study reveals new pathways that other types of HIV-1 may use to evolve resistance to drugs that make up the current recommended treatment for newly diagnosed individuals.

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Study on Suitable Analysis Method for HIV-1 Non-catalytic Integrase Inhibitor

10.21203/rs.3.rs-47642/v3 ◽

2020 ◽

Author(s):

Ki Hoon Park ◽

Minjee Kim ◽

Seoung Eun Bae ◽

Hee Jung Lee ◽

Kyung-Chang Kim ◽

...

Keyword(s):

Antiviral Activity ◽

Integrase Inhibitor ◽

Treatment Method ◽

Strand Transfer ◽

Inhibition Assay ◽

Analysis Method ◽

Catalytic Core ◽

Host Chromosome ◽

Inconsistent Result ◽

Hiv 1

Abstract Background: Integrase (IN) is an essential protein for HIV replication that catalyzes insertion of the reverse-transcribed viral genome into the host chromosome during the early steps of viral infection. Highly active anti-retroviral therapy (HAART) is a HIV/AIDS treatment method that combines three or more antiviral drugs often formulated from compounds that inhibit the activities of viral reverse transcriptase and protease enzymes. Early IN inhibitors (INIs) mainly serve as integrase strand transfer inhibitors (INSTI) that disrupt strand transfer by binding the catalytic core domain (CCD) of IN. However, mutations of IN can confer resistance to INSTI. Therefore, non-catalytic integrase inhibitors (NCINI) have been developed as next-generation INIs. Methods: In this study, we evaluated and compared the activity of INSTI and NCINI according to the analysis method. Antiviral activity was compared using p24 ELISA with MT2 cell and TZM-bl luciferase system with TZM-bl cell. Each drug was serially diluted and treated to MT2 and TZM-b1 cells, infected with HIV-1 AD8 strain and incubated for 5 and 2 days, respectively. Additionally, to analyze properties of INSTI and NCINI, transfer inhibition assay and 3'-processing inhibition assay were performed. Results: During screening of INIs using the p24 ELISA and TZM-bl luciferase systems, we found an inconsistent result with INSTI and NCINI drugs. Following infection of MT2 and TZM-bl cells with T-tropic HIV-1 strain, both INSTI and NCINI treatments induced significant p24 reduction in MT2 cells. However, NCINI showed no antiviral activity in the TZM-bl luciferase system, indicating that this widely used and convenient antiretroviral assay is not suitable for screening of NCINI compounds that target the second round of HIV-1 replication. Conclusion: Accordingly, we recommend application of other assay procedures, such as p24 ELISA or reverse transcription activity, in lieu of the TZM-bl luciferase system for preliminary NCINI drug screening. Utilization of appropriate analytical methods based on underlying mechanisms is necessary for accurate assessment of drug efficacy.

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Preclinical Profile of BI 224436, a Novel HIV-1 Non-Catalytic-Site Integrase Inhibitor

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02719-13 ◽

2014 ◽

Vol 58 (6) ◽

pp. 3233-3244 ◽

Cited By ~ 47

Author(s):

Craig Fenwick ◽

Ma'an Amad ◽

Murray D. Bailey ◽

Richard Bethell ◽

Michael Bös ◽

...

Keyword(s):

Antiviral Activity ◽

Phase 1 ◽

Integrase Inhibitor ◽

Parallel Synthesis ◽

Cell Permeability ◽

Strand Transfer ◽

Primary Resistance ◽

Catalytic Core ◽

Hiv 1

ABSTRACTBI 224436 is an HIV-1 integrase inhibitor with effective antiviral activity that acts through a mechanism that is distinct from that of integrase strand transfer inhibitors (INSTIs). This 3-quinolineacetic acid derivative series was identified using an enzymatic integrase long terminal repeat (LTR) DNA 3′-processing assay. A combination of medicinal chemistry, parallel synthesis, and structure-guided drug design led to the identification of BI 224436 as a candidate for preclinical profiling. It has antiviral 50% effective concentrations (EC50s) of <15 nM against different HIV-1 laboratory strains and cellular cytotoxicity of >90 μM. BI 224436 also has a low, ∼2.1-fold decrease in antiviral potency in the presence of 50% human serum and, by virtue of a steep dose-response curve slope, exhibits serum-shifted EC95values ranging between 22 and 75 nM. Passage of virus in the presence of inhibitor selected for either A128T, A128N, or L102F primary resistance substitutions, all mapping to a conserved allosteric pocket on the catalytic core of integrase. BI 224436 also retains full antiviral activity against recombinant viruses encoding INSTI resistance substitutions N155S, Q148H, and E92Q. In drug combination studies performed in cellular antiviral assays, BI 224436 displays an additive effect in combination with most approved antiretrovirals, including INSTIs. BI 224436 has drug-likein vitroabsorption, distribution, metabolism, and excretion (ADME) properties, including Caco-2 cell permeability, solubility, and low cytochrome P450 inhibition. It exhibited excellent pharmacokinetic profiles in rat (clearance as a percentage of hepatic flow [CL], 0.7%; bioavailability [F], 54%), monkey (CL, 23%;F, 82%), and dog (CL, 8%;F, 81%). Based on the excellent biological and pharmacokinetic profile, BI 224436 was advanced into phase 1 clinical trials.

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