scholarly journals GSK3640254 Is a Novel HIV-1 Maturation Inhibitor With an Optimized Virology Profile

2021 ◽  
Author(s):  
Ira Dicker ◽  
Jerry L. Jeffrey ◽  
Tricia Protack ◽  
Zeyu Lin ◽  
Mark Cockett ◽  
...  
Keyword(s):  
Antiviral Activity ◽  
Wild Type ◽  
Cleavage Rate ◽  
Virus Like Particles ◽  
Subtype B ◽  
Gastrointestinal Tolerability ◽  
Strong Antiviral Activity ◽  
Emergence Of Resistance ◽  
Hiv 1

HIV-1 maturation inhibitors (MIs) offer a novel mechanism of action and potential for use in HIV-1 treatment. Prior MIs displayed clinical efficacy but were associated with the emergence of resistance and some gastrointestinal tolerability events. Treatment with the potentially safer next-generation MI GSK3640254 (GSK’254) resulted in up to a 2-log 10 viral load reduction in a phase IIa proof-of-concept study. In vitro experiments have defined the antiviral and resistance profile for GSK’254. The compound displayed strong antiviral activity against a library of subtype B and C chimeric viruses containing Gag polymorphisms and site-directed mutants previously shown to affect potency of earlier-generation MIs, with a mean protein-binding adjusted 90% effective concentration of 33 nM. Furthermore, GSK’254 exhibited robust antiviral activity against a panel of HIV-1 clinical isolates, with a mean EC 50 of 9 nM. Mechanistic studies established that bound GSK’254 dissociated on average 7.1-fold more slowly from wild-type Gag virus-like particles (VLPs) compared with a previous-generation MI. In resistance studies, the previously identified A364V Gag region mutation was selected under MI pressure in cell culture and during the phase IIa clinical study. As expected, GSK’254 inhibited cleavage of p25 in a range of polymorphic HIV-1 Gag VLPs. Virus-like particles containing the A364V mutation exhibited a p25 cleavage rate 9.3 times faster than wild-type, providing a possible mechanism for MI resistance. The findings demonstrate that GSK’254 potently inhibits a broad range of HIV-1 strains expressing Gag polymorphisms.

scholarly journals The Retroviral Capsid Domain Dictates Virion Size, Morphology, and Coassembly of Gag into Virus-Like Particles

2005 ◽  
Vol 79 (21) ◽  
pp. 13463-13472 ◽  
Author(s):  
Danso Ako-Adjei ◽  
Marc C. Johnson ◽  
Volker M. Vogt
Keyword(s):  
Nuclear Export ◽  
Leukemia Virus ◽  
Particle Diameter ◽  
Particle Morphology ◽  
Structural Protein ◽  
Wild Type ◽  
Virus Like Particles ◽  
Gag Proteins ◽  
Hiv 1

ABSTRACT The retroviral structural protein, Gag, is capable of independently assembling into virus-like particles (VLPs) in living cells and in vitro. Immature VLPs of human immunodeficiency virus type 1 (HIV-1) and of Rous sarcoma virus (RSV) are morphologically distinct when viewed by transmission electron microscopy (TEM). To better understand the nature of the Gag-Gag interactions leading to these distinctions, we constructed vectors encoding several RSV/HIV-1 chimeric Gag proteins for expression in either insect cells or vertebrate cells. We used TEM, confocal fluorescence microscopy, and a novel correlative scanning EM (SEM)-confocal microscopy technique to study the assembly properties of these proteins. Most chimeric proteins assembled into regular VLPs, with the capsid (CA) domain being the primary determinant of overall particle diameter and morphology. The presence of domains between matrix and CA also influenced particle morphology by increasing the spacing between the inner electron-dense ring and the VLP membrane. Fluorescently tagged versions of wild-type RSV, HIV-1, or murine leukemia virus Gag did not colocalize in cells. However, wild-type Gag proteins colocalized extensively with chimeric Gag proteins bearing the same CA domain, implying that Gag interactions are mediated by CA. A dramatic example of this phenomenon was provided by a nuclear export-deficient chimera of RSV Gag carrying the HIV-1 CA domain, which by itself localized to the nucleus but relocalized to the cytoplasm in the presence of wild type HIV-1 Gag. Wild-type and chimeric Gag proteins were capable of coassembly into a single VLP as viewed by correlative fluorescence SEM if, and only if, the CA domain was derived from the same virus. These results imply that the primary selectivity of Gag-Gag interactions is determined by the CA domain.

Fluorine-Modifications Contribute to Potent Antiviral Activity against Highly Drug-Resistant HIV-1 and Favorable Blood-Brain Barrier (BBB) Penetration Property of Novel Central Nervous System (CNS)-targeting HIV-1 Protease Inhibitors In Vitro .

2022 ◽  
Author(s):  
Masayuki Amano ◽  
Ravikiran S. Yedidi ◽  
Pedro Miguel Salcedo-Gómez ◽  
Hironori Hayashi ◽  
Kazuya Hasegawa ◽  
...  
Keyword(s):  
Central Nervous System ◽  
Nervous System ◽  
Antiviral Activity ◽  
Protease Inhibitors ◽  
Blood Brain Barrier ◽  
Drug Resistant ◽  
Wild Type ◽  
Hiv 1 ◽  
Cns Targeting

To date, there are no specific treatment regimens for the HIV-1-related central nervous system (CNS) complications, such as HIV-1-associated neurocognitive disorders (HAND). In the present study, we report that two newly generated CNS-targeting HIV-1 protease inhibitors (PIs), GRL-08513 and GRL-08613, which have P1-3,5- bis -fluorophenyl- or P1- para -monofluorophenyl-ring, and P2-tetrahydropyrano-tetrahydrofuran ( Tp -THF) with a sulfonamide isostere, are potent against wild-type HIV-1s and multiple clinically isolated HIV-1s (EC 50 : 0.0001∼0.0032 μM). As assessed with HIV-1 variants that had been selected in vitro to propagate at 5 μM concentration of each HIV-1 PI (atazanavir, lopinavir, or amprenavir), GRL-08513 and GRL-08613 efficiently inhibited the replication of these highly-PI-resistant variants (EC 50 : 0.003∼0.006 μM). GRL-08513 and GRL-08613 also maintained their antiviral activity against HIV-2 ROD as well as severe multi-drug-resistant clinical HIV-1 variants. Additionally, when we assessed with the in vitro blood-brain barrier (BBB) reconstruction system, GRL-08513 and GRL-08613 showed the most promising properties of CNS-penetration among the evaluated compounds including the majority of FDA-approved cART drugs. In the crystallographic analysis of compound-protease (PR) complexes, it was demonstrated that the Tp -THF rings at the P2 moiety of GRL-08513 and GRL-08613 form robust hydrogen-bond interactions with the active-site of HIV-1 PR. Furthermore, both the P1-3,5- bis -fluorophenyl- and P1- para -monofluorophenyl-rings sustain greater contact surfaces and form stronger van der Waals interactions with PR compared to the case of darunavir-PR complex. Taken together, these results strongly suggest that GRL-08513 and GRL-08613 have favorable features for the patients infected with wild-type/multi-drug-resistant HIV-1s, and might serve as candidates of preventive and/or therapeutic for HAND and other CNS complications.

scholarly journals In VitroandIn VivoActivities of AIC292, a Novel HIV-1 Nonnucleoside Reverse Transcriptase Inhibitor

2013 ◽  
Vol 57 (11) ◽  
pp. 5320-5329 ◽  
Author(s):  
Steffen Wildum ◽  
Daniela Paulsen ◽  
Kai Thede ◽  
Helga Ruebsamen-Schaeff ◽  
Holger Zimmermann
Keyword(s):  
Antiviral Activity ◽  
Reverse Transcriptase ◽  
Highly Active Antiretroviral Therapy ◽  
Xenograft Model ◽  
Wild Type ◽  
Mouse Xenograft ◽  
Mouse Xenograft Model ◽  
Hiv 1

ABSTRACTNonnucleoside reverse transcriptase inhibitors (NNRTIs) are important and frequently used elements of highly active antiretroviral therapy (HAART) for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. However, the development of drug resistance, as well as the side effects of existing drugs, defines a medical need for novel NNRTIs with excellent tolerability, improved activity against NNRTI-resistant viruses, and a low barrier to resistance. Within the chemical class of diarylpyrazole-[imidazolidinone]-carboxamides, AIC292 was identified as a promising novel HIV-1 NNRTI and has successfully completed single-dose clinical phase I studies. Here, we report on the antiviral activity of AIC292, evaluatedin vitroagainst wild-type and NNRTI-resistant HIV-1 isolates andin vivousing an engineered mouse xenograft model. AIC292 inhibited wild-type HIV-1 laboratory strains at low nanomolar concentrations, was well tolerated in different cell lines, and showed excellent selectivity in a lead profiling screen. In addition, activity of AIC292 could be demonstrated against a broad panel of wild-type HIV-1 group M and group O clinical isolates. AIC292 also retained activity against viruses harboring NNRTI resistance-associated mutations (RAMs), including the most prevalent variants, K103N, Y181C, and G190A. Interestingly, viruses bearing the L100I RAM were hypersusceptible to AIC292. Two-drug combination assays showed no antagonistic interactions between AIC292 and representative marketed HIV drugs with regard to antiviral activity. Furthermore, AIC292 displayed potent antiviralin vivoefficacy in a mouse xenograft model when applied once daily. Taken together, these data show that AIC292 represents a molecule with the antiviral properties of a novel NNRTI for the treatment of HIV-1 infection.

scholarly journals P2′ Benzene Carboxylic Acid Moiety Is Associated with Decrease in Cellular Uptake: Evaluation of Novel Nonpeptidic HIV-1 Protease Inhibitors Containing P2bis-Tetrahydrofuran Moiety

2013 ◽  
Vol 57 (10) ◽  
pp. 4920-4927 ◽  
Author(s):  
Ravikiran S. Yedidi ◽  
Kenji Maeda ◽  
W. Sean Fyvie ◽  
Melinda Steffey ◽  
David A. Davis ◽  
...  
Keyword(s):  
Carboxylic Acid ◽  
Antiviral Activity ◽  
Protease Inhibitors ◽  
Cellular Uptake ◽  
Wild Type ◽  
Acid Moiety ◽  
Binding Profile ◽  
Cell Extracts ◽  
Hiv 1

ABSTRACTGRL007 and GRL008, two structurally related nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) containing 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF) as the P2 moiety and a sulfonamide isostere consisting of benzene carboxylic acid and benzene carboxamide as the P2′ moiety, respectively, were evaluated for their antiviral activity and interactions with wild-type protease (PRWT). Both GRL007 (Kiof 12.7 pM with PRWT) and GRL008 (Kiof 8.9 pM) inhibited PRWTwith high potencyin vitro. X-ray crystallographic analysis of PRWTin complex with GRL007 or GRL008 showed that thebis-THF moiety of both compounds has three direct polar contacts with the backbone amide nitrogen atoms of Asp29 and Asp30 of PRWT. The P2′ moiety of both compounds showed one direct contact with the backbone of Asp30′ and a bridging polar contact with Gly48′ through a water molecule. Cell-based antiviral assays showed that GRL007 was inactive (50% effective concentration [EC50] of >1 μM) while GRL008 was highly active (EC50of 0.04 μM) against wild-type HIV-1. High-performance liquid chromatography (HPLC)/mass spectrometry-based cellular uptake assays showed 8.1- and 84-fold higher intracellular concentrations of GRL008 than GRL007 in human MT-2 and MT-4 cell extracts, respectively. Thus, GRL007, in spite of its favorable enzyme-inhibitory activity and protease binding profile, exhibited a lack of antiviral activity in cell-based assays, most likely due to its compromised cellular uptake associated with its P2′ benzene carboxylic acid moiety. The anti-HIV-1 potency, favorable toxicity, and binding profile of GRL008 suggest that further optimization of the P2′ moiety may improve its antiretroviral features.

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

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.

scholarly journals Effect of Small Polyanions on In Vitro Assembly of Selected Members of Alpha-, Beta- and Gammaretroviruses

Viruses ◽  
2021 ◽  
Vol 13 (1) ◽  
pp. 129
Author(s):  
Alžběta Dostálková ◽  
Barbora Vokatá ◽  
Filip Kaufman ◽  
Pavel Ulbrich ◽  
Tomáš Ruml ◽  
...  
Keyword(s):  
Equine Infectious Anemia Virus ◽  
Electron Tomography ◽  
Leukemia Virus ◽  
Virus Like Particles ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Anemia Virus ◽  
Immature Virus ◽  
Hiv 1

The assembly of a hexameric lattice of retroviral immature particles requires the involvement of cell factors such as proteins and small molecules. A small, negatively charged polyanionic molecule, myo-inositol hexaphosphate (IP6), was identified to stimulate the assembly of immature particles of HIV-1 and other lentiviruses. Interestingly, cryo-electron tomography analysis of the immature particles of two lentiviruses, HIV-1 and equine infectious anemia virus (EIAV), revealed that the IP6 binding site is similar. Based on this amino acid conservation of the IP6 interacting site, it is presumed that the assembly of immature particles of all lentiviruses is stimulated by IP6. Although this specific region for IP6 binding may be unique for lentiviruses, it is plausible that other retroviral species also recruit some small polyanion to facilitate the assembly of their immature particles. To study whether the assembly of retroviruses other than lentiviruses can be stimulated by polyanionic molecules, we measured the effect of various polyanions on the assembly of immature virus-like particles of Rous sarcoma virus (RSV), a member of alpharetroviruses, Mason-Pfizer monkey virus (M-PMV) representative of betaretroviruses, and murine leukemia virus (MLV), a member of gammaretroviruses. RSV, M-PMV and MLV immature virus-like particles were assembled in vitro from truncated Gag molecules and the effect of selected polyanions, myo-inostol hexaphosphate, myo-inositol, glucose-1,6-bisphosphate, myo-inositol hexasulphate, and mellitic acid, on the particles assembly was quantified. Our results suggest that the assembly of immature particles of RSV and MLV was indeed stimulated by the presence of myo-inostol hexaphosphate and myo-inositol, respectively. In contrast, no effect on the assembly of M-PMV as a betaretrovirus member was observed.

scholarly journals A Conformational Transition Observed in Single HIV-1 Gag Molecules during In Vitro Assembly of Virus-Like Particles

2014 ◽  
Vol 88 (6) ◽  
pp. 3577-3585 ◽  
Author(s):  
J. B. Munro ◽  
A. Nath ◽  
M. Farber ◽  
S. A. K. Datta ◽  
A. Rein ◽  
...  
Keyword(s):  
Conformational Transition ◽  
Virus Like Particles ◽  
In Vitro Assembly ◽  
Hiv 1

scholarly journals In Vitro Infection Model Characterizing the Effect of Efflux Pump Inhibition on Prevention of Resistance to Levofloxacin and Ciprofloxacin in Streptococcus pneumoniae

2007 ◽  
Vol 51 (11) ◽  
pp. 3988-4000 ◽  
Author(s):  
Arnold Louie ◽  
David L. Brown ◽  
Weiguo Liu ◽  
Robert W. Kulawy ◽  
Mark R. Deziel ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Efflux Pump ◽  
Community Acquired Pneumonia ◽  
Fluoroquinolone Resistance ◽  
Infection Model ◽  
Wild Type ◽  
Efflux Pump Inhibition ◽  
Short Term Exposure ◽  
Emergence Of Resistance

ABSTRACT The prevalence of fluoroquinolone-resistant Streptococcus pneumoniae is slowly rising as a consequence of the increased use of fluoroquinolone antibiotics to treat community-acquired pneumonia. We tested the hypothesis that increased efflux pump (EP) expression by S. pneumoniae may facilitate the emergence of fluoroquinolone resistance. By using an in vitro pharmacodynamic infection system, a wild-type S. pneumoniae strain (Spn-058) and an isogenic strain with EP overexpression (Spn-RC2) were treated for 10 days with ciprofloxacin or levofloxacin in the presence or absence of the EP inhibitor reserpine to evaluate the effect of EP inhibition on the emergence of resistance. Cultures of Spn-058 and Spn-RC2 were exposed to concentration-time profiles simulating those in humans treated with a regimen of ciprofloxacin at 750 mg orally once every 12 h and with regimens of levofloxacin at 500 and 750 mg orally once daily (QD; with or without continuous infusions of 20 μg of reserpine/ml). The MICs of ciprofloxacin and levofloxacin for Spn-058 were both 1 μg/ml when susceptibility testing was conducted with each antibiotic alone and with each antibiotic in the presence of reserpine. For Spn-RC2, the MIC of levofloxacin alone and with reserpine was also 1 μg/ml; the MICs of ciprofloxacin were 2 and 1 μg/ml, respectively, when determined with ciprofloxacin alone and in combination with reserpine. Reserpine, alone, had no effect on the growth of Spn-058 and Spn-RC2. For Spn-058, simulated regimens of ciprofloxacin at 750 mg every 12 h or levofloxacin at 500 mg QD were associated with the emergence of fluoroquinolone resistance. However, the use of ciprofloxacin at 750 mg every 12 h and levofloxacin at 500 mg QD in combination with reserpine rapidly killed Spn-058 and prevented the emergence of resistance. For Spn-RC2, levofloxacin at 500 mg QD was associated with the emergence of resistance, but again, the resistance was prevented when this levofloxacin regimen was combined with reserpine. Ciprofloxacin at 750 mg every 12 h also rapidly selected for ciprofloxacin-resistant mutants of Spn-RC2. However, the addition of reserpine to ciprofloxacin therapy only delayed the emergence of resistance. Levofloxacin at 750 mg QD, with and without reserpine, effectively eradicated Spn-058 and Spn-RC2 without selecting for fluoroquinolone resistance. Ethidium bromide uptake and efflux studies demonstrated that, at the baseline, Spn-RC2 had greater EP expression than Spn-058. These studies also showed that ciprofloxacin was a better inducer of EP expression than levofloxacin in both Spn-058 and Spn-RC2. However, in these isolates, the increase in EP expression by short-term exposure to ciprofloxacin and levofloxacin was transient. Mutants of Spn-058 and Spn-RC2 that emerged under suboptimal antibiotic regimens had a stable increase in EP expression. Levofloxacin at 500 mg QD in combination with reserpine, an EP inhibitor, or at 750 mg QD alone killed wild-type S. pneumoniae and strains that overexpressed reserpine-inhibitable EPs and was highly effective in preventing the emergence of fluoroquinolone resistance in S. pneumoniae during therapy. Ciprofloxacin at 750 mg every 12 h, as monotherapy, was ineffective for the treatment of Spn-058 and Spn-RC2. Ciprofloxacin in combination with reserpine prevented the emergence of resistance in Spn-058 but not in Spn-RC2, the EP-overexpressing strain.

scholarly journals In Vitro Antiviral Activity and Cross-Resistance Profile of PL-100, a Novel Protease Inhibitor of Human Immunodeficiency Virus Type 1

2007 ◽  
Vol 51 (11) ◽  
pp. 4036-4043 ◽  
Author(s):  
Serge Dandache ◽  
Guy Sévigny ◽  
Jocelyn Yelle ◽  
Brent R. Stranix ◽  
Neil Parkin ◽  
...  
Keyword(s):  
Human Immunodeficiency Virus ◽  
Antiviral Activity ◽  
Highly Active Antiretroviral Therapy ◽  
Cross Resistance ◽  
Drug Resistant ◽  
Resistance Profile ◽  
Immunodeficiency Virus ◽  
Hiv 1

ABSTRACT Despite the success of highly active antiretroviral therapy, the current emergence and spread of drug-resistant variants of human immunodeficiency virus (HIV) stress the need for new inhibitors with distinct properties. We designed, produced, and screened a library of compounds based on an original l-lysine scaffold for their potentials as HIV type 1 (HIV-1) protease inhibitors (PI). One candidate compound, PL-100, emerged as a specific and noncytotoxic PI that exhibited potent inhibition of HIV-1 protease and viral replication in vitro (Ki , ∼36 pM, and 50% effective concentration [EC50], ∼16 nM, respectively). To confirm that PL-100 possessed a favorable resistance profile, we performed a cross-resistance study using a panel of 63 viral strains from PI-experienced patients selected for the presence of primary PI mutations known to confer resistance to multiple PIs now in clinical use. The results showed that PL-100 retained excellent antiviral activity against almost all of these PI-resistant viruses and that its performance in this regard was superior to those of atazanavir, amprenavir, indinavir, lopinavir, nelfinavir, and saquinavir. In almost every case, the increase in the EC50 for PL-100 observed with viruses containing multiple mutations in protease was far less than that obtained with the other drugs tested. These data underscore the potential for PL-100 to be used in the treatment of drug-resistant HIV disease and argue for its further development.

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

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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