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

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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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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Wild-Type-Like Viral Replication Potential of Human Immunodeficiency Virus Type 1 Envelope Mutants Lacking Palmitoylation Signals

Journal of Virology ◽

10.1128/jvi.79.13.8374-8387.2005 ◽

2005 ◽

Vol 79 (13) ◽

pp. 8374-8387 ◽

Cited By ~ 30

Author(s):

Woan-Eng Chan ◽

Hui-Hua Lin ◽

Steve S.-L. Chen

Keyword(s):

T Cells ◽

Lipid Rafts ◽

Virus Type ◽

Lipid Raft ◽

Transmembrane Protein ◽

Cytoplasmic Tail ◽

Cell Surface Expression ◽

Wild Type ◽

Hiv 1

ABSTRACT Palmitoylation of the cytoplasmic domain of the human immunodeficiency type virus type 1 (HIV-1) envelope (Env) transmembrane protein, gp41, has been implicated in Env targeting to detergent-resistant lipid rafts, Env incorporation into the virus, and viral infectivity. In contrast, we provide evidence here to show that HIV-1 infectivity, Env targeting to lipid rafts, and Env incorporation into the virus are independent of cytoplasmic tail palmitoylation. The T-cell (T)-tropic HXB2-based virus, which utilizes CXCR4 as the entry coreceptor, carrying a Cys-to-Ser mutation at residue 764 or 837 or at both replicated with wild-type (WT) virus replication kinetics in CD4+ T cells. The properties of Env expression, precursor processing, cell surface expression, and Env incorporation of these three mutant viruses were normal compared to those of the WT virus. These three mutant Env proteins all effectively mediated one-cycle virus infection. When the Cys residues were replaced by Ala residues, all single and double mutants still retained the phenotypes of infectivity, Env incorporation, and lipid raft localization of the WT Env. When Cys-to-Ala substitutions were introduced into the macrophage (M)-tropic ConB virus, which utilizes CCR5 as the coreceptor, these mutations did not affect the replication potential, Env phenotypes, lipid raft targeting, or Env assembly into the virus of the WT Env. These T- and M-tropic mutants also productively replicated in human primary CD4+ T cells. Moreover, mutations at both Cys residues significantly reduced the level of palmitoylation of the Env. Our results together support the notion that palmitoylation of the cytoplasmic tail of the HIV-1 Env is not essential for the HIV-1 virus life cycle.

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Subtype-Specific Differences in the Human Immunodeficiency Virus Type 1 Reverse Transcriptase Connection Subdomain of CRF01_AE Are Associated with Higher Levels of Resistance to 3′-Azido-3′-Deoxythymidine

Journal of Virology ◽

10.1128/jvi.00859-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8502-8513 ◽

Cited By ~ 25

Author(s):

Krista A. Delviks-Frankenberry ◽

Galina N. Nikolenko ◽

Frank Maldarelli ◽

Saiki Hase ◽

Yutaka Takebe ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Rnase H ◽

Wild Type ◽

Dna Templates ◽

Subtype B ◽

Immunodeficiency Virus ◽

Azt Resistance ◽

Hiv 1 ◽

Rna And Dna

ABSTRACT 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.

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Interaction between Hck and HIV-1 Nef negatively regulates cell surface expression of M-CSF receptor

Blood ◽

10.1182/blood-2007-04-086017 ◽

2008 ◽

Vol 111 (1) ◽

pp. 243-250 ◽

Cited By ~ 28

Author(s):

Masateru Hiyoshi ◽

Shinya Suzu ◽

Yuka Yoshidomi ◽

Ranya Hassan ◽

Hideki Harada ◽

...

Keyword(s):

Cell Surface ◽

Direct Interaction ◽

Surface Expression ◽

Inhibitory Effect ◽

Cell Surface Expression ◽

Down Regulation ◽

Critical Determinant ◽

Src Tyrosine Kinase ◽

Macrophage Colony ◽

Hiv 1

Nef is a multifunctional pathogenetic protein of HIV-1, the interaction of which with Hck, a Src tyrosine kinase highly expressed in macrophages, has been shown to be responsible for the development of AIDS. However, how the Nef-Hck interaction leads to the functional aberration of macrophages is poorly understood. We recently showed that Nef markedly inhibited the activity of macrophage colony-stimulating factor (M-CSF), a primary cytokine for macrophages. Here, we show that the inhibitory effect of Nef is due to the Hck-dependent down-regulation of the cell surface expression of M-CSF receptor Fms. In the presence of Hck, Nef induced the accumulation of an immature under–N-glycosylated Fms at the Golgi, thereby down-regulating Fms. The activation of Hck by the direct interaction with Nef was indispensable for the down-regulation. Unexpectedly, the accumulation of the active Hck at the Golgi where Nef prelocalized was likely to be another critical determinant of the function of Nef, because the expression of the constitutive-active forms of Hck alone did not fully down-regulate Fms. These results suggest that Nef perturbs the intracellular maturation and the trafficking of nascent Fms, through a unique mechanism that required both the activation of Hck and the aberrant spatial regulation of the active Hck.

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Differences in the Fitness of Two Diverse Wild-Type Human Immunodeficiency Virus Type 1 Isolates Are Related to the Efficiency of Cell Binding and Entry

Journal of Virology ◽

10.1128/jvi.79.11.7121-7134.2005 ◽

2005 ◽

Vol 79 (11) ◽

pp. 7121-7134 ◽

Cited By ~ 71

Author(s):

Andre J. Marozsan ◽

Dawn M. Moore ◽

Michael A. Lobritz ◽

Erika Fraundorf ◽

Awet Abraha ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Human Immunodeficiency Virus Type ◽

Virus Type ◽

Host Cells ◽

Replicative Capacity ◽

Wild Type ◽

Subtype B ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT The ability of one primary human immunodeficiency virus type 1 (HIV-1) isolate to outcompete another in primary CD4+ human lymphoid cells appears to be mediated by the efficiency of host cell entry. This study was designed to test the role of entry on fitness of wild-type HIV-1 isolates (e.g., replicative capacity) and to examine the mechanism(s) involved in differential entry efficiency. The gp120 coding regions of two diverse HIV-1 isolates (the more-fit subtype B strain, B5-91US056, and less-fit C strain, C5-97ZA003) were cloned into a neutral HIV-1 backbone by using a recently described yeast cloning technique. The fitness of the primary B5 HIV-1 isolates and its env gene cloned into the NL4-3 laboratory strain had similar fitness, and both were more fit than the C5 primary isolate and its env/NL4-3 chimeric counterpart. Increased fitness of the B5 over C5 virus was mediated by the gp120 coding region of the env gene. An increase in binding/fusion, as well as decreased sensitivity to entry inhibitors (PSC-RANTES and T-20), was observed in cell fusion assays mediated by B5 gp120 compared to C5 gp120. Competitive binding assays using a novel whole virus-cell system indicate that the primary or chimeric B5 had a higher avidity for CD4/CCR5 on host cells than the C5 counterpart. This increased avidity of an HIV-1 isolate for its cell receptors may be a significant factor influencing overall replicative capacity or fitness.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01876-21 ◽

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.

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Role for CCR5Δ32 Protein in Resistance to R5, R5X4, and X4 Human Immunodeficiency Virus Type 1 in Primary CD4+ Cells

Journal of Virology ◽

10.1128/jvi.78.5.2277-2287.2004 ◽

2004 ◽

Vol 78 (5) ◽

pp. 2277-2287 ◽

Cited By ~ 67

Author(s):

Lokesh Agrawal ◽

Xihua Lu ◽

Jin Qingwen ◽

Zainab VanHorn-Ali ◽

Ioan Vlad Nicolescu ◽

...

Keyword(s):

Human Immunodeficiency Virus ◽

Mononuclear Cells ◽

Genetic Resistance ◽

Surface Expression ◽

Mutant Protein ◽

Cell Surface Expression ◽

Wild Type ◽

Immunodeficiency Virus ◽

Hiv 1

ABSTRACT CCR5Δ32 is a loss-of-function mutation that abolishes cell surface expression of the human immunodeficiency virus (HIV) coreceptor CCR5 and provides genetic resistance to HIV infection and disease progression. Since CXCR4 and other HIV coreceptors also exist, we hypothesized that CCR5Δ32-mediated resistance may be due not only to the loss of CCR5 function but also to a gain-of-function mechanism, specifically the active inhibition of alternative coreceptors by the mutant CCR5Δ32 protein. Here we demonstrate that efficient expression of the CCR5Δ32 protein in primary CD4+ cells by use of a recombinant adenovirus (Ad5/Δ32) was able to down-regulate surface expression of both wild-type CCR5 and CXCR4 and to confer broad resistance to R5, R5X4, and X4 HIV type 1 (HIV-1). This may be important clinically, since we found that CD4+ cells purified from peripheral blood mononuclear cells of individuals who were homozygous for CCR5Δ32, which expressed the mutant protein endogenously, consistently expressed lower levels of CXCR4 and showed less susceptibility to X4 HIV-1 isolates than cells from individuals lacking the mutation. Moreover, CD4+ cells from individuals who were homozygous for CCR5Δ32 expressed the mutant protein in five of five HIV-exposed, uninfected donors tested but not in either of two HIV-infected donors tested. The mechanism of inhibition may involve direct scavenging, since we were able to observe a direct interaction of CCR5 and CXCR4 with CCR5Δ32, both by genetic criteria using the yeast two-hybrid system and by biochemical criteria using the coimmunoprecipitation of heterodimers. Thus, these results suggest that at least two distinct mechanisms may account for genetic resistance to HIV conferred by CCR5Δ32: the loss of wild-type CCR5 surface expression and the generation of CCR5Δ32 protein, which functions as a scavenger of both CCR5 and CXCR4.

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04274-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 310-316 ◽

Cited By ~ 10

Author(s):

Vincent Cutillas ◽

Thibault Mesplede ◽

Kaitlin Anstett ◽

Said Hassounah ◽

Mark A. Wainberg

Keyword(s):

Dna Binding ◽

Integrase Inhibitor ◽

Strand Transfer ◽

Wild Type ◽

Transfer Activity ◽

Subtype B ◽

Integrase Strand Transfer Inhibitors ◽

Dna Binding Affinity ◽

Hiv 1 ◽

Level Resistance

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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Thermostable HIV-1 group O reverse transcriptase variants with the same fidelity as murine leukaemia virus reverse transcriptase

Biochemical Journal ◽

10.1042/bj20101852 ◽

2011 ◽

Vol 436 (3) ◽

pp. 599-607 ◽

Cited By ~ 20

Author(s):

Verónica Barrioluengo ◽

Mar Álvarez ◽

Daniela Barbieri ◽

Luis Menéndez-Arias

Keyword(s):

Reverse Transcriptase ◽

Polymerase Activity ◽

Wild Type ◽

Wild Type Enzyme ◽

Leukaemia Virus ◽

Murine Leukaemia Virus ◽

Subtype B ◽

Rna Targets ◽

Forward Mutation ◽

Hiv 1

Wild-type HIV-1 group O RT (reverse transcriptase) shows increased thermostability in comparison with HIV-1 group M subtype B RT and MLV (murine leukaemia virus) RT. However, its utility in the amplification of RNA targets is limited by the reduced accuracy of lentiviral RTs compared with oncoretroviral RTs (i.e. MLV RT). The effects of the mutations K65R, R78A and K65R/V75I on the fidelity of HIV-1 group O RTs were studied using gel-based and M13mp2 lacZ forward-mutation fidelity assays. Forward-mutation assays demonstrated that mutant RTs K65R, R78A and K65R/V75I showed >9-fold increased accuracy in comparison with the wild-type enzyme and were approximately two times more faithful than the MLV RT. Compared with MLV RT, all of the tested HIV-1 group O RT variants showed decreased frameshift fidelity. However, K65R RT showed a higher tendency to introduce one-nucleotide deletions in comparison with other HIV-1 group O RT variants. R78A had a destabilizing effect on the RT, either in the presence or absence of V75I. At temperatures above 52 °C, K65R and K65R/V75I retained similar levels of DNA polymerase activity to the wild-type HIV-1 group O RT, but were more efficient than HIV-1 group M subtype B and MLV RTs. K65R, K65R/V75I and R78A RTs showed decreased misinsertion and mispair extension fidelity in comparison with the wild-type enzyme for most base pairs studied. These assays revealed that nucleotide selection is mainly governed by kpol (pol is polymerization) in the case of K65R, whereas both kpol and Kd affect nucleotide discrimination in the case of K65R/V75I.

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