Limited HIV-associated neuropathologies and lack of immune activation in sub-saharan African individuals with late-stage subtype C HIV-1 infection.

Although previous studies have suggested that subtype B HIV-1 proviruses in the brain are associated with physiological changes and immune activation accompanied with microgliosis and astrogliosis, and indicated that both HIV-1 subtype variation and geographical location might influence the neuropathogenicity of HIV-1 in the brain. The natural course of neuropathogenesis of the most widespread subtype C HIV-1 has not been adequately investigated, especially for people living with HIV (PLWH) in sub-Saharan Africa. To characterize the natural neuropathology of subtype C HIV-1, postmortem frontal lobe and basal ganglia tissues were collected from nine ART-naïve individuals who died of late-stage AIDS with subtype C HIV-1 infection, and eight uninfected deceased individuals as controls. Histological staining was performed on all brain tissues to assess brain pathologies. Immunohistochemistry (IHC) against CD4, p24, Iba-1, GFAP, and CD8 in all brain tissues was conducted to evaluate potential viral production and immune activation. Histological results showed mild perivascular cuffs of lymphocytes only in a minority of the infected individuals. Viral capsid p24 protein was only detected in circulating immune cells of one infected individual, suggesting a lack of productive HIV-1 infection of the brain even at the late-stage of AIDS. Notably, similar levels of Iba-1 or GFAP between HIV + and HIV- brain tissues indicated a lack of microgliosis and astrogliosis, respectively. Similar levels of CD8 + cytotoxic T lymphocyte (CTL) infiltration between HIV + and HIV- brain tissues indicated CTL were not likely to be involved within subtype C HIV-1 infected participants of this cohort. Results from this subtype C HIV-1 study suggest that there is a lack of productive infection and limited neuropathogenesis by subtype C HIV-1 even at late-stage disease, which is in contrast to what was reported for subtype B HIV-1 by other investigators.

Impact of subtype C-specific amino acid variants on HIV-1 Tat-TAR interaction: insights from molecular modelling and dynamics.

BACKGROUND: HIV-1 produces Tat, a crucial protein for transcription, viral replication, and CNS neurotoxicity. Tat interacts with TAR, enhancing HIV reverse transcription. Subtype C Tat variants (C31S, R57S, Q63E) are associated with reduced transactivation and neurovirulence compared to subtype B. However, their precise impact on Tat-TAR binding is unclear. This study investigates how these substitutions affect Tat-TAR interaction. METHODS: We utilized molecular modelling techniques, including MODELLER, to produce precise three-dimensional structures of HIV-1 Tat protein variants. We utilized Tat subtype B as the reference or wild type, and generated Tat variants to mirror those amino acid variants found in Tat subtype C. Subtype C-specific amino acid substitutions were selected based on their role in the neuropathogenesis of HIV-1. Subsequently, we conducted molecular docking of each Tat protein variant to TAR using HDOCK, followed by molecular dynamic simulations. RESULTS: Molecular docking results indicated that Tat subtype B (TatWt) showed the highest affinity for the TAR element (-262.07), followed by TatC31S (-261.61), TatQ63E (-256.43), TatC31S/R57S/Q63E (-238.92), and TatR57S (-222.24). However, binding free energy analysis showed higher affinities for single variants TatQ63E (-349.2 ± 10.4 kcal/mol) and TatR57S (-290.0 ± 9.6 kcal/mol) compared to TatWt (-247.9 ± 27.7 kcal/mol), while TatC31S and TatC31S/R57SQ/63E showed lower values. Interactions over the protein trajectory were also higher for TatQ63E and TatR57S compared to TatWt, TatC31S, and TatC31S/R57SQ/63E, suggesting that modifying amino acids within the Arginine/Glutamine-rich region notably affects TAR interaction. Single amino acid mutations TatR57S and TatQ63E had a significant impact, while TatC31S had minimal effect. Introducing single amino acid variants from TatWt to a more representative Tat subtype C (TatC31S/R57SQ/63E) resulted in lower predicted binding affinity, consistent with previous findings. CONCLUSIONS: These identified amino acid positions likely contribute significantly to Tat-TAR interaction and the differential pathogenesis and neuropathogenesis observed between subtype B and subtype C. Additional experimental investigations should prioritize exploring the influence of these amino acid signatures on TAR binding to gain a comprehensive understanding of their impact on viral transactivation, potentially identifying them as therapeutic targets.

The emergence and circulation of human immunodeficiency virus (HIV)-1 subtype C.

Introduction. Human immunodeficiency virus (HIV)-1 subtype C is the most prevalent globally and is thought to have originated in non-human primates in the Democratic Republic of Congo.Hypothesis/Gap Statement. Although the global dominance of HIV-1 subtype C is well established, a thorough understanding of its evolutionary history and transmission dynamics across various risk populations remains elusive. The current knowledge is insufficient to fully capture the global diversification and dissemination of this subtype.Aim. We for the first time sought to investigate the global evolutionary history and spatiotemporal dynamics of HIV-1 subtype C using a selection of maximum-likelihood-based phylodynamic approaches on a total of 1210 near full-length genomic sequences sampled from 32 countries, collected in 4 continents, with sampling dates between 1986-2019 among various risk groups were analysed.Methodology. We subsampled the HIV-1 subtype C genomic datasets based on continent and risk group traits, and performed nucleotide substitution model selection analysis, maximum likelihood (ML) phylogenetic reconstruction, phylogenetic tree topology similarity analysis, temporal signal analysis and traced the timings of viral spread both geographically and by risk group.Results. Based on the phylodynamic analyses of four datasets (full1210, locrisk626, loc562 and risk393), we inferred the time to the most recent common ancestor (TMRCA) in the 1930s and an evolutionary rate of 0.0023 substitutions per site per year. The total number of introduction events of HIV-1 subtype C between continents and between risk groups is estimated to be 71 and 115, respectively. The largest number of introductions occurred from Africa to Europe (n=32), from not-recorded to heterosexual (n=40) and from heterosexual to not-recorded (n=51) risk groups.Conclusion. Our results emphasize that HIV subtype C has mainly spread from Africa to Europe, likely through heterosexual transmission.

Mechanism of drug resistance in HIV-1 protease subtype C in the presence of Atazanavir.

AIDS is one of the deadliest diseases in the history of humankind caused by HIV. Despite the technological development, curtailing the viral infection inside human host still remains a challenge. Therapies such as HAART uses a combination of drugs to inhibit the viral activity. One of the important targets includes HIV protease and inhibiting its activity will minimize the production of mature structural proteins. However, the genetic diversity and the occurrence of drug resistant mutations adds complexity to effective drug design. In this study, we aimed at understanding the drug binding mechanism of one such subtype, namely subtype C and its insertion variant L38HL. We performed multiple molecular dynamics simulations along with binding free energy analysis of wild-type and L38HL bound to Atazanavir (ATV). From the analysis, we revealed that the insertion alters the hydrogen bond and hydrophobic interaction networks. The alterations in the interaction networks increase flexibility at the hinge-fulcrum interface. Further, the effects of these changes affect flap tip curling. Moreover, the changes in the hinge-fulcrum-cantilever interface alters the concerted motion of the functional regions leading to change in the direction of flap movement thus causing a subtle change in the active site volume. Additionally, formation of intramolecular hydrogen bonds in the ATV docked to L38HL restricted the movement of R1 and R2 groups thereby altering the interactions. Overall, the changes in the flexibility of flap together with the changes in the active site volume and compactness of the ligand provide insights for increased binding affinity of ATV with L38HL.

Cannabis Use Associates With Reduced Proviral Burden and Inflammatory Cytokine in Tissues From Men With Clade C HIV-1 on Suppressive Antiretroviral Therapy.

BACKGROUND: Human immunodeficiency virus 1 (HIV-1) tissue reservoirs remain the main obstacle against an HIV cure. Limited information exists regarding cannabis's effects on HIV-1 infections in vivo, and the impact of cannabis use on HIV-1 parenchymal tissue reservoirs is unexplored. METHODS: To investigate whether cannabis use alters HIV-1 tissue reservoirs, we systematically collected 21 postmortem brain and peripheral tissues from 20 men with subtype C HIV-1 and with suppressed viral load enrolled in Zambia, 10 of whom tested positive for cannabis use. The tissue distribution and copies of subtype C HIV-1 LTR, gag, env DNA and RNA, and the relative mRNA levels of cytokines IL-1ß, IL-6, IL-10, and TGF-ß1 were quantified using PCR-based approaches. Utilizing generalized linear mixed models we compared persons with HIV-1 and suppressed viral load, with and without cannabis use. RESULTS: The odds of tissues harboring HIV-1 DNA and the viral DNA copies in those tissues were significantly lower in persons using cannabis. Moreover, the transcription levels of proinflammatory cytokines IL-1ß and IL-6 in lymphoid tissues of persons using cannabis were also significantly lower. CONCLUSIONS: Our findings suggested that cannabis use is associated with reduced sizes and inflammatory cytokine expression of subtype C HIV-1 reservoirs in men with suppressed viral load.

Delayed identification of treatment failure causes high levels of acquired drug resistance and less future drug options among HIV-1-infected South Indians.

PURPOSE: HIV-1 Drug Resistance Mutations (DRMs) among Immunological failure (IF) on NRTI based first-line regimens, Thymidine analogue (TA) - AZT & D4T and Non-Thymidine Analogue (NTA) -TDF; and predict viral drug susceptibility to gain vision about optimal treatment strategies for second-line. METHODS: Cross-sectionally, 300 HIV-1 infected patients, failing first-line HAART were included. HIV-1 pol gene spanning 20-240 codons of RT was genotyped and mutation pattern was examined, (IAS-USA 2014 and Stanford HIV drug resistance database v7.0). RESULTS: The median age of the participants was 35 years (IQR 29-40), CD4 T cell count of TDF failures was low at 172 cells/µL (IQR 80-252), and treatment duration was low among TDF failures (24 months vs. 61 months) (p < 0.0001). Majority of the TDF failures were on EFV based first-line (89 % vs 45 %) (p < 0.0001). Level of resistance for TDF and AZT shows, that resistance to TDF was about one-third (37 %) of TDF participants and onefourth (23 %) of AZT participants; resistance to AZT was 17 % among TDF participants and 47 % among AZT participants; resistance to both AZT and TDF was significantly high among AZT participants [21 % vs. 8 %, OR 3.057 (95 % CI 1.4-6.8), p < 0.0001]. CONCLUSION: Although delayed identification of treatment failure caused high levels of acquired drug resistance in our study. Thus, we must include measures to regularize virological monitoring with integrated resistance testing in LMIC (Low and Middle Income Countries) like in India; this will help to preserve the effectiveness of ARV and ensure the success of ending AIDS as public health by 2030.

Molecular and Phylogenetic Analysis of HIV-1 Subtype C in Bahia State, Northeastern Brazil.

HIV-1 subtype C is associated with more than half of infections in southern Brazil and has been increasing in other regions of the country. In a previous study carried out in northeastern Brazil, we found a prevalence of 4.1% of subtype C. This work investigates the origin of subtype C in the state of Bahia based on five new viral sequences. The phylogenetic analysis showed that subtype C viruses found in Bahia descend from the main lineage that circulates in other Brazilian regions.

HIV-1 Vif protein sequence variations in South African people living with HIV and their influence on Vif-APOBEC3G interaction.

PURPOSE: Despite extensive research, HIV-1 remains a global epidemic with variations in pathogenesis across regions and subtypes. The Viral Infectivity Factor (Vif) protein, which neutralizes the host protein APOBEC3G, has been implicated in differences in clinical outcomes among people living with HIV (PLHIV). Most studies on Vif sequence diversity have focused on subtype B, leaving gaps in understanding Vif variations in HIV-1C regions like South Africa. This study aimed to identify and compare Vif sequence diversity in a cohort of 51 South African PLHIV and other HIV-1C prevalent regions. METHODS: Sanger sequencing was used for Vif analysis in the cohort, and additional sequences were obtained from the Los Alamos database. Molecular modeling and docking techniques were employed to study the influence of subtype-specific variants on Vif-APOBEC3G binding affinity. RESULTS: The findings showed distinct genetic variations between Vif sequences from India and Uganda, while South African sequences had wider distribution and closer relatedness to both. Specific amino acid substitutions in Vif were associated with geographic groups. Molecular modeling and docking analyses consistently identified specific residues (ARGR19, LYS26, TYR30, TYR44, and TRP79) as primary contributors to intermolecular contacts between Vif and APOBEC3G, essential for their interaction. The Indian Vif variant exhibited the highest predicted binding affinity to APOBEC3G among the studied groups. CONCLUSIONS: These results provide insights into Vif sequence diversity in HIV-1C prevalent regions and shed light on differential pathogenesis observed in different geographical areas. The identified Vif amino acid residues warrant further investigation for their diagnostic, prognostic, and therapeutic potential.

Optimizing ancestral trait reconstruction of large HIV Subtype C datasets through multiple-trait subsampling.

Large datasets along with sampling bias represent a challenge for phylodynamic reconstructions, particularly when the study data are obtained from various heterogeneous sources and/or through convenience sampling. In this study, we evaluate the presence of unbalanced sampled distribution by collection date, location, and risk group of human immunodeficiency virus Type 1 Subtype C using a comprehensive subsampling strategy and assess their impact on the reconstruction of the viral spatial and risk group dynamics using phylogenetic comparative methods. Our study shows that a most suitable dataset for ancestral trait reconstruction can be obtained through subsampling by all available traits, particularly using multigene datasets. We also demonstrate that sampling bias is inflated when considerable information for a given trait is unavailable or of poor quality, as we observed for the trait risk group. In conclusion, we suggest that, even if traits are not well recorded, including them deliberately optimizes the representativeness of the original dataset rather than completely excluding them. Therefore, we advise the inclusion of as many traits as possible with the aid of subsampling approaches in order to optimize the dataset for phylodynamic analysis while reducing the computational burden. This will benefit research communities investigating the evolutionary and spatio-temporal patterns of infectious diseases.

Safety and immunogenicity of a recombinant oligomeric gp145 subtype C Env protein (gp145 C.6980) HIV vaccine candidate in healthy, HIV-1-uninfected adult participants in the US.

BACKGROUND: An approach to a preventive HIV vaccine is induction of effective broadly neutralizing antibodies (bnAbs) and effector binding antibodies (bAbs). Preclinical studies suggest that trimeric envelope (Env) proteins may elicit nAbs, which led to the development of the recombinant gp145 subtype C Env protein (gp145 C.6980) immunogen. HVTN 122 was a Phase 1 trial that evaluated the safety, tolerability, and immunogenicity of gp145 C.6980 in adults. METHODS: Healthy, HIV-1 seronegative adults received three intramuscular injections of gp145 C.6980 with aluminum hydroxide (alum) at months 0, 2, and 6 at either 300 mcg (high dose, n = 25) or 100 mcg (low dose, n = 15), or placebo/saline (placebo, n = 5). Participants were followed for 12 months. RESULTS: Forty-five participants were enrolled. High and low doses of the study protein were well-tolerated, with mild or moderate reactogenicity commonly reported. Only one adverse event (mild injection site pruritis) in one participant (low dose) was considered product-related; there were no dose-limiting toxicities. High and low dose recipients demonstrated robust bAb responses to vaccine-matched consensus gp140 Env and subtype-matched gp120 Env proteins two weeks post-last vaccination (response rates >90 %), while no responses were detected to a heterologous subtype-matched V1V2 antigen. No significant differences were seen between high and low dose groups. Participants in both experimental arms demonstrated nAb response rates of 76.5 % to a tier 1 virus (MW9635.26), but no responses to tier 2 isolates. Env-specific CD4 + T-cell responses were elicited in 36.4 % of vaccine recipients, without significant differences between groups; no participants demonstrated CD8 + T-cell responses. CONCLUSIONS: Three doses of novel subtype C gp145 Env protein with alum were safe and well-tolerated. Participants demonstrated bAb, Env-specific CD4 + T-cell, and tier 1 nAb responses, but the regimen failed to induce tier 2 or heterologous nAb responses. CLINICAL TRIALS REGISTRATION: NCT03382418.

Longitudinal Assessment of Virological Failure and Antiretroviral Drug Resistance among Treatment-naive Subjects Living with HIV.

INTRODUCTION: Human immunodeficiency virus (HIV) infection, the etiological agent of acquired immunodeficiency syndrome (AIDS), is a serious public health issue. Therapeutic measures have been successful in increasing the survival and improving the quality of life. However, some treatment-naive subjects living with HIV present resistance-associated mutations as a result of late diagnosis and/or mutant strain infections. The objective of this study was to identify the virus genotype and assess the antiretroviral resistance profile based on the results of HIV genotyping in treatment-naive subjects living with HIV, after six months of taking antiretroviral therapy. METHODS: This was a prospective cohort study on treatment-naive adults living with HIV attending a specialized outpatient clinic in southern Santa Catarina State, Brazil. The participants were interviewed and had blood samples drawn. The genotypic antiretroviral drug resistance profile was examined in patients with detectable viral loads. RESULTS: 65 treatment-naive subjects living with HIV were recruited for this study. After six months of taking antiretroviral therapy, resistance-associated mutations were observed in 3 (4.6%) subjects living with HIV. CONCLUSION: Subtype C was identified as the circulating subtype in southern Santa Catarina State, and L10V, K103N, A98G, and Y179D were the most common mutations found in treatment-naive subjects.

Generation and characterization of infectious molecular clones of transmitted/founder HIV-1 subtype C viruses.

The genetic diversity of HIV impedes vaccine development. Identifying the viral properties of transmitted/founder (T/F) variants may provide a common vaccine target. To study the biological nature of T/F viruses, we constructed full-length clones from women detected during Fiebig stage I acute HIV-1 infection (AHI) from heterosexual male-to-female (MTF) transmission; and clones after one year of infection using In-Fusion-based cloning. Eighteen full-length T/F clones were generated from 9 women and six chronic infection clones were from 2 individuals. All clones but one were non-recombinant subtype C. Three of the 5 T/F clones and 3 chronic clones tested replicated efficiently in PBMCs and utilised CCR5 coreceptor for cell entry. Transmitted/founder and chronic infection clones displayed heterogenous in vitro replicative capacity and resistance to type I interferon. T/F viruses had shorter Env glycoproteins and fewer N-linked glycosylation sites in Env. Our findings suggest MTF transmission may select viruses with compact envelopes.

HIV-1 subtype C Nef-mediated SERINC5 down-regulation significantly contributes to overall Nef activity.

BACKGROUND: Nef performs multiple cellular activities that enhance HIV-1 pathogenesis. The role of Nef-mediated down-regulation of the host restriction factor SERINC5 in HIV-1 pathogenesis is not well-defined. We aimed to investigate if SERINC5 down-regulation activity contributes to HIV-1 subtype C disease progression, to assess the relative contribution of this activity to overall Nef function, and to identify amino acids required for optimal activity. We measured the SERINC5 down-regulation activity of 106 subtype C Nef clones, isolated from individuals in early infection, for which the Nef activities of CD4 and HLA-I down-regulation as well as alteration of TCR signalling were previously measured. The relationship between SERINC5 down-regulation and markers of disease progression, and the relative contribution of SERINC5 down-regulation to a Nef fitness model-derived E value (a proxy for overall Nef fitness in vivo), were assessed. RESULTS: No overall relationship was found between SERINC5 down-regulation and viral load set point (p = 0.28) or rate of CD4+ T cell decline (p = 0.45). CD4 down-regulation (p = 0.02) and SERINC5 down-regulation (p = 0.003) were significant determinants of E values in univariate analyses, with the greatest relative contribution for SERINC5 down-regulation, and only SERINC5 down-regulation remained significant in the multivariate analysis (p = 0.003). Using a codon-by-codon analysis, several amino acids were significantly associated with increased (10I, 11V, 38D, 51T, 65D, 101V, 188H and, 191H) or decreased (10K, 38E, 65E, 135F, 173T, 176T and, 191R) SERINC5 down-regulation activity. Site-directed mutagenesis experiments of selected mutants confirmed a substantial reduction in SERINC5 down-regulation activity associated with the mutation 173T, while mutations 10K, 135F, and 176T were associated with more modest reductions in activity that were not statistically significant. CONCLUSIONS: These results suggest that SERINC5 down-regulation is a significant contributor to overall Nef function and identify potential genetic determinants of this Nef function that may have relevance for vaccines or therapeutics.

Understanding Drug Resistance of Wild-Type and L38HL Insertion Mutant of HIV-1 C Protease to Saquinavir.

Acquired immunodeficiency syndrome (AIDS) is one of the most challenging infectious diseases to treat on a global scale. Understanding the mechanisms underlying the development of drug resistance is necessary for novel therapeutics. HIV subtype C is known to harbor mutations at critical positions of HIV aspartic protease compared to HIV subtype B, which affects the binding affinity. Recently, a novel double-insertion mutation at codon 38 (L38HL) was characterized in HIV subtype C protease, whose effects on the interaction with protease inhibitors are hitherto unknown. In this study, the potential of L38HL double-insertion in HIV subtype C protease to induce a drug resistance phenotype towards the protease inhibitor, Saquinavir (SQV), was probed using various computational techniques, such as molecular dynamics simulations, binding free energy calculations, local conformational changes and principal component analysis. The results indicate that the L38HL mutation exhibits an increase in flexibility at the hinge and flap regions with a decrease in the binding affinity of SQV in comparison with wild-type HIV protease C. Further, we observed a wide opening at the binding site in the L38HL variant due to an alteration in flap dynamics, leading to a decrease in interactions with the binding site of the mutant protease. It is supported by an altered direction of motion of flap residues in the L38HL variant compared with the wild-type. These results provide deep insights into understanding the potential drug resistance phenotype in infected individuals.

Subtle Longitudinal Alterations in Env Sequence Potentiate Differences in Sensitivity to Broadly Neutralizing Antibodies following Acute HIV-1 Subtype C Infection.

Broadly neutralizing antibodies (bNAbs) for HIV-1 prevention or cure strategies must inhibit transmitted/founder and reservoir viruses. Establishing sensitivity of circulating viruses to bNAbs and genetic patterns affecting neutralization variability may guide rational bNAbs selection for clinical development. We analyzed 326 single env genomes from nine individuals followed longitudinally following acute HIV-1 infection, with samples collected at ~1 week after the first detection of plasma viremia; 300 to 1,709 days postinfection but prior to initiating antiretroviral therapy (ART) (median = 724 days); and ~1 year post ART initiation. Sequences were assessed for phylogenetic relatedness, potential N- and O-linked glycosylation, and variable loop lengths (V1 to V5). A total of 43 env amplicons (median = 3 per patient per time point) were cloned into an expression vector and the TZM-bl assay was used to assess the neutralization profiles of 15 bNAbs targeting the CD4 binding site, V1/V2 region, V3 supersite, MPER, gp120/gp41 interface, and fusion peptide. At 1 µg/mL, the neutralization breadths were as follows: VRC07-LS and N6.LS (100%), VRC01 (86%), PGT151 (81%), 10-1074 and PGT121 (80%), and less than 70% for 10E8, 3BNC117, CAP256.VRC26, 4E10, PGDM1400, and N123-VRC34.01. Features associated with low sensitivity to V1/V2 and V3 bNAbs were higher potential glycosylation sites and/or relatively longer V1 and V4 domains, including known "signature" mutations. The study shows significant variability in the breadth and potency of bNAbs against circulating HIV-1 subtype C envelopes. VRC07-LS, N6.LS, VRC01, PGT151, 10-1074, and PGT121 display broad activity against subtype C variants, and major determinants of sensitivity to most bNAbs were within the V1/V4 domains. IMPORTANCE Broadly neutralizing antibodies (bNAbs) have potential clinical utility in HIV-1 prevention and cure strategies. However, bNAbs target diverse epitopes on the HIV-1 envelope and the virus may evolve to evade immune responses. It is therefore important to identify antibodies with broad activity in high prevalence settings, as well as the genetic patterns that may lead to neutralization escape. We investigated 15 bNAbs with diverse biophysical properties that target six epitopes of the HIV-1 Env glycoprotein for their ability to inhibit viruses that initiated infection, viruses circulating in plasma at chronic infection before antiretroviral treatment (ART), or viruses that were archived in the reservoir during ART in subtype C infected individuals in South Africa, a high burden country. We identify the antibodies most likely to be effective for clinical use in this setting and describe mutational patterns associated with neutralization escape from these antibodies.

Molecular Dynamics Study on Selected Bioactive Phytochemicals as Potential Inhibitors of HIV-1 Subtype C Protease.

Acquired immunodeficiency syndrome (AIDS), one of the deadliest global diseases, is caused by the Human Immunodeficiency Virus (HIV). To date, there are no known conventional drugs that can cure HIV/AIDS, and this has prompted continuous scientific efforts in the search for novel and potent anti-HIV therapies. In this study, molecular dynamics simulation (MDS) and computational techniques were employed to investigate the inhibitory potential of bioactive compounds from selected South African indigenous plants against HIV-1 subtype C protease (HIVpro). Of the eight compounds (CMG, MA, UA, CA, BA, UAA, OAA and OA) evaluated, only six (CMG (-9.9 kcal/mol), MA (-9.3 kcal/mol), CA (-9.0 kcal/mol), BA (-8.3 kcal/mol), UAA (-8.5 kcal/mol), and OA (-8.6 kcal/mol)) showed favourable activities against HIVpro and binding landscapes like the reference FDA-approved drugs, Lopinavir (LPV) and Darunavir (DRV), with CMG and MA having the highest binding affinities. Using the structural analysis (root-mean-square deviation (RMSD), fluctuation (RMSF), and radius of gyration (RoG) of the bound complexes with HIVpro after 350 ns, structural evidence was observed, indicating that the six compounds are potential lead candidates for inhibiting HIVpro. This finding was further corroborated by the structural analysis of the enzyme-ligand complexe systems, where structural mechanisms of stability, flexibility, and compactness of the study metabolites were established following binding with HIVpro. Furthermore, the ligand interaction plots revealed that the metabolites interacted hydrophobically with the active site amino residues, with identification of other key residues implicated in HIVpro inhibition for drug design. Overall, this is the first computational report on the anti-HIV-1 activities of CMG and MA, with efforts on their in vitro and in vivo evaluations underway. Judging by the binding affinity, the degree of stability, and compactness of the lead metabolites (CMG, MA, CA, BA, OA, and UAA), they could be concomitantly explored with conventional HIVpro inhibitors in enhancing their therapeutic activities against the HIV-1 serotype.

Systematic post-mortem analysis of brain tissue from an HIV-1 subtype C viremic decedent revealed a paucity of infection and pathology.

Whether the human brain is a robust reservoir for HIV-1 subtype C has yet to be established. We aimed to determine whether HIV-1 subtype C infection can be detected in the brain tissue of a viremic individual at post-mortem and whether the viral burden was differential between different brain regions. This study reports a 38-year-old Zambian female decedent with severe wasting who was on Atripla for antiretroviral therapy. The cause of death was determined to be HIV/AIDS end-stage disease. The QuantStudio 3 Real-Time PCR System analyzed formalin-fixed paraffin-embedded tissue DNA from a systematic sampling of the entire left-brain hemisphere. Plasma and cerebral spinal fluid HIV-1 RNA loads were 576,123 and 14,962 copies/mL, respectively. The lymph node DNA viral load was 2316 copies per 106 cells. Two hundred and six (96.3%) tissue blocks had amplifiable DNA. HIV-1 viral DNA was detected in 35.9% of the blocks, the highest in the basal ganglia (66.7%) and the frontal lobe (46%). Overall, HIV detection was random, with low viral copies detected by quantitative polymerase chain reaction (qPCR); the lowest was observed in the occipital (median, IQR, range) 0.0 [0.0-0.0], 0.0-31.3, and the highest in the basal ganglia (mean ± SD, range, 125.1149.5, 0.0-350.0). Significant differences in HIV-1 DNA distribution were observed between the occipital versus parietal (p = 0.049), occipital versus frontal (p = 0.019), occipital versus basal ganglia (p = 0.005), cerebellum versus frontal (p = 0.021), cerebellum versus basal ganglia (p = 0.007), and temporal versus frontal (p = 0.034).

Non-active site mutations in the HIV protease: Diminished drug binding affinity is achieved through modulating the hydrophobic sliding mechanism.

The global HIV/AIDS epidemic still currently affects approximately 38 million individuals globally. The protease enzyme of the human immunodeficiency virus is a major drug target in antiviral therapy, however, under the influence of reverse transcriptase and in the context of drug pressure, the rapid PR mutation rate contributes significantly to clinical failure. The set of cooperative non-active site mutations, I13V/I62V/V77I, have been associated with reduced inhibitor susceptibility and are the focus of the current study. When compared to the wild-type protease the mutant protease exhibited decreased binding affinities towards ATV and DRV by 64- and 12-fold, respectively, and decreased the overall favourable Gibbs free energy for ATV, DRV, RTV and SQV. Moreover, these mutations decreased the thermal stability of the protease when in complex with ATV and DRV by approximately 6.4 and 4.2 °C, respectively. The crystal structure of the mutant protease revealed that the location of these mutations and their effect on the hydrophobic sliding mechanism may be crucial in their role in resistance.

Covalent binding of human two-domain CD4 to an HIV-1 subtype C SOSIP.664 trimer modulates its structural dynamics.

The human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) mediates host cell infection by binding to the cellular receptor CD4. Recombinant Env bound to CD4 has been explored for its potential as an HIV vaccine immunogen as receptor binding exposes otherwise shielded, conserved functional sites. Previous preclinical studies showed an interchain disulphide linkage facilitated between Env and 2dCD4S60C generates an immunogenic complex that elicits potent, broadly neutralizing antibodies (bNAbs) against clinically relevant HIV-1. This study investigated conformational dynamics of 2dCD4WT and 2dCD4S60C bound to an HIV-1C SOSIP.664 Env trimer using hydrogen-deuterium exchange mass spectrometry. The Env:2dCD4S60C complex maintains key contact residues required for MHCII and Env/gp120 binding and the residues encompassing Ibalizumab's epitope. Important residues remaining anchored, with an increased flexibility in surrounding regions, evidenced by the higher exchange seen in flanking residues compared to Env:2dCD4WT. While changes in Env:2dCD4S60C dynamics in domain 1 were moderate, domain 2 exhibited greater variation. Lack of stability-inducing H-bonds in these allosteric sites suggest the improved immunogenicity of Env:2dCD4S60C result from exposed CD4 residues providing diverse/novel antigenic targets for the development of potent, broadly neutralizing Ibalizumab-like antibodies.

Construction and characterization of a full-length, replication-competent and infectious enhanced green fluorescence protein-tagged HIV-1 subtype C molecular clone.

HIV-1 subtype C virus accounts for nearly 50% of the total HIV infections globally. Despite this high prevalence, our understanding of subtype C specific infections remains limited due to lack of an in vitro model system. This is the first report of construction and characterization of a full-length and infectious EGFP-tagged HIV-1 subtype C molecular clone. The EGFP gene was inserted in-frame between the Nef and Env sequence in the HIV genome. The recombinant virus displayed expression of viral genes, infectivity and replication kinetics similar to the parental virus. VSV-G pseudotyping of the recombinant virus led to enhancement of HIV infection. The presence of the EGFP gene provides a rapid, easy and quantitative measure of HIV infection by flow cytometry and fluorescence microscopy. This clone will serve as an extremely beneficial tool to study HIV-1 subtype C specific infections.