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.

Impact of Cannabis Use on Immune Cell Populations and the Viral Reservoir in People With HIV on Suppressive Antiretroviral Therapy.

BACKGROUND: Human immunodeficiency virus (HIV) infection remains incurable due to the persistence of a viral reservoir despite antiretroviral therapy (ART). Cannabis (CB) use is prevalent amongst people with HIV (PWH), but the impact of CB on the latent HIV reservoir has not been investigated. METHODS: Peripheral blood cells from a cohort of PWH who use CB and a matched cohort of PWH who do not use CB on ART were evaluated for expression of maturation/activation markers, HIV-specific T-cell responses, and intact proviral DNA. RESULTS: CB use was associated with increased abundance of naive T cells, reduced effector T cells, and reduced expression of activation markers. CB use was also associated with reduced levels of exhausted and senescent T cells compared to nonusing controls. HIV-specific T-cell responses were unaffected by CB use. CB use was not associated with intact or total HIV DNA frequency in CD4 T cells. CONCLUSIONS: This analysis is consistent with the hypothesis that CB use reduces activation, exhaustion, and senescence in the T cells of PWH, and does not impair HIV-specific CD8 T-cell responses. Longitudinal and interventional studies with evaluation of CB exposure are needed to fully evaluate the impact of CB use on the HIV reservoir.

Curcuma Longa Induces the Transcription Factor FOXP3 to Downregulate Human Chemokine CCR5 Expression and Inhibit HIV-1 Infection.

HIV mutations occur frequently despite the substantial success of combination antiretroviral therapy, which significantly impairs HIV progression. Failure to develop specific vaccines, the occurrence of drug-resistant strains, and the high incidence of adverse effects due to combination antiviral therapy regimens call for novel and safer antivirals. Natural products are an important source of new anti-infective agents. For instance, curcumin inhibits HIV and inflammation in cell culture assays. Curcumin, the principal constituent of the dried rhizomes of Curcuma longa L. (turmeric), is known as a strong anti-oxidant and anti-inflammatory agent with different pharmacological effects. This work aims to assess curcumin's inhibitory effects on HIV in vitro and to explore the underpinning mechanism, focusing on CCR5 and the transcription factor forkhead box protein P3 (FOXP3). First, curcumin and the RT inhibitor zidovudine (AZT) were evaluated for their inhibitory properties. HIV-1 pseudovirus infectivity was determined by green fluorescence and luciferase activity measurements in HEK293T cells. AZT was used as a positive control that inhibited HIV-1 pseudoviruses dose-dependently, with IC50 values in the nanomolar range. Then, a molecular docking analysis was carried out to assess the binding affinities of curcumin for CCR5 and HIV-1 RNase H/RT. The anti-HIV activity assay showed that curcumin inhibited HIV-1 infection, and the molecular docking analysis revealed equilibrium dissociation constants of [Formula: see text]9.8[Formula: see text]kcal/mol and [Formula: see text]9.3[Formula: see text]kcal/mol between curcumin and CCR5 and HIV-1 RNase H/RT, respectively. To examine curcumin's anti-HIV effect and its mechanism in vitro, cell cytotoxicity, transcriptome sequencing, and CCR5 and FOXP3 amounts were assessed at different concentrations of curcumin. In addition, human CCR5 promoter deletion constructs and the FOXP3 expression plasmid pRP-FOXP3 (with an EGFP tag) were generated. Whether FOXP3 DNA binding to the CCR5 promoter was blunted by curcumin was examined using transfection assays employing truncated CCR5 gene promoter constructs, a luciferase reporter assay, and a chromatin immunoprecipitation (ChIP) assay. Furthermore, micromolar concentrations of curcumin inactivated the nuclear transcription factor FOXP3, which resulted in decreased expression of CCR5 in Jurkat cells. Moreover, curcumin inhibited PI3K-AKT activation and its downstream target FOXP3. These findings provide mechanistic evidence encouraging further assessment of curcumin as a dietary agent used to reduce the virulence of CCR5-tropic HIV-1. Curcumin-mediated FOXP3 degradation was also reflected in its functions, namely, CCR5 promoter transactivation and HIV-1 virion production. Furthermore, curcumin inhibition of CCR5 and HIV-1 might constitute a potential therapeutic strategy for reducing HIV progression.

Genetic characterization of HIV-1 viruses among cases with antiretroviral therapy failure in Suzhou City, China.

BACKGROUND: This retrospective study aimed to characterize the distribution of HIV-1 genotypes and the prevalence of drug resistance mutations in people with antiretroviral treatment (ART) failure in Suzhou City, China. METHODS: Pol gene of HIV-1 viruses in blood samples of EDTA anticoagulants from 398 patients with failed antiviral treatment was successfully amplified by using an in-house assay. Drug resistance mutations were analyzed by using the Stanford HIV Drug Resistance Database system ( https://hivdb.stanford.edu/hivdb/by-mutations/ ). HIV-1 genotypes were determined by the REGA HIV subtyping tool (version 3.46, https://www.genomedetective.com/app/typingtool/hiv ). Near full-length genomes (NFLG) of HIV-1 viruses were obtained by next generation sequencing method. RESULTS: Sequences analysis of the pol gene revealed that CRF 01_AE (57.29%, 228/398) was the dominant subtype circulating in Suzhou City, followed by CRF 07_BC (17.34%, 69/398), subtype B (7.54%, 30/398), CRF 08_BC (6.53%, 26/398), CRF 67_01B (3.02%, 12/398) and CRF55_01B (2.51%, 10/398). The overall prevalence of drug-resistant mutations in cases with ART failure was 64.57% (257/398), including 45.48% (181/398) for nucleotide reverse transcriptase inhibitors (NRTIs) mutations, 63.32% (252/398) for non-nucleoside reverse transcriptase inhibitors (NNRTIs) mutations, and 3.02% (12/398) for protease inhibitors (PIs) mutations. Ten near full-length genomes (NFLG) of HIV-1 viruses were identified, including six recombinants of CRF 01_AE and subtype B, two recombinants of CRF 01_AE, subtype B and subtype C sequences, one recombinant of CRF 01_AE and subtype C and one recombinant of CRF 01_AE, subtype A1 and subtype C. CONCLUSIONS: The high prevalence of drug-resistant HIV-1 viruses was a serious challenge for HIV prevention and treatment of people with HIV infection. Treatment regimens for ART failure patients should be adjusted over time based on the outcome of drug resistance tests. NFLG sequencing facilitates the identification of new recombinants of HIV-1.

Anti-human immunodeficiency virus-1 activity of MoMo30 protein isolated from the traditional African medicinal plant Momordica balsamina.

BACKGROUND: Plants are used in traditional healing practices of many cultures worldwide. Momordica balsamina is a plant commonly used by traditional African healers as a part of a treatment for HIV/AIDS. It is typically given as a tea to patients with HIV/AIDS. Water-soluble extracts of this plant were found to contain anti-HIV activity. METHODS: We employed cell-based infectivity assays, surface plasmon resonance, and a molecular-cell model of the gp120-CD4 interaction to study the mechanism of action of the MoMo30-plant protein. Using Edman degradation results of the 15 N-terminal amino acids, we determined the gene sequence of the MoMo30-plant protein from an RNAseq library from total RNA extracted from Momordica balsamina. RESULTS: Here, we identify the active ingredient of water extracts of the leaves of Momordica balsamina as a 30 kDa protein we call MoMo30-plant. We have identified the gene for MoMo30 and found it is homologous to a group of plant lectins known as Hevamine A-like proteins. MoMo30-plant is distinct from other proteins previously reported agents from the Momordica species, such as ribosome-inactivating proteins such as MAP30 and Balsamin. MoMo30-plant binds to gp120 through its glycan groups and functions as a lectin or carbohydrate-binding agent (CBA). It inhibits HIV-1 at nanomolar levels and has minimal cellular toxicity at inhibitory levels. CONCLUSIONS: CBAs like MoMo30 can bind to glycans on the surface of the enveloped glycoprotein of HIV (gp120) and block entry. Exposure to CBAs has two effects on the virus. First, it blocks infection of susceptible cells. Secondly, MoMo30 drives the selection of viruses with altered glycosylation patterns, potentially altering their immunogenicity. Such an agent could represent a change in the treatment strategy for HIV/AIDS that allows a rapid reduction in viral loads while selecting for an underglycosylated virus, potentially facilitating the host immune response.

Polyphenol Extracts from Grape Seeds and Apple Can Reactivate Latent HIV-1 Transcription through Promoting P-TEFb Release from 7SK snRNP.

The principal barrier for the eradication of HIV/AIDS is the virus latency. One of the effective strategies so called "shock and kill" is to use latency-reversing agents (LRAs) to activate the latent HIV reservoirs and then combine them with the highly active antiretroviral therapy (HAART) to eradicate the virus. However, most of the current LRAs are too toxic; therefore, they have not been used clinically. Our preliminary data indicated that polyphenols from grape seeds can activate HIV in latently infected Jurkat T cells. Owing to a lot of food containing polyphenols and based on a reasoning whether all of these kinds of polyphenols contain the latency-reversing function, in this study, we screened 22 fruits/vegetables to see whether polyphenols from these can reactivate latent HIV-1 transcription. We finally proved that the polyphenols from grape seeds, apple, pomegranate, and bilberry can reactivate latent HIV-1 transcription. The activation of which can be detected on the level of protein and mRNA. The activation of which is in a dose- and time-dependent manner, while the activated polyphenol extracts have the effects to stimulate Tat-independent HIV-1 transcription. The mechanism shows that polyphenol extracts from grape seeds and apple can stimulate P-TEFb's release from 7SK snRNP to induce HIV gene transcription. These results indicate that using a few food of high-content polyphenols as latent activators and combining HARRT may be of great use for the treatment of HIV/AIDS in the future.

Suicide gene therapy in cancer and HIV-1 infection: An alternative to conventional treatments.

Suicide Gene Therapy (SGT) aims to introduce a gene encoding either a toxin or an enzyme making the targeted cell more sensitive to chemotherapy. SGT represents an alternative approach to combat pathologies where conventional treatments fail such as pancreatic cancer or the high-grade glioblastoma which are still desperately lethal. We review the possibility to use SGT to treat these cancers which have shown promising results in vitro and in preclinical trials. However, SGT has so far failed in phase III clinical trials thus further improvements are awaited. We can now take advantages of the many advances made in SGT for treating cancer to combat other pathologies such as HIV-1 infection. In the review we also discuss the feasibility to add SGT to the therapeutic arsenal used to cure HIV-1-infected patients. Indeed, preliminary results suggest that both productive and latently infected cells are targeted by the SGT. In the last section, we address the limitations of this approach and how we might improve it.

Sliding of HIV-1 reverse transcriptase over DNA creates a transient P pocket - targeting P-pocket by fragment screening.

HIV-1 reverse transcriptase (RT) slides over an RNA/DNA or dsDNA substrate while copying the viral RNA to a proviral DNA. We report a crystal structure of RT/dsDNA complex in which RT overstepped the primer 3'-end of a dsDNA substrate and created a transient P-pocket at the priming site. We performed a high-throughput screening of 300 drug-like fragments by X-ray crystallography that identifies two leads that bind the P-pocket, which is composed of structural elements from polymerase active site, primer grip, and template-primer that are resilient to drug-resistance mutations. Analogs of a fragment were synthesized, two of which show noticeable RT inhibition. An engineered RT/DNA aptamer complex could trap the transient P-pocket in solution, and structures of the RT/DNA complex were determined in the presence of an inhibitory fragment. A synthesized analog bound at P-pocket is further analyzed by single-particle cryo-EM. Identification of the P-pocket within HIV RT and the developed structure-based platform provide an opportunity for the design new types of polymerase inhibitors.

MOF@COF Heterostructure Hybrid for Dual-Mode Photoelectrochemical-Electrochemical HIV-1 DNA Sensing.

We developed a novel metal-organic framework (MOF)@covalent-organic framework (COF) hybrid with a hierarchical nanostructure and excellent photoactivity, which further acted as the bifunctional platform of a dual-mode photoelectrochemical (PEC) and electrochemical (EC) biosensor for detecting HIV-1 DNA via immobilizing the HIV-1 DNA probe. First, the presynthesized Cu-MOF nanoellipsoids were used as the template for the in situ growth of the COF network, which was synthesized using copper-phthalocyanine tetra-amine (CoPc-TA) and 2,9-bis[p-(formyl)phenyl]-1,10-phenanthroline as building blocks through the Schiff base condensation. In view of the large specific surface area, abundant reserved amino group, excellent electrochemical activity, and high photoactivity, the obtained Cu-MOF@CuPc-TA-COF heterostructure not only can serve as the sensitive platform for anchoring the HIV-1 DNA probe strands but also can be utilized as the signal transducers for PEC and EC biosensors. Thereby, the constructed biosensor shows the sensitive and selective analysis ability toward the HIV-1 target DNA via the complementary hybridization between probe and target DNA strands. The dual-mode PEC and EC measurements revealed that the Cu-MOF@CuPc-TA-COF-based biosensor displayed a wide linear detection range from 1 fM to 1 nM and an extremely low limit of detection (LOD) of 0.07 and 0.18 fM, respectively. In addition, the dual-mode PEC-EC biosensor also demonstrated remarkable selectivity, high stability, good reproducibility, and preferable regeneration ability, as well as acceptable applicability, for which the detected HIV-1 DNA in human serum showed good consistency with real concentrations. Thereby, the present work can open a new dual-mode PEC-EC platform for detecting HIV-1 DNA based on the porous-organic framework heterostructure.

HIV-1 Nucleic Acids Identify Rectal HIV Exposures in Self-Collected Rectal Swabs, Whereas Y-Chromosome Single Tandem Repeat Mixtures Are Not Reliable Biomarkers of Condomless Receptive Anal Intercourse.

BACKGROUND: To focus interventions, biomarkers of HIV-1 exposure could help in identifying subpopulations at highest risk of acquisition. We assessed whether Y-chromosome single tandem repeat (YSTR) mixtures obtained from rectal swabs could serve as a biomarker of condomless receptive anal intercourse (CRAI) among men who have sex with men and transgender women and evaluated the feasibility of detecting HIV-1 virions to assess exposures. METHODS: Twenty-nine sexually active HIV-seronegative men who have sex with men and one transgender woman from New York City answered on-site and mobile app sexual behavior questionnaires. They were randomized to collecting self-administered rectal swabs every morning or after receptive anal intercourse (RAI). YSTR profiles were assessed from blood sample and swabs; HIV-1 exposure was measured by conducting quantitative polymerase chain reaction in swabs. RESULTS: After 2 months, the daily mobile survey had 135%-201% more instances of anal sex acts and 170%-193% more RAI than on-site surveys. Daily mobile reporting had 11%-35% less CRAI events than those reported on-site (Pdaily = 0.001; Pper-sex = 0.047). The daily swabbing arm reported less RAI (P < 0.001) and CRAI (P < 0.038) and had 2.95 lower odds of detecting YSTR mixtures (P = 0.021) than the per-sex-event arm. Surprisingly, YSTR detection was not significantly modified by report of bowel movements and lubricant, enema, or condom use. No participant became HIV-1 infected, yet HIV-1 total nucleic acids were detected in 6 independent episodes of CRAI in 2 participants taking pre-exposure prophylaxis. CONCLUSIONS: YSTR mixtures demonstrated 80% specificity but only 30% sensitivity as a biomarker of CRAI in self-collected rectal swabs. However, detection of HIV-1 exposures in self-collected swabs may help in identifying those needing further HIV risk reduction strategies.

Array-based dynamic allele specific hybridization (Array-DASH): Optimization-free microarray processing for multiple simultaneous genomic assays.

We report proof-of-principle experiments regarding a dynamic microarray protocol enabling accurate and semi-quantitative DNA analysis for re-sequencing, fingerprinting and genotyping. Single-stranded target molecules hybridise to surface-bound probes during initial gradual cooling with high-fidelity. Real-time tracking of target denaturation (via fluorescence) during a 'dynamic' gradual heating phase permits 'melt-curve' analysis. The probe most closely matching the target sequence is identified based on the highest melting temperature. We demonstrated a >99% re-sequencing accuracy and a potential detection rate of 1% for SNPs. Experiments employing Hypericum ribosomal ITS regions and HIV genomes illustrated a reliable detection level of 5% plus simultaneous re-sequencing and genotyping. Such performance suggests a range of potential real-world applications involving rapid sequence interrogation, for example, in the Covid-19 pandemic. Guidance is offered towards the development of a commercial platform and dedicated software required to bring this technique into mainstream science.

Field evaluation of HIV-1 viral load monitoring in adults and children receiving antiretroviral treatment in Nigeria by dried blood spot testing with RealTime HIV-1 on m2000.

In resource-limited settings, use of dried blood spots (DBS) could be a pragmatic alternative to plasma for VL monitoring in people living with HIV (PLWH). We compared results from DBS to standard plasma VL testing under field conditions in patients receiving antiretroviral therapy (ART). DBS cards were prepared from venous blood (V-DBS), finger-pricks using micro-capillary tubes (M-DBS), and direct spotting (D-DBS). DBS and matched EDTA plasma were tested on the Abbott m2000 platform using the appropriate RealTime HIV-1 quantitative CE protocol. Matched plasma samples were also tested on the Roche COBAS Ampliprep/COBAS TaqMan version 2.0. Diagnostic accuracy indicators (sensitivity, specificity, misclassification rate, and kappa coefficient) for viral failure (VF) based on different VL threshold levels and agreement of absolute VL were calculated. A total of 669 participants provided 2676 samples. V-DBS had a peak sensitivity for VF of 89.1 % [95 % CI: 85.5-92.7] at the 1000 copies/mL threshold and a peak specificity of 97.4 % [95 % CI: 95.9-99.0] at the 5000 copies/mL threshold. The lowest proportion of upward misclassification (patients classified with VF who actually had viral suppression) for V-DBS was 3.1 % [95 % CI: 1.4-4.8] at the 5000 copies/mL threshold, whereas the lowest proportion of downward misclassification (patients classified as undetectable who actually had VF) was 10.9 % [95 % CI: 7.2-14.5] at the 1000 copies/mL threshold. Abbott RealTime HIV-1 VL results from all 3 DBS types for adults and children showed strong correlation with the gold standard plasma-based assay. DBS could be useful for monitoring VL in resource limited settings such as Nigeria.

A Protocol for Studying HIV-1 Envelope Glycoprotein Function.

HIV-1 envelope glycoproteins (Envs) bind to CD4 receptor and CCR5/CXCR4 coreceptor and mediate viral entry (Feng et al., 1996; Herschhorn et al., 2016, 2017; Kwong et al., 1998). HIV-1 Envs are the sole target of neutralizing antibodies and a main focus of vaccine development (Flemming et al., 2018). Here, we provide a step-by-step protocol to measure Env sensitivity to ligands, cold, and small molecules, as well as to study viral infectivity and to dissect parameters affecting HIV-1 Env function. For complete details on the use and execution of this protocol, please refer to Harris et al. (2020).

HIV X4 Variants Increase Arachidonate 5-Lipoxygenase in the Pulmonary Microenvironment and are associated with Pulmonary Arterial Hypertension.

Pulmonary Arterial Hypertension (PAH) is overrepresented in People Living with Human Immunodeficiency Virus (PLWH). HIV protein gp120 plays a key role in the pathogenesis of HIV-PAH. Genetic changes in HIV gp120 determine viral interactions with chemokine receptors; specifically, HIV-X4 viruses interact with CXCR4 while HIV-R5 interact with CCR5 co-receptors. Herein, we leveraged banked samples from patients enrolled in the NIH Lung HIV studies and used bioinformatic analyses to investigate whether signature sequences in HIV-gp120 that predict tropism also predict PAH. Further biological assays were conducted in pulmonary endothelial cells in vitro and in HIV-transgenic rats. We found that significantly more persons living with HIV-PAH harbor HIV-X4 variants. Multiple HIV models showed that recombinant gp120-X4 as well as infectious HIV-X4 remarkably increase arachidonate 5-lipoxygenase (ALOX5) expression. ALOX5 is essential for the production of leukotrienes; we confirmed that leukotriene levels are increased in bronchoalveolar lavage fluid of HIV-infected patients. This is the first report associating HIV-gp120 genotype to a pulmonary disease phenotype, as we uncovered X4 viruses as potential agents in the pathophysiology of HIV-PAH. Altogether, our results allude to the supplementation of antiretroviral therapy with ALOX5 antagonists to rescue patients with HIV-X4 variants from fatal PAH.

Next-Generation Sequencing for HIV Drug Resistance Testing: Laboratory, Clinical, and Implementation Considerations.

Higher accessibility and decreasing costs of next generation sequencing (NGS), availability of commercial kits, and development of dedicated analysis pipelines, have allowed an increasing number of laboratories to adopt this technology for HIV drug resistance (HIVDR) genotyping. Conventional HIVDR genotyping is traditionally carried out using population-based Sanger sequencing, which has a limited capacity for reliable detection of variants present at intra-host frequencies below a threshold of approximately 20%. NGS has the potential to improve sensitivity and quantitatively identify low-abundance variants, improving efficiency and lowering costs. However, some challenges exist for the standardization and quality assurance of NGS-based HIVDR genotyping. In this paper, we highlight considerations of these challenges as related to laboratory, clinical, and implementation of NGS for HIV drug resistance testing. Several sources of variation and bias occur in each step of the general NGS workflow, i.e., starting material, sample type, PCR amplification, library preparation method, instrument and sequencing chemistry-inherent errors, and data analysis options and limitations. Additionally, adoption of NGS-based HIVDR genotyping, especially for clinical care, poses pressing challenges, especially for resource-poor settings, including infrastructure and equipment requirements and cost, logistic and supply chains, instrument service availability, personnel training, validated laboratory protocols, and standardized analysis outputs. The establishment of external quality assessment programs may help to address some of these challenges and is needed to proceed with NGS-based HIVDR genotyping adoption.

Oregano Oil and Its Principal Component, Carvacrol, Inhibit HIV-1 Fusion into Target Cells.

Oregano essential oil has long been known for its health-promoting benefits. Here, we report its activity against viral replication. Oregano oil was found to specifically inhibit lentiviruses, such as human and simian immunodeficiency viruses (HIV and SIV), irrespective of virus tropism, but not hepatitis C virus, adenovirus 5 (ADV5), Zika virus, and influenza (H1N1) virus. Oregano oil's most abundant components, carvacrol and its isomer, thymol, were shown to block virus-target cell fusion while not perturbing other stages of the virus life cycle. We detected changes in virus particle density, suggesting that cholesterol depletion from the HIV-1 envelope membrane reduces virus entry. Furthermore, infection was rescued by adding exogenous cholesterol. The evolution of viral resistance to carvacrol supported this mechanism of action with the identification of mutations in the viral gp41 fusion protein that counteracted cholesterol depletion. In addition, resistance to carvacrol emerged later than typically observed for other clinically used drugs, strengthening its antiviral potential. Structure-activity relationship studies revealed key motifs of carvacrol and thymol required for HIV neutralization and identified previously unknown active analogs. Carvacrol was also shown to additively cooperate with antiretroviral therapy. In sum, oregano oil and improved carvacrol and thymol analogs could be considered to supplement current HIV therapeutics.IMPORTANCE Oregano essential oil has multiple benefits in traditional medicine, cosmetics, and food industries. Carvacrol and its analog, thymol, are well-described components of oregano oil. Here, we show that these compounds inhibit HIV-target cell fusion independently of viral tropism. Our results suggest that carvacrol and thymol alter the cholesterol content of the viral membrane, blocking HIV-1 entry into the target cell. Resistance to carvacrol has selected for viruses with mutations in the viral envelope glycoprotein, gp41. This protein is known for its interaction with cholesterol present in membrane lipid rafts. Together, these results demonstrate the potential of therapies targeting the viral envelope membrane, and oregano oil is a safe supplement to antiretrovirals, potentially delaying disease progression and resistance development.

Discordance between Etravirine Phenotype and Genotype-Based Predicted Phenotype for Subtype C HIV-1 from First-Line Antiretroviral Therapy Failures in South Africa.

Etravirine (ETR) is a nonnucleoside reverse transcriptase inhibitor (NNRTI) used in treatment-experienced individuals. Genotypic resistance test-interpretation systems can predict ETR resistance; however, genotype-based algorithms are derived primarily from HIV-1 subtype B and may not accurately predict resistance in non-B subtypes. The frequency of ETR resistance among recombinant subtype C HIV-1 and the accuracy of genotypic interpretation systems were investigated. HIV-1LAI containing full-length RT from HIV-1 subtype C-positive individuals experiencing virologic failure (>10,000 copies/ml and >1 NNRTI resistance-associated mutation) were phenotyped for ETR susceptibility. Fold change (FC) was calculated against a composite 50% effective concentration (EC50) from treatment-naive individuals and three classifications were assigned: (i) <2.9-FC, susceptible; (ii) ≥2.9- to 10-FC, partially resistant; and (iii) >10-FC, fully resistant. The Stanford HIVdb-v8.4 was used for genotype predictions merging the susceptible/potential low-level and low-level/intermediate groups for 3 × 3 comparison. Fifty-four of a hundred samples had reduced ETR susceptibility (≥2.9-FC). The FC correlated with HIVdb-v8.4 (Spearman's rho = 0.62; P < 0.0001); however, 44% of samples were partially (1 resistance classification difference) and 4% completely discordant (2 resistance classification differences). Of the 34 samples with an FC of >10, 26 were HIVdb-v8.4 classified as low-intermediate resistant. Mutations L100I, Y181C, or M230L were present in 27/34 (79%) of samples with an FC of >10 but only in 2/46 (4%) of samples with an FC of <2.9. No other mutations were associated with ETR resistance. Viruses containing the mutation K65R were associated with reduced ETR susceptibility, but 65R reversions did not increase ETR susceptibility. Therefore, genotypic interpretation systems were found to misclassify ETR susceptibility in HIV-1 subtype C samples. Modifications to genotypic algorithms are needed to improve the prediction of ETR resistance for the HIV-1 subtype C.

RT-qPCR Detection of Low-Copy HIV RNA with Yin-Yang Probes.

Accurate monitoring of low levels of viral load (the number of viral particles per milliliter of plasma) in HIV-infected patients is important in terms of evaluation of the progress of antiretroviral therapy. The general approach for detection of low copy HIV RNA is reverse transcription combined with quantitative real-time PCR based on fluorescence detection. The selection of primers and the structure of fluorogenic oligonucleotide probes are crucial for sensitivity and accuracy of the assay. In this chapter, we report the RT-qPCR protocol for detection of low copy HIV RNA using double stranded Yin-Yang DNA probes containing identical fluorescent dyes on each strand of the probe. Dye residues attached to the 3'-end of an oligonucleotide and 5'-end of the complementary oligonucleotide form a self-quenched aggregate in a Yin-Yang duplex probe, and display fluorescence light up upon probe strand displacement with the target sequence amplified in the course of PCR. Among several fluorescent dyes tested (R6G, ROX, Cy5) the ROX labeled Yin-Yang probes showed better fluorescence increase and lower Ct values. All the homo Yin-Yang probes were superior to corresponding dye-quencher probes and allowed reliable detection of 10-10,000 copies of HIV RNA per mL.

Human transcription factor YY1 could upregulate the HIV-1 gene expression.

Gene expression in HIV-1 is regulated by the promoters in 5' long-terminal repeat (LTR) element, which contain multiple DNA regulatory elements that serve as binding sites for cellular transcription factors. YY1 could repress HIV-1 gene expression and latent infection. Here, however, we observed that virus production can be increased by YY1 over-expression and decreased under YY1 depleted condition by siRNA treatment. To identify functional domain(s) of YY1 activation, we constructed a number of YY1 truncated mutants. Our data show that full-length YY1 enhances the viral transcription both through U3 and U3RU5 promoters. Moreover, the C-terminal region (296-414 residues) of YY1 is responsible for the transcriptional upregulation, which could be enhanced further in the presence of the viral Tat protein. The central domain of YY1 (155-295 residues) does not affect LTR activity but has a negative effect on HIV-1 gene expression. Taken together, our study shows that YY1 could act as a transcriptional activator in HIV-1 replication, at least in the early stages of infection. [BMB Reports 2020; 53(5): 248-253].

Selective Estrogen Receptor ß Agonists: a Therapeutic Approach for HIV-1 Associated Neurocognitive Disorders.

The persistence of HIV-1 associated neurocognitive disorders (HAND) in the post-cART era, afflicting between 40 and 70% of HIV-1 seropositive individuals, supports a critical need for the development of adjunctive therapeutic treatments. Selective estrogen receptor ß agonists, including S-Equol (SE), have been implicated as potential therapeutic targets for the treatment of neurocognitive disorders. In the present study, the therapeutic efficacy of 0.2 mg SE for the treatment of HAND was assessed to address two key questions in the HIV-1 transgenic (Tg) rat. First, does SE exhibit robust therapeutic efficacy when treatment is initiated relatively early (i.e., between 2 and 3 months of age) in the course of viral protein exposure? Second, does the therapeutic utility of SE generalize across multiple neurocognitive domains? Treatment with SE enhanced preattentive processes and stimulus-response learning to the level of controls in all (i.e., 100%) HIV-1 Tg animals. For sustained and selective attention, statistically significant effects were not observed in the overall analyses (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). However, given our a priori hypothesis, subsequent analyses were conducted, revealing enhanced sustained and selective attention, approximating controls, in a subset (i.e., 50%, n = 5 and 80%, n = 8, respectively) of HIV-1 Tg animals treated with SE. Thus, the therapeutic efficacy of SE is greater when treatment is initiated relatively early in the course of viral protein exposure and generalizes across neurocognitive domains, supporting an adjunctive therapeutic for HAND in the post-cART era. Graphical Abstract HIV-1 transgenic (Tg) and control animals were treated with either 0.2 mg S-Equol (SE) or placebo between 2 and 3 months of age (Control: Placebo, n = 10, SE, n = 10; HIV-1 Tg: Placebo, n = 10, SE, n = 10). Neurocognitive assessments, tapping preattentive processes, stimulus response learning, sustained attention and selective attention, were conducted to evaluate the utility of SE as a therapeutic for HIV-1 associated neurocognitive disorders (HAND). Planned comparisons between HIV-1 Tg and control animals treated with placebo were utilized to establish a genotype effect, revealing prominent neurocognitive impairments (NCI) in the HIV-1 Tg rat across all domains. Furthermore, to establish the utility of SE, HIV-1 Tg animals treated with SE were compared to control animals treated with placebo. Treatment with 0.2 mg SE ameliorated NCI, to levels that were indistinguishable from controls, in at least a subset (i.e., 50-100%) of HIV-1 Tg animals. Thus, SE supports an efficacious, adjunctive therapeutic for HAND.