Direct Analysis of HIV mRNA m6A Methylation by Nanopore Sequencing.

The post-transcriptional processing and chemical modification of HIV RNA are understudied aspects of HIV virology, primarily due to the limited ability to accurately map and quantify RNA modifications. Modification-specific antibodies or modification-sensitive endonucleases coupled with short-read RNA sequencing technologies have allowed for low-resolution or limited mapping of important regulatory modifications of HIV RNA such as N6-methyladenosine (m6A). However, a high-resolution map of where these sites occur on HIV transcripts is needed for detailed mechanistic understanding. This has recently become possible with new sequencing technologies. Here, we describe the direct RNA sequencing of HIV transcripts using an Oxford Nanopore Technologies sequencer and the use of this technique to map m6A at near single nucleotide resolution. This technology also provides the ability to identify splice variants with long RNA reads and thus, can provide high-resolution RNA modification maps that distinguish between overlapping splice variants. The protocols outlined here for m6A also provide a powerful paradigm for studying any other RNA modifications that can be detected on the nanopore platform.

CRISPR/Cas9 genome editing of CCR5 combined with C46 HIV-1 fusion inhibitor for cellular resistant to R5 and X4 tropic HIV-1.

Hematopoietic stem-cell (HSC) transplantation using a donor with a homozygous mutation in the HIV co-receptor CCR5 (CCR5Δ32/Δ32) holds great promise as a cure for HIV-1. Previously, there were three patients that had been reported to be completely cured from HIV infection by this approach. However, finding a naturally suitable Human Leukocyte Antigen (HLA)-matched homozygous CCR5Δ32 donor is very difficult. The prevalence of this allele is only 1% in the Caucasian population. Therefore, additional sources of CCR5Δ32/Δ32 HSCs are required. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is one method to mediate CCR5 knockout in HSCs that has been successfully employed as a gene editing tool in clinical trials. Additional anti-HIV-1 strategies are still required for broad-spectrum inhibition of HIV-1 replication. Here in this study, we combined an additional anti-HIV-1 therapy, which is C46, a cell membrane-anchored HIV-1 fusion inhibitor with the CRISPR/Cas9 mediated knockout CCR5. The combined HIV-1 therapeutic genes were investigated for the potential prevention of both CCR5 (R5)- and CXCR4 (X4)-tropic HIV-1 infections in the MT4CCR5 cell line. The combinatorial CRISPR/Cas9 therapies were superior compared to single method therapy for achieving the HIV-1 cure strategy and shows potential for future applications.

Genetic Variations in EIF2AK3 are Associated with Neurocognitive Impairment in People Living with HIV.

Based on emerging evidence on the role for specific single-nucleotide variants (SNVs) in EIF2AK3 encoding the integrated stress response kinase PERK, in neurodegeneration, we assessed the association of EIF2AK3 SNVs with neurocognitive performance in people with HIV (PWH) using a candidate gene approach. This retrospective study included the CHARTER cohort participants, excluding those with severe neuropsychiatric comorbidities. Genome-wide data previously obtained for 1047 participants and targeted sequencing of 992 participants with available genomic DNA were utilized to interrogate the association of three noncoding and three coding EIF2AK3 SNVs with the continuous global deficit score (GDS) and global neurocognitive impairment (NCI; GDS ≥ 0.5) using univariable and multivariable methods, with demographic, disease-associated, and treatment characteristics as covariates. The cohort characteristics were as follows: median age, 43.1 years; females, 22.8%; European ancestry, 41%; median CD4 + T cell counts, 175/µL (nadir) and 428/µL (current). At first assessment, 70.5% used ART and 68.3% of these had plasma HIV RNA levels ≤ 200 copies/mL. All three noncoding EIF2AK3 SNVs were associated with GDS and NCI (all p < 0.05). Additionally, 30.9%, 30.9%, and 41.2% of participants had at least one risk allele for the coding SNVs rs1805165 (G), rs867529 (G), and rs13045 (A), respectively. Homozygosity for all three coding SNVs was associated with significantly worse GDS (p < 0.001) and more NCI (p < 0.001). By multivariable analysis, the rs13045 A risk allele, current ART use, and Beck Depression Inventory-II value > 13 were independently associated with GDS and NCI (p < 0.001) whereas the other two coding SNVs did not significantly correlate with GDS or NCI after including rs13045 in the model. The coding EIF2AK3 SNVs were associated with worse performance in executive functioning, motor functioning, learning, and verbal fluency. Coding and non-coding SNVs of EIF2AK3 were associated with global NC and domain-specific performance. The effects were small-to-medium in size but present in multivariable analyses, raising the possibility of specific SNVs in EIF2AK3 as an important component of genetic vulnerability to neurocognitive complications in PWH.

Bioinformatics and system biology approach to identify the influences among COVID-19, influenza, and HIV on the regulation of gene expression.

Background: Coronavirus disease (COVID-19), caused by SARS-CoV-2, has emerged as a infectious disease, coexisting with widespread seasonal and sporadic influenza epidemics globally. Individuals living with HIV, characterized by compromised immune systems, face an elevated risk of severe outcomes and increased mortality when affected by COVID-19. Despite this connection, the molecular intricacies linking COVID-19, influenza, and HIV remain unclear. Our research endeavors to elucidate the shared pathways and molecular markers in individuals with HIV concurrently infected with COVID-19 and influenza. Furthermore, we aim to identify potential medications that may prove beneficial in managing these three interconnected illnesses. Methods: Sequencing data for COVID-19 (GSE157103), influenza (GSE185576), and HIV (GSE195434) were retrieved from the GEO database. Commonly expressed differentially expressed genes (DEGs) were identified across the three datasets, followed by immune infiltration analysis and diagnostic ROC analysis on the DEGs. Functional enrichment analysis was performed using GO/KEGG and Gene Set Enrichment Analysis (GSEA). Hub genes were screened through a Protein-Protein Interaction networks (PPIs) analysis among DEGs. Analysis of miRNAs, transcription factors, drug chemicals, diseases, and RNA-binding proteins was conducted based on the identified hub genes. Finally, quantitative PCR (qPCR) expression verification was undertaken for selected hub genes. Results: The analysis of the three datasets revealed a total of 22 shared DEGs, with the majority exhibiting an area under the curve value exceeding 0.7. Functional enrichment analysis with GO/KEGG and GSEA primarily highlighted signaling pathways associated with ribosomes and tumors. The ten identified hub genes included IFI44L, IFI44, RSAD2, ISG15, IFIT3, OAS1, EIF2AK2, IFI27, OASL, and EPSTI1. Additionally, five crucial miRNAs (hsa-miR-8060, hsa-miR-6890-5p, hsa-miR-5003-3p, hsa-miR-6893-3p, and hsa-miR-6069), five essential transcription factors (CREB1, CEBPB, EGR1, EP300, and IRF1), and the top ten significant drug chemicals (estradiol, progesterone, tretinoin, calcitriol, fluorouracil, methotrexate, lipopolysaccharide, valproic acid, silicon dioxide, cyclosporine) were identified. Conclusion: This research provides valuable insights into shared molecular targets, signaling pathways, drug chemicals, and potential biomarkers for individuals facing the complex intersection of COVID-19, influenza, and HIV. These findings hold promise for enhancing the precision of diagnosis and treatment for individuals with HIV co-infected with COVID-19 and influenza.

A simultaneous knockout knockin genome editing strategy in HSPCs potently inhibits CCR5- and CXCR4-tropic HIV-1 infection.

Allogeneic hematopoietic stem and progenitor cell transplant (HSCT) of CCR5 null (CCR5Δ32) cells can be curative for HIV-1-infected patients. However, because allogeneic HSCT poses significant risk, CCR5Δ32 matched bone marrow donors are rare, and CCR5Δ32 transplant does not confer resistance to the CXCR4-tropic virus, it is not a viable option for most patients. We describe a targeted Cas9/AAV6-based genome editing strategy for autologous HSCT resulting in both CCR5- and CXCR4-tropic HIV-1 resistance. Edited human hematopoietic stem and progenitor cells (HSPCs) maintain multi-lineage repopulation capacity in vivo, and edited primary human T cells potently inhibit infection by both CCR5-tropic and CXCR4-tropic HIV-1. Modification rates facilitated complete loss of CCR5-tropic replication and up to a 2,000-fold decrease in CXCR4-tropic replication without CXCR4 locus disruption. This multi-factor editing strategy in HSPCs could provide a broad approach for autologous HSCT as a functional cure for both CCR5-tropic and CXCR4-tropic HIV-1 infections.

The CRISPR-Cas9 induced CCR5 Δ32 mutation as a potent gene therapy methodology for resistance to HIV-1 variant: a review.

Human Immunodeficiency Virus (HIV) has continuously been the greatest epidemic for humanity over a period spanning almost five decades. With no specific cure or treatment available to date despite extensive research, the C-C Chemokine Receptor 5, Delta 32 (CCR5 Δ32) allele genetic point mutation plays an imperative role in the prevention of acquired immunodeficiency syndrome (AIDS). This comprehensive study aims to review the induction of the homozygous recessive deletion genotype using the Clustered Regularly Interspaced Short Palindromic Repeats, Cas 9 Enzyme (CRISPR-Cas9), and hematopoietic stem cell transplantation under positive selection pressure for active immunity in seropositive patients' populations as the phenotype. A methodology is proposed to trigger a significant increase in the expression of Delta 32 beneficial mutant alleles within controlled modern healthcare facilities utilizing totipotent stem cells through somatic gene therapy. It acts upon two dysfunctional CCR5 genes, translating mutant G protein-coupled co-receptors, whose primary function is similar to that of C-X-C Motif Chemokine receptor 4 (CXCR4), by blocking the entry of viral RNA into the CD4+ T helper lymphocytes, halting infection and seizing viral life cycle. This modification is endemic in Northern Europe, where it naturally pertains to the Caucasian descent population samples in the form of polymorphism, p (X=0.01), where X is the probability of frequency of complete immunity against HIV-1 in population samples. The epigenetics of the single nucleotide polymorphism (SNP) are analyzed as they play a significant role in immunity distribution. Furthermore, a comparative analysis within the ethical boundaries of CRISPR-Cas9 is conducted to discuss the practical aspects and challenges of the presented methodologies and treatment alternatives. Additionally, the study assembles all available data and summarizes preexisting research while providing a promising solution to this ethical dilemma. Finally, a methodology is devised to answer the question of whether the variant-specific epidemic of AIDS caused by HIV-1 can be cured via artificially inducing immunity by CRISPR-Cas9.

Molecular mechanisms behind the generation of pro-oncogenic HIV-1 matrix protein p17 variants.

HIV-1 matrix protein p17 variants (vp17s), characterized by amino acid insertions at the COOH-terminal region of the viral protein, have been recently identified and studied for their biological activity. Different from their wild-type counterpart (refp17), vp17s display a potent B cell growth and clonogenic activity. Recent data have highlighted the higher prevalence of vp17s in people living with HIV-1 (PLWH) with lymphoma compared with those without lymphoma, suggesting that vp17s may play a key role in lymphomagenesis. Molecular mechanisms involved in vp17 development are still unknown. Here we assessed the efficiency of HIV-1 Reverse Transcriptase (RT) in processing this genomic region and highlighted the existence of hot spots of mutation in Gag, at the end of the matrix protein and close to the matrix-capsid junction. This is possibly due to the presence of inverted repeats and palindromic sequences together with a high content of Adenine in the 322-342 nucleotide portion, which constrain HIV-1 RT to pause on the template. To define the recombinogenic properties of hot spots of mutation in the matrix gene, we developed plasmid vectors expressing Gag and a minimally modified Gag variant, and measured homologous recombination following cell co-nucleofection by next-generation sequencing. Data obtained allowed us to show that a wide range of recombination events occur in concomitance with the identified hot spots of mutation and that imperfect events may account for vp17s generation.

The ISG15-Protease USP18 Is a Pleiotropic Enhancer of HIV-1 Replication.

The innate immune response to viruses is formed in part by interferon (IFN)-induced restriction factors, including ISG15, p21, and SAMHD1. IFN production can be blocked by the ISG15-specific protease USP18. HIV-1 has evolved to circumvent host immune surveillance. This mechanism might involve USP18. In our recent studies, we demonstrate that HIV-1 infection induces USP18, which dramatically enhances HIV-1 replication by abrogating the antiviral function of p21. USP18 downregulates p21 by accumulating misfolded dominant negative p53, which inactivates wild-type p53 transactivation, leading to the upregulation of key enzymes involved in de novo dNTP biosynthesis pathways and inactivated SAMHD1. Despite the USP18-mediated increase in HIV-1 DNA in infected cells, it is intriguing to note that the cGAS-STING-mediated sensing of the viral DNA is abrogated. Indeed, the expression of USP18 or knockout of ISG15 inhibits the sensing of HIV-1. We demonstrate that STING is ISGylated at residues K224, K236, K289, K347, K338, and K370. The inhibition of STING K289-linked ISGylation suppresses its oligomerization and IFN induction. We propose that human USP18 is a novel factor that potentially contributes in multiple ways to HIV-1 replication.

Schlafen14 Impairs HIV-1 Expression in a Codon Usage-Dependent Manner.

Schlafen (SLFN) is a family of proteins upregulated by type I interferons with a regulatory role in translation. Intriguingly, SLFN14 associates with the ribosome and can degrade rRNA, tRNA, and mRNA in vitro, but a role in translation is still unknown. Ribosomes are important regulatory hubs during translation elongation of mRNAs rich in rare codons. Therefore, we evaluated the potential role of SLFN14 in the expression of mRNAs enriched in rare codons, using HIV-1 genes as a model. We found that, in a variety of cell types, including primary immune cells, SLFN14 regulates the expression of HIV-1 and non-viral genes based on their codon adaptation index, a measurement of the synonymous codon usage bias; consequently, SLFN14 inhibits the replication of HIV-1. The potent inhibitory effect of SLFN14 on the expression of the rare codon-rich transcript HIV-1 Gag was minimized by codon optimization. Mechanistically, we found that the endoribonuclease activity of SLFN14 is required, and that ribosomal RNA degradation is involved. Therefore, we propose that SLFN14 impairs the expression of HIV-1 transcripts rich in rare codons, in a catalytic-dependent manner.

The Inhibition of Gag-Pol Expression by the Restriction Factor Shiftless Is Dispensable for the Restriction of HIV-1 Infection.

The interferon-induced host cell protein Shiftless (SFL) inhibits -1 programmed ribosomal frameshifting (-1PRF) required for the expression of HIV-1 Gal-Pol and the formation of infectious HIV-1 particles. However, the specific regions in SFL required for antiviral activity and the mechanism by which SFL inhibits -1PRF remain unclear. Employing alanine scanning mutagenesis, we found that basic amino acids in the predicted zinc ribbon motif of SFL are essential for the suppression of Gag-Pol expression but dispensable for anti-HIV-1 activity. We have shown that SFL inhibits the expression of the murine leukemia virus (MLV) Gag-Pol polyprotein and the formation of infectious MLV particles, although Gag-Pol expression of MLV is independent of -1PRF but requires readthrough of a stop codon. These findings indicate that SFL might inhibit HIV-1 infection by more than one mechanism and that SFL might target programmed translational readthrough as well as -1PRF signals, both of which are regulated by mRNA secondary structure elements.

Genetic haute couture to block HIV-1 at front doors.

The chemokine receptors CCR5 and CXCR4 are "front doors" for HIV-1 infection in host cells, and their targeting represents a potential solution for a cure. Dudek et al.1 now propose a new gene editing strategy to simultaneously block CCR5- and CXCR4-mediated HIV-1 entry in autologous hematopoietic stem and progenitor cells (HSPCs).

Endogenous retroelement expression in the gut microenvironment of people living with HIV-1.

BACKGROUND: Endogenous retroelements (EREs), including human endogenous retroviruses (HERVs) and long interspersed nuclear elements (LINEs), comprise almost half of the human genome. Our previous studies of the interferome in the gut suggest potential mechanisms regarding how IFNb may drive HIV-1 gut pathogenesis. As ERE activity is suggested to partake in type 1 immune responses and is incredibly sensitive to viral infections, we sought to elucidate underlying interactions between ERE expression and gut dynamics in people living with HIV-1 (PLWH). METHODS: ERE expression profiles from bulk RNA sequencing of colon biopsies and PBMC were compared between a cohort of PLWH not on antiretroviral therapy (ART) and uninfected controls. FINDINGS: 59 EREs were differentially expressed in the colon of PLWH when compared to uninfected controls (padj <0.05 and FC ≤ -1 or ≥ 1) [Wald's Test]. Of these 59, 12 EREs were downregulated in PLWH and 47 were upregulated. Colon expression of the ERE loci LTR19_12p13.31 and L1FLnI_1q23.1s showed significant correlations with certain gut immune cell subset frequencies in the colon. Furthermore L1FLnI_1q23.1s showed a significant upregulation in peripheral blood mononuclear cells (PBMCs) of PLWH when compared to uninfected controls suggesting a common mechanism of differential ERE expression in the colon and PBMC. INTERPRETATION: ERE activity has been largely understudied in genomic characterizations of human pathologies. We show that the activity of certain EREs in the colon of PLWH is deregulated, supporting our hypotheses that their underlying activity could function as (bio)markers and potential mediators of pathogenesis in HIV-1 reservoirs. FUNDING: US NIH grants NCI CA260691 (DFN) and NIAID UM1AI164559 (DFN).

Computationally inferred cell-type specific epigenome-wide DNA methylation analysis unveils distinct methylation patterns among immune cells for HIV infection in three cohorts.

BACKGROUND: Epigenome-wide association studies (EWAS) have identified CpG sites associated with HIV infection in blood cells in bulk, which offer limited knowledge of cell-type specific methylation patterns associated with HIV infection. In this study, we aim to identify differentially methylated CpG sites for HIV infection in immune cell types: CD4+ T-cells, CD8+ T-cells, B cells, Natural Killer (NK) cells, and monocytes. METHODS: Applying a computational deconvolution method, we performed a cell-type based EWAS for HIV infection in three independent cohorts (Ntotal = 1,382). DNA methylation in blood or in peripheral blood mononuclear cells (PBMCs) was profiled by an array-based method and then deconvoluted by Tensor Composition Analysis (TCA). The TCA-computed CpG methylation in each cell type was first benchmarked by bisulfite DNA methylation capture sequencing in a subset of the samples. Cell-type EWAS of HIV infection was performed in each cohort separately and a meta-EWAS was conducted followed by gene set enrichment analysis. RESULTS: The meta-analysis unveiled a total of 2,021 cell-type unique significant CpG sites for five inferred cell types. Among these inferred cell-type unique CpG sites, the concordance rate in the three cohorts ranged from 96% to 100% in each cell type. Cell-type level meta-EWAS unveiled distinct patterns of HIV-associated differential CpG methylation, where 74% of CpG sites were unique to individual cell types (false discovery rate, FDR <0.05). CD4+ T-cells had the largest number of unique HIV-associated CpG sites (N = 1,624) compared to any other cell type. Genes harboring significant CpG sites are involved in immunity and HIV pathogenesis (e.g. CD4+ T-cells: NLRC5, CX3CR1, B cells: IFI44L, NK cells: IL12R, monocytes: IRF7), and in oncogenesis (e.g. CD4+ T-cells: BCL family, PRDM16, monocytes: PRDM16, PDCD1LG2). HIV-associated CpG sites were enriched among genes involved in HIV pathogenesis and oncogenesis that were enriched among interferon-α and -γ, TNF-α, inflammatory response, and apoptotic pathways. CONCLUSION: Our findings uncovered computationally inferred cell-type specific modifications in the host epigenome for people with HIV that contribute to the growing body of evidence regarding HIV pathogenesis.

ABCG2 polymorphisms and susceptibility to ARV-associated hepatotoxicity.

BACKGROUND: The ABCG2 421C/A polymorphism contributes significantly to the distribution and absorption of antiretroviral (ARV) regimens and is associated with the undesirable side effects of efavirenz. METHODS: To investigate this, we examined ABCG2 34G/A (rs2231137) and 421C/A (rs2231142) genetic variations in 149 HIV-infected patients (116 without hepatotoxicity, 33 with ARV-induced hepatotoxicity) and 151 healthy controls through the PCR-restriction fragment length polymorphism (PCR-RFLP) technique. RESULTS AND DISCUSSION: The ABCG2 34GA genotype and 34A allele indicated a risk for antiretroviral therapy-associated hepatotoxicity development (p = 0.09, OR = 1.58, 95% CI: 0.93-2.69; p = 0.06, OR = 1.50, 95% CI: 0.98-2.30). The haplotype GA was associated with hepatotoxicity (p = 0.042, OR = 2.37, 95% CI: 1.04-5.43; p = 0.042, OR = 2.49, 95% CI: 1.04-5.96). Moreover, when comparing HIV patients with hepatotoxicity to healthy controls, the haplotype GA had an association with an elevated risk for the development of hepatotoxicity (p = 0.041, OR = 1.73, 95% CI: 1.02-2.93). Additionally, the association of the ABCG2 34GA genotype with the progression of HIV (p = 0.02, OR = 1.97, 95% CI: 1.07-3.63) indicated a risk for advanced HIV infection. Furthermore, the ABCG2 421AA genotype was linked to tobacco users and featured as a risk factor for the progression of HIV disease (p = 0.03, OR = 11.07, 95% CI: 1.09-270.89). CONCLUSION: The haplotype GA may enhance the risk of hepatotoxicity development and its severity. Individuals with the ABCG2 34A allele may also be at risk for the development of hepatotoxicity. Additionally, individuals with an advanced stage of HIV and the ABCG2 34GA genotype may be at risk for disease progression.

Legacy of a magic gene-CCR5-∆32: From discovery to clinical benefit in a generation.

The discovery of the 32-bp deletion allele of the chemokine receptor gene CCR5 showed that homozygous carriers display near-complete resistance to HIV infection, irrespective of exposure. Algorithms of molecular evolutionary theory suggested that the CCR5-∆32 mutation occurred but once in the last millennium and rose by strong selective pressure relatively recently to a ~10% allele frequency in Europeans. Several lines of evidence support the hypothesis that CCR5-∆32 was selected due to its protective influence to resist Yersinia pestis, the agent of the Black Death/bubonic plague of the 14th century. Powerful anti-AIDS entry inhibitors targeting CCR5 were developed as a treatment for HIV patients, particularly those whose systems had developed resistance to powerful anti-retroviral therapies. Homozygous CCR5-∆32/∆32 stem cell transplant donors were used to produce HIV-cleared AIDS patients in at least five "cures" of HIV infection. CCR5 has also been implicated in regulating infection with Staphylococcus aureus, in recovery from stroke, and in ablation of the fatal graft versus host disease (GVHD) in cancer transplant patients. While homozygous CCR5-∆32/32 carriers block HIV infection, alternatively they display an increased risk for encephalomyelitis and death when infected with the West Nile virus.

HIV-1 mRNA knockdown with CRISPR/CAS9 enhances neurocognitive function.

Mixed glia are infiltrated with HIV-1 virus early in the course of infection leading to the development of a persistent viral reservoir in the central nervous system. Modification of the HIV-1 genome using gene editing techniques, including CRISPR/Cas9, has shown great promise towards eliminating HIV-1 viral reservoirs; whether these techniques are capable of removing HIV-1 viral proteins from mixed glia, however, has not been systematically evaluated. Herein, the efficacy of adeno-associated virus 9 (AAV9)-CRISPR/Cas9 gene editing for eliminating HIV-1 messenger RNA (mRNA) from cortical mixed glia was evaluated in vitro and in vivo. In vitro, a within-subjects experimental design was utilized to treat mixed glia isolated from neonatal HIV-1 transgenic (Tg) rats with varying doses (0, 0.9, 1.8, 2.7, 3.6, 4.5, or 5.4 µL corresponding to a physical titer of 0, 4.23 × 109, 8.46 × 109, 1.269 × 1010, 1.692 × 1010, 2.115 × 1010, and 2.538 × 1010 gc/µL) of CRISPR/Cas9 for 72 h. Dose-dependent decreases in the number of HIV-1 mRNA, quantified using an innovative in situ hybridization technique, were observed in a subset (i.e., n = 5 out of 8) of primary mixed glia. In vivo, HIV-1 Tg rats were retro-orbitally inoculated with CRISPR/Cas9 for two weeks, whereby treatment resulted in profound excision (i.e., approximately 53.2%) of HIV-1 mRNA from the medial prefrontal cortex. Given incomplete excision of the HIV-1 viral genome, the clinical relevance of HIV-1 mRNA knockdown for eliminating neurocognitive impairments was evaluated via examination of temporal processing, a putative neurobehavioral mechanism underlying HIV-1-associated neurocognitive disorders (HAND). Indeed, treatment with CRISPR/Cas9 protractedly, albeit not permanently, restored the developmental trajectory of temporal processing. Proof-of-concept studies, therefore, support the susceptibility of mixed glia to gene editing and the potential of CRISPR/Cas9 to serve as a novel therapeutic strategy for HAND, even in the absence of full viral eradication.

Optimization of HIV Sequencing Method Using Vela Sentosa Library on Miseq Ilumina Platform.

Genotypic testing is often recommended to improve the management of patients infected with human immunodeficiency virus (HIV). To help combat this major pandemic, next-generation sequencing (NGS) techniques are widely used to analyse resistance to antiretroviral drugs. In this study, we used a Vela Sentosa kit (Vela Diagnostics, Kendall, Singapore), which is usually used for the Ion Torrent personal genome machine (PGM) platform, to sequence HIV using the Illumina Miseq platform. After RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR), minor modifications were applied to the Vela Sentosa kit to adapt it to the Illumina Miseq platform. Analysis of the results showed the same mutations present in the samples using both sequencing platforms. The total number of reads varied from 185,069 to 752,343 and from 642,162 to 2,074,028 in the Ion Torrent PGM platform and the Illumina Miseq platform, respectively. The average depth was 21,955 and 46,856 for Ion Torrent PGM and Illumina Miseq platforms, respectively. The cost of sequencing a run of eight samples was quite similar between the two platforms (about USD 1790 for Illumina Miseq and about USD 1833 for Ion Torrent PGM platform). We have shown for the first time that it is possible to adapt and use the Vela Sentosa kit for the Illumina Miseq platform to obtain high-quality results with a similar cost.

Impact of HLA class I functional divergence on HIV control.

Heterozygosity of Human leukocyte antigen (HLA) class I genes is linked to beneficial outcomes after HIV infection, presumably through greater breadth of HIV epitope presentation and cytotoxic T cell response. Distinct allotype pairs, however, differ in the extent to which they bind shared sets of peptides. We developed a functional divergence metric that measures pairwise complementarity of allotype-associated peptide binding profiles. Greater functional divergence for pairs of HLA-A and/or HLA-B allotypes was associated with slower AIDS progression and independently with enhanced viral load control. The metric predicts immune breadth at the peptide level rather than gene level and redefines HLA heterozygosity as a continuum differentially affecting disease outcome. Functional divergence may affect response to additional infections, vaccination, immunotherapy, and other diseases where HLA heterozygote advantage occurs.

Association of polymorphism of IL-17A, IL-17F, and IL-6 with Toxoplasma gondii infection susceptibility in HIV/AIDS patients in Shiraz, southern Iran.

INTRODUCTION: Toxoplasma gondii infection is considered as one of the most important opportunistic infections and cause of death in HIV patients. METHODS: In this cross-sectional study, 334 HIV positive patients were included. The molecular test was performed by the restriction fragment length polymorphism-polymerase chain reaction method. Allelic frequency, haplotype analyses, and linkage disequilibrium were calculated. The odds ratio was calculated. The linear regression model was used to analysis of interleukin (IL)-17A, IL-17F, and IL-6 single-nucleotide polymorphism genotypes in HIV patients with and without toxoplasmosis. RESULTS: In total, 95 tested'patients (28.4%) were positive for toxoplasmosis. The risk of toxoplasma infection in the current study did not correlate with IL-17 and IL-6 polymorphism and the risk of contracting toxoplasma was also not significantly correlated in this study. There was no association between the frequency of alleles and the risk of toxoplasma infection in IL-17 haplotype analysis. CONCLUSION: The findings of this study revealed that there were significant differences in the serum levels of IL-6 and IL-17A, but not IL-17F, between the case and control groups in various genetic models. However, these polymorphisms did not show a significant relationship with toxoplasma infection in HIV-positive patients. This study represents the first investigation in Iran to explore the role of IL-6 and IL-17 polymorphisms in toxoplasma infection among HIV-positive patients.

Chronic HIV-1 Tat action induces HLA-DR downregulation in B cells: A mechanism for lymphoma immune escape in people living with HIV.

Despite the success of combination antiretroviral therapy, people living with human immunodeficiency virus (HIV) still have an increased risk of Epstein-Barr virus (EBV)-associated B cell malignancies. In the HIV setting, B cell physiology is altered by coexistence with HIV-infected cells and the chronic action of secreted viral proteins, for example, HIV-1 Tat that, once released, efficiently penetrates noninfected cells. We modeled the chronic action of HIV-1 Tat on B cells by ectopically expressing Tat or TatC22G mutant in two lymphoblastoid B cell lines. The RNA-sequencing analysis revealed that Tat deregulated the expression of hundreds of genes in B cells, including the downregulation of a subset of major histocompatibility complex (MHC) class II-related genes. Tat-induced downregulation of HLA-DRB1 and HLA-DRB5 genes led to a decrease in HLA-DR surface expression; this effect was reproduced by coculturing B cells with Tat-expressing T cells. Chronic Tat presence decreased the NF-ᴋB pathway activity in B cells; this downregulated NF-ᴋB-dependent transcriptional targets, including MHC class II genes. Notably, HLA-DRB1 and surface HLA-DR expression was also decreased in B cells from people with HIV. Tat-induced HLA-DR downregulation in B cells impaired EBV-specific CD4+ T cell response, which contributed to the escape from immune surveillance and could eventually promote B cell lymphomagenesis in people with HIV.