HIV latency is a major obstacle to curing infection. Current strategies to eradicate HIV aim at increasing transcription of the latent provirus. In the present study we observed that latently ...infected CD4+ T cells from HIV-infected individuals failed to produce viral particles upon ex vivo exposure to SAHA (vorinostat), despite effective inhibition of histone deacetylases. To identify steps that were not susceptible to the action of SAHA or other latency reverting agents, we used a primary CD4+ T cell model, joint host and viral RNA sequencing, and a viral-encoded reporter. This model served to investigate the characteristics of latently infected cells, the dynamics of HIV latency, and the process of reactivation induced by various stimuli. During latency, we observed persistence of viral transcripts but only limited viral translation. Similarly, the reactivating agents SAHA and disulfiram successfully increased viral transcription, but failed to effectively enhance viral translation, mirroring the ex vivo data. This study highlights the importance of post-transcriptional blocks as one mechanism leading to HIV latency that needs to be relieved in order to purge the viral reservoir.
Abstract
Bovine leukemia virus (BLV)-induced tumoral development is a multifactorial phenomenon that remains incompletely understood. Here, we highlight the critical role of the cellular ...CCCTC-binding factor (CTCF) both in the regulation of BLV transcriptional activities and in the deregulation of the three-dimensional (3D) chromatin architecture surrounding the BLV integration site. We demonstrated the in vivo recruitment of CTCF to three conserved CTCF binding motifs along the provirus. Next, we showed that CTCF localized to regions of transitions in the histone modifications profile along the BLV genome and that it is implicated in the repression of the 5′Long Terminal Repeat (LTR) promoter activity, thereby contributing to viral latency, while favoring the 3′LTR promoter activity. Finally, we demonstrated that BLV integration deregulated the host cellular 3D chromatin organization through the formation of viral/host chromatin loops. Altogether, our results highlight CTCF as a new critical effector of BLV transcriptional regulation and BLV-induced physiopathology.
Cellular composition and the responsiveness of the immune system evolve upon aging and are influenced by biological sex. CD4+ T cells from women living with HIV exhibit a decreased viral replication ...ex vivo compared to men's. We, thus, hypothesized that these findings could be recapitulated in vitro and infected primary CD4+ T cells with HIV-based vectors pseudotyped with VSV-G or HIV envelopes. We used cells isolated from twenty donors to interrogate the effect of sex and age on permissiveness over a six-day activation kinetics. Our data identified an increased permissiveness to HIV between 24 and 72 h post-stimulation. Sex- and age-based analyses at these time points showed an increased susceptibility to HIV of the cells isolated from males and from donors over 50 years of age, respectively. A parallel assessment of surface markers' expression revealed higher frequencies of activation marker CD69 and of immune checkpoint inhibitors (PD-1 and CTLA-4) in the cells from highly permissive donors. Furthermore, positive correlations were identified between the expression kinetics of CD69, PD-1 and CTLA-4 and HIV expression kinetics. The cell population heterogeneity was assessed using a single-cell RNA-Seq analysis and no cell subtype enrichment was identified according to sex. Finally, transcriptomic analyses further highlighted the role of activation in those differences with enriched activation and cell cycle gene sets in male and older female cells. Altogether, this study brought further evidence about the individual features affecting HIV replication at the cellular level and should be considered in latency reactivation studies for an HIV cure.
Retroviruses differ in their preferences for sites for viral DNA integration in the chromosomes of infected cells. Human immunodeficiency virus (HIV) integrates preferentially within active ...transcription units, whereas murine leukemia virus (MLV) integrates preferentially near transcription start sites and CpG islands. We investigated the viral determinants of integration-site selection using HIV chimeras with MLV genes substituted for their HIV counterparts. We found that transferring the MLV integrase (IN) coding region into HIV (to make HIVmIN) caused the hybrid to integrate with a specificity close to that of MLV. Addition of MLV gag (to make HIVmGagmIN) further increased the similarity of target-site selection to that of MLV. A chimeric virus with MLV Gag only (HIVmGag) displayed targeting preferences different from that of both HIV and MLV, further implicating Gag proteins in targeting as well as IN. We also report a genome-wide analysis indicating that MLV, but not HIV, favors integration near DNase I-hypersensitive sites (i.e., +/- 1 kb), and that HIVmIN and HIVmGagmIN also favored integration near these features. These findings reveal that IN is the principal viral determinant of integration specificity; they also reveal a new role for Gag-derived proteins, and strengthen models for integration targeting based on tethering of viral IN proteins to host proteins.
The stable insertion of a copy of their genome into the host cell genome is an essential step of the life cycle of retroviruses. The site of viral DNA integration, mediated by the viral-encoded ...integrase enzyme, has important consequences for both the virus and the host cell. The analysis of retroviral integration site distribution was facilitated by the availability of the human genome sequence, revealing the non-random feature of integration site selection and identifying different favored and disfavored genomic locations for individual retroviruses. This review will summarize the current knowledge about retroviral differences in their integration site preferences as well as the mechanisms involved in this process.
The identification of associations between interleukin‐28B (IL‐28B) variants and the spontaneous clearance of hepatitis C virus (HCV) raises the issues of causality and the net contribution of host ...genetics to the trait. To estimate more precisely the net effect of IL‐28B genetic variation on HCV clearance, we optimized genotyping and compared the host contributions in multiple‐ and single‐source cohorts to control for viral and demographic effects. The analysis included individuals with chronic or spontaneously cleared HCV infections from a multiple‐source cohort (n = 389) and a single‐source cohort (n = 71). We performed detailed genotyping in the coding region of IL‐28B and searched for copy number variations to identify the genetic variant or haplotype carrying the strongest association with viral clearance. This analysis was used to compare the effects of IL‐28B variation in the two cohorts. Haplotypes characterized by carriage of the major alleles at IL‐28B single‐nucleotide polymorphisms (SNPs) were highly overrepresented in individuals with spontaneous clearance versus those with chronic HCV infections (66.1% versus 38.6%, P = 6 × 10−9). The odds ratios for clearance were 2.1 95% confidence interval (CI) = 1.6‐3.0 and 3.9 (95% CI = 1.5‐10.2) in the multiple‐ and single‐source cohorts, respectively. Protective haplotypes were in perfect linkage (r2 = 1.0) with a nonsynonymous coding variant (rs8103142). Copy number variants were not detected. Conclusion: We identified IL‐28B haplotypes highly predictive of spontaneous HCV clearance. The high linkage disequilibrium between IL‐28B SNPs indicates that association studies need to be complemented by functional experiments to identify single causal variants. The point estimate for the genetic effect was higher in the single‐source cohort, which was used to effectively control for viral diversity, sex, and coinfections and, therefore, offered a precise estimate of the net host genetic contribution. (HEPATOLOGY 2011;)
Advances in large-scale analysis of human genomic variability provide unprecedented opportunities to study the genetic basis of susceptibility to infectious agents. We report here the use of an in ...vitro system for the identification of a locus on HSA8q24.3 associated with cellular susceptibility to HIV-1. This locus was mapped through quantitative linkage analysis using cell lines from multigeneration families, validated in vitro, and followed up by two independent association studies in HIV-positive individuals. Single nucleotide polymorphism rs2572886, which is associated with cellular susceptibility to HIV-1 in lymphoblastoid B cells and in primary T cells, was also associated with accelerated disease progression in one of two cohorts of HIV-1-infected patients. Biological analysis suggests a role of the rs2572886 region in the regulation of the LY6 family of glycosyl-phosphatidyl-inositol (GPI)-anchored proteins. Genetic analysis of in vitro cellular phenotypes provides an attractive approach for the discovery of susceptibility loci to infectious agents.
There is an ever-increasing volume of data on host genes that are modulated during HIV infection, influence disease susceptibility or carry genetic variants that impact HIV infection. We created ...GuavaH (Genomic Utility for Association and Viral Analyses in HIV, http://www.GuavaH.org), a public resource that supports multipurpose analysis of genome-wide genetic variation and gene expression profile across multiple phenotypes relevant to HIV biology.
We included original data from 8 genome and transcriptome studies addressing viral and host responses in and ex vivo. These studies cover phenotypes such as HIV acquisition, plasma viral load, disease progression, viral replication cycle, latency and viral-host genome interaction. This represents genome-wide association data from more than 4,000 individuals, exome sequencing data from 392 individuals, in vivo transcriptome microarray data from 127 patients/conditions, and 60 sets of RNA-seq data. Additionally, GuavaH allows visualization of protein variation in ~8,000 individuals from the general population. The publicly available GuavaH framework supports queries on (i) unique single nucleotide polymorphism across different HIV related phenotypes, (ii) gene structure and variation, (iii) in vivo gene expression in the setting of human infection (CD4+ T cells), and (iv) in vitro gene expression data in models of permissive infection, latency and reactivation.
The complexity of the analysis of host genetic influences on HIV biology and pathogenesis calls for comprehensive motors of research on curated data. The tool developed here allows queries and supports validation of the rapidly growing body of host genomic information pertinent to HIV research.
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