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Analysis of HIV-1 transcriptome in different cell models with nanopore sequencing [HIVTCD4_A2018]

ABSTRACT: To explore in depth and in a quantitative manner the complexity of the HIV-1 splicing landscape we used nanopore long-read cDNA (ONT) sequencing in NL4-3 HIV-1 infected primary CD4+ T cells and transfected/infected HeLa cells. Mean read lengths were between 1286 and 2626 nucleotides with maximum sizes of up to 9182 nucleotides, sufficiently long to span all possible splice junctions and to be assigned to a total of 229 exon combinations. Seventy isoforms were above the threshold of 5 copies in infected T cells and a core of 36 isoforms were found in common in infected T cells, infected HeLa cells and transfected HeLa cells. Quantification of both all viral isoforms as well as splice site (SS) usage were compiled to build “splice trees”, a quantitative representation of the splicing pathways leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2, using over-expression of U1 D2UpEx snRNA in transfected HeLa cells and its impact on viral RNA expression. Furthermore, we produced the first dynamic picture of the cascade of events occuring between 12 and 24 hours of CD4+ T cells HIV-1 infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. Altogether, our results show that ONT sequencing allows one to grasp the dynamic of splicing events modulating the viral RNA landscape in infected cells.

ORGANISM(S): Homo sapiens

PROVIDER: GSE138423 | GEO | 2020/08/24

REPOSITORIES: GEO

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Analysis of HIV-1 transcriptome in different cell models with nanopore sequencing [KHI-1_A2019]

Project description:To explore in depth and in a quantitative manner the complexity of the HIV-1 splicing landscape we used nanopore long-read cDNA (ONT) sequencing in NL4-3 HIV-1 infected primary CD4+ T cells and transfected/infected HeLa cells. Mean read lengths were between 1286 and 2626 nucleotides with maximum sizes of up to 9182 nucleotides, sufficiently long to span all possible splice junctions and to be assigned to a total of 229 exon combinations. Seventy isoforms were above the threshold of 5 copies in infected T cells and a core of 36 isoforms were found in common in infected T cells, infected HeLa cells and transfected HeLa cells. Quantification of both all viral isoforms as well as splice site (SS) usage were compiled to build “splice trees”, a quantitative representation of the splicing pathways leading to the different viral isoforms. This approach allowed visualizing the complete rewiring of SS usages upon perturbation of SS D2, using over-expression of U1 D2UpEx snRNA in transfected HeLa cells and its impact on viral RNA expression. Furthermore, we produced the first dynamic picture of the cascade of events occuring between 12 and 24 hours of CD4+ T cells HIV-1 infection. In particular, our data highlighted the importance of non-coding exons in viral RNA transcriptome regulation. Altogether, our results show that ONT sequencing allows one to grasp the dynamic of splicing events modulating the viral RNA landscape in infected cells.

2020-08-24 | GSE138424 | GEO

Analysis of HIV-1 transcriptome in different cell models with nanopore sequencing [HIVTA_A2017]

2020-08-24 | GSE138422 | GEO

Analysis of HIV-1 transcriptome in different cell models with nanopore sequencing [HIVEXTA_A2018]

2020-08-24 | GSE138421 | GEO

miRNA profiling in HIV-1 latently infected cells

Project description:Herein expression trends of host miRNA were measured in HIV-1 latently infected and persistent replication cells, as well as the control cells. HIV-1 latency infection was established by infecting CEM-SS lymphocytes with HIV-1 Bru strain. After selection and long-term culture, the chronically infected cell showed the characteristics of latency definition: 1. The provirus was intergrated in to the host genome.2. No viral expression could be detected during culture.3 .Cell stimulators, such as TNFa,PMA, etc, reactivate the viral expression. As expected, miRNA trend was different in HIV-1 latency when compared to the control or HIV-1 replication. A subset of miRNAs is enriched in HIV-1 latency model. The observation reinforces the concept of active HIV-1 interplay with host small RNAs that modulate HIV-1 infection mode.

2012-12-01 | GSE38388 | GEO

miRNA profiling in HIV-1 latently infected cells

2012-12-01 | E-GEOD-38388 | biostudies-arrayexpress

Complementation can maintain a quasispecies of drug sensitive and resistant HIV

Project description:The goal of this study was to determine how an HIV quasispecies is maintained in the face of selection. We deep sequenced the HIV provirus from cell populations as well as single cells at different time points from in vitro evolution experiments and found that when a less fit and more fit infect the same cell, they share components (complmentation) and therefore allow the less fit to perpetuate. We reproduced a quasispecies to an HIV reverse transcriptase inhibitor. The drug resistant genotype never completely supplanted the drug sensitive genotype, which stabilized at about 20% of viral sequences. Single-cell sequencing showed that resistant genotype frequency plateaued when cells were co-infected with sensitive and resistant genotypes, suggesting a sharing of viral proteins in co-infected cells (complementation), masking genotypic differences. To test if complementation can confer phenotypic drug resistance, we co-transfected fluorescently labelled molecular clones of sensitive and resistant HIV and observed drug resistance in genotypically sensitive virus from co-transfected cells. Resistant virus preferentially co-infected cells with drug sensitive HIV, explaining initiation of co-infections. Modelling showed that a stable quasispecies could form at the experimental multiplicities of infection. Conclusions: Complementation can lead to a quasispecies in infection environments where multiple infections per cell are common

2020-05-13 | GSE150390 | GEO

HeLa cells: control vs IFN-gamma-treated

Project description:Transcriptional profiling of HeLa cells comparing control untreated HeLa cells with IFN-gamma-treated HeLa cells To infect host cells, many viruses use small cellular compartments, called endosomes, for entry, and the thiol/disulfide interchange in viral envelope glycoproteins (Envs) is crucial for infection. By screening cysteine-reacting chemicals, we found a compound, 4,4’-dithiopyridine, which is active at acidic pH, efficiently restricts retrovirus vector infection. We thus hypothesized that some products of endosome-localized, interferon-stimulated genes (ISGs) exhibit anti-viral activity by inhibiting thiol/disulfide interchange in viral Envs. Among the hundreds of ISGs, gamma-interferon (IFN)-inducible lysosomal thiolreductase (GILT) is the only molecule that resides in the endosomes/lysosomes and digests the S-S bonds of proteins under acidic conditions. We now report that GILT significantly inhibits the replication of retroviruses, including HIV-1 and MLV, by restricting both the early and late phases of their life cycles. Using the VSV-G pseudotyped HIV-1 model, we found that GILT digests the S-S bonds of viral Env proteins. GILT also inhibits HIV-1 viral release by digesting the S-S bond of CD63, an endosome-localized molecule reportedly involved in HIV-1 particle release. The effect of -IFN on HIV-1 is limited, although GILT induced by gamma-IFN is supposed to have anti-HIV-1 activity. We found that while gamma-IFN effectively inhibits MLV replication through GILT, the gamma-IFN signaling is remarkably inhibited by the HIV-1 Env protein, but not the MLV Env protein. These findings suggest that GILT functions as an anti-viral host factor induced by gamma-IFN, however, HIV-1 may have evolved to inhibit gamma-IFN signaling by the Env protein.

2017-01-01 | GSE79568 | GEO

Epigenome analysis in several cohorts of HIV progession and in uninfected individuals

Project description:Genome wide DNA methylation profiling of CD4 T cells from uninfected and HIV-infected individuals (viremic, ART-suppressed and elite controllers [EC]) The Illumina Infinium 450k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 485,577 CpGs in DNA from peripheral CD4 T cells samples. Samples included: 22 from HIV-uninfected individuals (uninfected group), 42 from HIV-infected individuals (21 from HIV-infected viremic (viremic group) and 21 from the same participants after viral suppression (viral load< 50 copies HIV-1 RNA/plasma) by antiretroviral therapy administrarion (ART group), and 21 from elite controllers (EC group)

2023-01-16 | GSE143942 | GEO

Dynamics of HIV Latency and Reactivation in a Primary CD4+ T Cell Model

Project description: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.

2017-02-24 | GSE95297 | GEO

Proteomic and metabolic changes in cancer cells after alternation of SHMT2 expressions

Project description:In order to understand the underline mechanism of SHMT2 (serine hydroxymethyltransferase 2) effect on tumor growth, proteome and metabolome analysis were carried on an engineered HeLa cell line (HeLa-SHMT2-shSHMT2, short as HeLa-Ss), which has inducible SHMT2 over-expression or suppression by treating cell with tetracycline or IPTG, respectively. SHMT2 over-expression in HeLa-ss cell increased cell proliferation in vitro and in vivo, deceased expression of several mitochondrial complex I and III proteins, and increased glycine and glutathione levels in cells. BioID method identified more than 20 SHMT2 associated proteins that are involved in oxidation-reduction process. These results indicate SHMT2 involves in the regulation of cellular redox balance. SHMT2 suppression only reduced growth of cells under glycine depletion condition in cell culture. It increased expression of several proteins involved in glutaminolysis and amino acid transporters, and elevated metabolites related to glutamine metabolism. These results indicate tumor cells have a compensatory reaction after SHMT2 suppression. Further reducing glycine levels in cells by sodium benzoate caused cell death in cultured cell and slightly reduced tumor growth in vivo. Benzoate treatment induces more changes in protein expressions and metabolite levels, and it may provide a new addition for tumor treatment.

2020-08-20 | MTBLS405 | MetaboLights

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