HIV Modifies the m6A and m5C Epitranscriptomic Landscape of the Host Cell

The study of RNA modifications, today known as epitranscriptomics, is of growing interest. The N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications are abundantly present on mRNA molecules, and impact RNA interactions with other proteins or molecules, thereby affecting cellular processes, such as RNA splicing, export, stability, and translation. Recently m6A and m5C marks were found to be present on human immunodeficiency (HIV) transcripts as well and affect viral replication. Therefore, the discovery of RNA methylation provides a new layer of regulation of HIV expression and replication, and thus offers novel array of opportunities to inhibit replication. However, no study has been performed to date to investigate the impact of HIV replication on the transcript methylation level in the infected cell. We used a productive HIV infection model, consisting of the CD4+ SupT1 T cell line infected with a VSV-G pseudotyped HIVeGFP-based vector, to explore the temporal landscape of m6A and m5C epitranscriptomic marks upon HIV infection, and to compare it to mock-treated cells. Cells were collected at 12, 24, and 36 h post-infection for mRNA extraction and FACS analysis. M6A RNA modifications were investigated by methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-Seq). M5C RNA modifications were investigated using a bisulfite conversion approach followed by high-throughput sequencing (BS-Seq). Our data suggest that HIV infection impacted the methylation landscape of HIV-infected cells, inducing mostly increased methylation of cellular transcripts upon infection. Indeed, differential methylation (DM) analysis identified 59 m6A hypermethylated and only 2 hypomethylated transcripts and 14 m5C hypermethylated transcripts and 7 hypomethylated ones. All data and analyses are also freely accessible on an interactive web resource (http://sib-pc17.unil.ch/HIVmain.html). Furthermore, bothm6A andm5Cmethylations were detected on viral transcripts and viral particle RNA genomes, as previously described, but additional patterns were identified. This work used differential epitranscriptomic analysis to identify novel players involved in HIV life cycle, thereby providing innovative opportunities for HIV regulation

Characterization of the epitranscriptomic landscape of HIV-infected cells

The study of RNA modifications, today known as epitranscriptomics, is of growing interest. The N6-methyladenosine (m6A) and 5-methylcytosine (m5C) RNA modifications are abundantly present on mRNA molecules, and impact RNA interactions with other proteins or molecules, thereby affecting cellular processes, such as RNA splicing, export, stability and translation. Recently m6A and m5C marks were found to be present on human immunodeficiency (HIV) transcripts as well and affect viral replication. Therefore, the discovery of RNA methylation provides a new layer of regulation of HIV expression and replication, and thus a novel array of opportunities to inhibit replication. However, no study has been performed to date to investigate the impact of HIV replication on the transcript methylation level in the infected cell. To achieve this goal, we used a productive HIV infetion model, consisting of the CD4+ SupT1 T cell line infected with a VSV-G pseudotyped HIVeGFP-based vector, to explore the temporal landscape of m6A and m5C epitranscriptomic marks upon HIV infection, and compare it to mock-treated cells. Cells were collected at 12, 24 and 36h post-infection for mRNA extraction and FACS analysis. M6A RNA modifications were investigated by methylated RNA immunoprecipitation followed by high-throughput sequencing (MeRIP-Seq). M5C RNA modifications were investigated using a bisulfite conversion approach followed by high-throughput sequencing (BS-Seq). Our data suggest that HIV Infection impacted the methylation landscape of HIV-infected cells, inducing mostly increased methylation upon infection. Indeed, differential methylation (DM) analysis identified 62 m6A hypermethylated and only 2 hypomethylated transcripts and 14 m5C hypermethylated transcripts and 7 hypomethylated ones. Similar to differential expression analysis, we reasoned that differential methylation analysis may reveal novel factors potentially acting as HIV dependency factors (HDF) or HIV inhibitory factors (HIF), therefore impacting viral replication success. Further characterization of putative HDF/HIF DM genes, using CRISPR-Cas9-mediated knock-out approach followed by HIV_GFP infection, is necessary to validate their role on viral replication. Furthermore, both m6A and m5C methylation was detected on viral transcripts, as previously described, but additional patterns were identified. Altogether, our study provides the first overview of the epitrantranscriptome landscape of HIV-infected cells and offers novel opportunities to identify cellular factors impacting the viral replication success. -- L'étude des modifications de l'ARN, aujourd'hui appelée épitranscriptomique, suscite un intérêt croissant. Les modifications N6-méthyladénosine (m6A) et 5-méthylcytosine (m5C) de l'ARN sont abondamment présentes sur les molécules d'ARN messager (ARNm). Elles peuvent avoir un impact sur les interactions entre l'ARN et les autres molécules ou protéines, et être affectées ainsi lors de processus cellulaires, tels que l'épissage, l'exportation, la stabilité et la traduction des ARNm. Les modifications m6A et m5C ont récemment été identifiées également sur les transcrits du virus de l’immunodéficience humaine (VIH) avec un rôle sur la réplication virale. La découverte de la méthylation de l'ARN du VIH offre un niveau additionnel de régulation de l'expression et de la réplication du VIH, et donc une nouvelle gamme d'opportunités pour inhiber la réplication. Cependant, aucune étude n'a été réalisée à ce jour visant à étudier l'impact du VIH sur le niveau de méthylation des transcrits dans la cellule infectée. Pour ce faire, nous avons utilisé un modèle productif d'infection à VIH, soit une lignée de cellules T, SupT1, infectées par le vecteur viral HIVeGFP pseudotypé à l’aide d’une enveloppe VSV-G, afin d’explorer le profil temporel des marques épitranscriptomiques m6A et m5C lors de l’infection VIH et de le comparer à celui des cellules non-infectées. Des cellules ont donc été collectées 12, 24 et 36 heures après l'infection en vue de l'extraction de leur ARNm et de leur analyse par FACS. Les modifications m6A ont été investiguées à l’aide de l’immunoprécipitation de l'ARN méthylé, suivie d'un séquençage à haut-débit (MeRIP-Seq). Les modifications m5C ont été étudiées en utilisant une approche de conversion au bisulfite suivie d'un séquençage à haut-débit (BS-Seq). Nos données révèlent que l'infection du VIH impacte le profil de méthylation des cellules infectées, en induisant principalement une augmentation de la proportion de méthylation lors de l'infection. L'analyse de méthylation différentielle (DM) a identifié 62 transcrits hyperméthylés et 2 transcrits hypométhylés pour la marque m6A et 14 transcrits hyperméthylés et 7 hypométhylés pour la marque m5C. Comme pour les analyses d’expression différentielle des gènes, nous avons émis l’hypothèse que l’analyse différentielle du taux de méthylation des transcrits pouvait révéler de nouveaux acteurs cellulaires, agissant potentiellement en tant que facteurs facilitant l’infection virale (HIV Dependency Factors, HDF) ou au contraire l’inhibant (HIV Inhibitory Factors, HIF). La caractérisation de ces HDF/HIF potentiels, par une invalidation génique par approche de type CRISPR-Cas9, est donc nécessaire pour valider leur rôle sur la réplication virale. De plus, les méthylations m6A et m5C ont également été détectés sur les transcrits viraux, comme décrit précédemment, mais de nouvelles régions ont été identifiées. En conclusion, notre étude présente la première vue d’ensemble du profil épitranscriptomique des cellules infectées par le VIH et offre de nouvelles opportunités d’identifier des facteurs cellulaires impliqués dans la réplication virale

The Impact of Epitranscriptomics on Antiviral Innate Immunity

Epitranscriptomics, i.e., chemical modifications of RNA molecules, has proven to be a new layer of modulation and regulation of protein expression, asking for the revisiting of some aspects of cellular biology. At the virological level, epitranscriptomics can thus directly impact the viral life cycle itself, acting on viral or cellular proteins promoting replication, or impacting the innate antiviral response of the host cell, the latter being the focus of the present review

Sex and Age Impact CD4+ T Cell Susceptibility to HIV In Vitro through Cell Activation Dynamics

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

T-Cells Subsets in Castleman Disease: Analysis of 28 Cases Including Unicentric, Multicentric and HHV8-Related Clinical Forms

Castleman disease (CD) is a rare lymphoproliferative disorder that includes various clinico-pathological subtypes. According to clinical course, CD is divided into unicentric CD (UCD) and multicentric CD (MCD). MCD is further distinguished based on the etiological driver in herpes virus-8-related MCD (that can occur in the setting of HIV); in MCD associated with POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, and skin changes); and idiopathic MCD (iMCD). The latter can also be divided in iMCD-TAFRO (thrombocytopenia, anasarca, fever, myelofibrosis, organomegaly) and iMCD not otherwise specified. To date, CD pathogenesis is still uncertain, but CD may represent the histological and clinical result of heterogeneous pathomechanisms. Transcriptome investigations in CD lymph nodes have documented the expression and up-regulation of different cytokines; furthermore, few recent studies have shown alterations of different T-cell subsets in CD patients, suggesting a possible role of the nodal microenvironment in CD development. On this basis, our study aimed to investigate the distribution of T-cell subsets in the clinico-pathological spectrum of CD. We evaluated the CD4/CD8 ratio and the number of T-regulatory (T-reg) FOXP3+ cells in 28 CD cases. In total, 32% of cases showed a decreased CD4/CD8 ratio due to increased CD8+ T-cells, including both UCD, iMCD, and HHV8+ MCD cases. The T-reg subset analysis revealed a statistically significant (p < 0.0001) lower mean number of FOXP3+ T-reg cells in CD cases when compared with non-specific reactive lymph nodes. We did not find statistically significant differences in T-reg numbers between the different CD subtypes. These findings may suggest that alterations in T-cell subpopulations that can lead to disruption of immune system control may contribute to the numerous changes in different cellular compartments that characterize CD

Intragenic PU-boxes control HIV-1 transcription and replication in myeloid infected cells

info:eu-repo/semantics/publishe

Role of the pol gene enhancer in HIV-1 transcription and replication in myeloid infected cells

info:eu-repo/semantics/publishe

Role of the pol gene enhancer in HIV-1 transcription and replication in myeloid infected cells

info:eu-repo/semantics/nonPublishe

A highly conserved toxo1 haplotype directs resistance to toxoplasmosis and its associated caspase-1 dependent killing of parasite and host macrophage.

International audienceNatural immunity or resistance to pathogens most often relies on the genetic make-up of the host. In a LEW rat model of refractoriness to toxoplasmosis, we previously identified on chromosome 10 the Toxo1 locus that directs toxoplasmosis outcome and controls parasite spreading by a macrophage-dependent mechanism. Now, we narrowed down Toxo1 to a 891 kb interval containing 29 genes syntenic to human 17p13 region. Strikingly, Toxo1 is included in a haplotype block strictly conserved among all refractory rat strains. The sequencing of Toxo1 in nine rat strains (5 refractory and 4 susceptible) revealed resistant-restricted conserved polymorphisms displaying a distribution gradient that peaks at the bottom border of Toxo1, and highlighting the NOD-like receptor, Nlrp1a, as a major candidate. The Nlrp1 inflammasome is known to trigger, upon pathogen intracellular sensing, pyroptosis programmed-cell death involving caspase-1 activation and cleavage of IL-1β. Functional studies demonstrated that the Toxo1-dependent refractoriness in vivo correlated with both the ability of macrophages to restrict T. gondii growth and a T. gondii-induced death of intracellular parasites and its host macrophages. The parasite-induced cell death of infected macrophages bearing the LEW-Toxo1 alleles was found to exhibit pyroptosis-like features with ROS production, the activation of caspase-1 and IL1-β secretion. The pharmacological inactivation of caspase-1 using YVAD and Z-VAD inhibitors prevented the death of both intravacuolar parasites and host non-permissive macrophages but failed to restore parasite proliferation. These findings demonstrated that the Toxo1-dependent response of rat macrophages to T. gondii infection may trigger two pathways leading to the control of parasite proliferation and the death of parasites and host macrophages. The NOD-like receptor NLRP1a/Caspase-1 pathway is the best candidate to mediate the parasite-induced cell death. These data represent new insights towards the identification of a major pathway of innate resistance to toxoplasmosis and the prediction of individual resistance