Despite effective treatment, HIV can persist in latent reservoirs, which represent a major obstacle toward HIV eradication. Targeting and reactivating latent cells is challenging due to the heterogeneous nature of HIV-infected cells. Here, we used a primary model of HIV latency and single-cell RNA sequencing to characterize transcriptional heterogeneity during HIV latency and reactivation. Our analysis identified transcriptional programs leading to successful reactivation of HIV expression.

Natural 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.

Single-cell analyses allow uncovering cellular heterogeneity, not only per se, but also in response to viral infection. Similarly, single cell transcriptome analyses (scRNA-Seq) can highlight specific signatures, identifying cell subsets with particular phenotypes, which are relevant in the understanding of virus-host interactions.

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, both m6A and m5C methylations 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.