Project description:MiRNAs and other small noncoding RNAs (sncRNAs) are key players in post-transcriptional gene regulation. HIV-1 derived small noncoding RNAs (sncRNAs) have been described in HIV-1 infected cells, but their biological functions still remain to be elucidated. Here, we approached the question whether viral sncRNAs may play a role in the RNA interference (RNAi) pathway or whether viral mRNAs are targeted by cellular miRNAs in human monocyte-derived macrophages (MDM). The incorporation of viral sncRNAs and/or their target RNAs into RNA-induced silencing complex was investigated using photoactivatable ribonucleoside-induced cross-linking and immunoprecipitation (PAR-CLIP) as well as high-throughput sequencing of RNA isolated by cross-linking immunoprecipitation (HITS-CLIP), which capture Argonaute2-bound miRNAs and their target RNAs. HIV-1 infected monocyte-derived macrophages (MDM) were chosen as target cells, as they have previously been shown to express HIV-1 sncRNAs. In addition, we applied small RNA deep sequencing to study differential cellular miRNA expression in HIV-1 infected versus non-infected MDMs. PAR-CLIP and HITS-CLIP data demonstrated the absence of HIV-1 RNAs in Ago2-RISC, although the presence of a multitude of HIV-1 sncRNAs in HIV-1 infected MDMs was confirmed by small RNA sequencing. Small RNA sequencing revealed that 1.4% of all sncRNAs were of HIV-1 origin. However, neither HIV-1 derived sncRNAs nor putative HIV-1 target sequences incorporated into Ago2-RISC were identified, suggesting that HIV-1 sncRNAs are not involved in the canonical RNAi pathway nor is HIV-1 targeted by this pathway in HIV-1 infected macrophages.
Project description:Macrophages are a major target for human immunodeficiency virus type 1 (HIV-1) infection. However, macrophages are largely heterogeneous and may exhibit differences in permissiveness to HIV-1 infection. This study highlights the interplay of macrophage heterogeneity in HIV-1 pathogenesis. We show that monocyte-derived macrophages (MDM) could be divided into two distinct subsets: CD14+Siglec-1hiCD4+ (non-adherent MDM), and CD14+Siglec-1LoCD4- (adherent MDM). The CD14+Siglec-1hiCD4+MDM subset represented the smaller proportion in the macrophage pool, and varied among different donors. Fractionation and subsequent exposure of the two MDM subsets to HIV-1 revealed opposite outcomes in terms of HIV-1 capture and infection. Although the CD14+Siglec-1hiCD4+MDM captured significantly more HIV-1, infection was significantly higher in the CD14+Siglec-1LoCD4-MDM subset. Thus, CD14+Siglec-1hiCD4+MDM were less permissive to infection. Depletion of CD14+Siglec-1hiCD4+MDM or a decrease in their percentage, resulted in increased infection of MDM, suggestive of a capacity of these cells to capture and sequester HIV-1 in an environment that hinders its infectivity. Increased expression of innate restriction factors and cytokine genes were observed in the non-adherent CD14+Siglec-1hiCD4+MDM, both before and after HIV-1 infection, compared to the adherent CD14+Siglec-1LoCD4-MDM. The differential expression of gene expression profiles in the two macrophage subsets may provide an explanation for the differences observed in HIV-1 infectivity.
Project description:The heterogeneity and rarity of HIV-1-infected cells hampers effective cure strategies. We used single-cell DOGMA-seq to simultaneously capture transcription factor accessibility, transcriptome, 156 surface proteins, HIV-1 DNA, and HIV-1 RNA from six HIV-1+ individuals during viremia and after suppressive antiretroviral therapy. We identified 252 transcriptionally inactive (HIV-1 DNA+ RNA–) and 270 transcriptionally active (HIV-1 RNA+) HIV-1-infected cells from 82,549 memory CD4+ T cells. We identified increased transcription factor accessibility in HIV-1 DNA+ RNA– cells (RORC) and HIV-1 RNA+ cells (IRF and AP-1), in addition to CNC and MAF in both. Both HIV-1 DNA+ RNA– and HIV-1 RNA+ cells upregulate IKZF3 (Aiolos) that correlates with proliferation of HIV-1-infected cells. We revealed that the heterogeneous HIV-1-infected T cells comprise four distinct immune programs driven by epigenetic regulators – IRF-activation, Eomes-cytotoxic effector, AP-1-migration, and cell death. Our study revealed the single-cell epigenetic, transcriptional, and protein states of transcriptionally inactive and active HIV-1-infected cells.