The Autophagy Nucleation Factor ATG9 Forms Nanoclusters with the HIV-1 Receptor DC-SIGN and Regulates Early Antiviral Autophagy in Human Dendritic Cells.

Dendritic cells (DC) are critical cellular mediators of host immunity, notably by expressing a broad panel of pattern recognition receptors. One of those receptors, the C-type lectin receptor DC-SIGN, was previously reported as a regulator of endo/lysosomal targeting through functional connections with the autophagy pathway. Here, we confirmed that DC-SIGN internalization intersects with LC3+ autophagy structures in primary human monocyte-derived dendritic cells (MoDC). DC-SIGN engagement promoted autophagy flux which coincided with the recruitment of ATG-related factors. As such, the autophagy initiation factor ATG9 was found to be associated with DC-SIGN very early upon receptor engagement and required for an optimal DC-SIGN-mediated autophagy flux. The autophagy flux activation upon DC-SIGN engagement was recapitulated using engineered DC-SIGN-expressing epithelial cells in which ATG9 association with the receptor was also confirmed. Finally, Stimulated emission depletion (STED) microscopy performed in primary human MoDC revealed DC-SIGN-dependent submembrane nanoclusters formed with ATG9, which was required to degrade incoming viruses and further limit DC-mediated transmission of HIV-1 infection to CD4+ T lymphocytes. Our study unveils a physical association between the Pattern Recognition Receptor DC-SIGN and essential components of the autophagy pathway contributing to early endocytic events and the host's antiviral immune response.

A frequent SNP in TRIM5α strongly enhances the innate immune response against LINE-1 elements.

The intracellular restriction factor TRIM5α inhibits endogenous LINE-1 retroelements. It induces innate immune signaling cascades upon sensing of cytoplasmic LINE-1 complexes, thereby underlining its importance for protecting the human genome from harmful retrotransposition events. Here, we show that a frequent SNP within the RING domain of TRIM5α, resulting in the variant H43Y, blocks LINE-1 retrotransposition with higher efficiency compared to TRIM5α WT. Upon sensing of LINE-1 complexes in the cytoplasm, TRIM5α H43Y activates both NF-κB and AP-1 signaling pathways more potently than TRIM5α WT, triggering a strong block of the LINE-1 promoter. Interestingly, the H43Y allele lost its antiviral function suggesting that its enhanced activity against endogenous LINE-1 elements is the driving force behind its maintenance within the population. Thus, our study suggests that the H43Y variant of the restriction factor and sensor TRIM5α persists within the human population since it preserves our genome from uncontrolled LINE-1 retrotransposition with higher efficiency.

Nsp16 shields SARS-CoV-2 from efficient MDA5 sensing and IFIT1-mediated restriction.

Methylation of the mRNA 5' cap by cellular methyltransferases enables efficient translation and avoids recognition by innate immune factors. Coronaviruses encode viral 2'-O-methyltransferases to shield their RNA from host factors. Here, we generate recombinant SARS-CoV-2 harboring a catalytically inactive 2'-O-methyltransferase Nsp16, Nsp16mut, and analyze viral replication in human lung epithelial cells. Although replication is only slightly attenuated, we find SARS-CoV-2 Nsp16mut to be highly immunogenic, resulting in a strongly enhanced release of type I interferon upon infection. The elevated immunogenicity of Nsp16mut is absent in cells lacking the RNA sensor MDA5. In addition, we report that Nsp16mut is highly sensitive to type I IFN treatment and demonstrate that this strong antiviral effect of type I IFN is mediated by the restriction factor IFIT1. Together, we describe a dual role for the 2'-O-methyltransferase Nsp16 during SARS-CoV-2 replication in avoiding efficient recognition by MDA5 and in shielding its RNA from interferon-induced antiviral responses, thereby identifying Nsp16 as a promising target for generating attenuated and highly immunogenic SARS-CoV-2 strains and as a potential candidate for therapeutic intervention.

Identifying enhancers of innate immune signaling as broad-spectrum antivirals active against emerging viruses.

The increasingly frequent outbreaks of pathogenic viruses have underlined the urgent need to improve our arsenal of antivirals that can be deployed for future pandemics. Innate immunity is a powerful first line of defense against pathogens, and compounds that boost the innate response have high potential to act as broad-spectrum antivirals. Here, we harnessed localization-dependent protein-complementation assays (called Alpha Centauri) to measure the nuclear translocation of interferon regulatory factors (IRFs), thus providing a readout of innate immune activation following viral infection that is applicable to high-throughput screening of immunomodulatory molecules. As proof of concept, we screened a library of kinase inhibitors on severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and identified Gilteritinib as a powerful enhancer of innate responses to viral infection. This immunostimulatory activity of Gilteritinib was found to be dependent on the AXL-IRF7 axis and results in a broad and potent antiviral activity against unrelated RNA viruses.

Alarmin S100A9 restricts retroviral infection by limiting reverse transcription in human dendritic cells.

Dendritic cells (DC) subsets, like Langerhans cells (LC), are immune cells involved in pathogen sensing. They express specific antimicrobial cellular factors that are able to restrict infection and limit further pathogen transmission. Here, we identify the alarmin S100A9 as a novel intracellular antiretroviral factor expressed in human monocyte-derived and skin-derived LC. The intracellular expression of S100A9 is decreased upon LC maturation and inversely correlates with enhanced susceptibility to HIV-1 infection of LC. Furthermore, silencing of S100A9 in primary human LC relieves HIV-1 restriction while ectopic expression of S100A9 in various cell lines promotes intrinsic resistance to both HIV-1 and MLV infection by acting on reverse transcription. Mechanistically, the intracellular expression of S100A9 alters viral capsid uncoating and reverse transcription. S100A9 also shows potent inhibitory effect against HIV-1 and MMLV reverse transcriptase (RTase) activity in vitro in a divalent cation-dependent manner. Our findings uncover an unexpected intracellular function of the human alarmin S100A9 in regulating antiretroviral immunity in Langerhans cells.

Recognize Yourself-Innate Sensing of Non-LTR Retrotransposons.

Although mobile genetic elements, or transposons, have played an important role in genome evolution, excess activity of mobile elements can have detrimental consequences. Already, the enhanced expression of transposons-derived nucleic acids can trigger autoimmune reactions that may result in severe autoinflammatory disorders. Thus, cells contain several layers of protective measures to restrict transposons and to sense the enhanced activity of these "intragenomic pathogens". This review focuses on our current understanding of immunogenic patterns derived from the most active elements in humans, the retrotransposons long interspersed element (LINE)-1 and Alu. We describe the role of known pattern recognition receptors in nucleic acid sensing of LINE-1 and Alu and the possible consequences for autoimmune diseases.

Human TRIM5α senses and restricts LINE-1 elements.

Mobile genetic elements have significantly shaped our genomic landscape. LINE-1 retroelements are the only autonomously active elements left in the human genome. Since new insertions can have detrimental consequences, cells need to efficiently control LINE-1 retrotransposition. Here, we demonstrate that the intrinsic immune factor TRIM5α senses and restricts LINE-1 retroelements. Previously, rhesus TRIM5α has been shown to efficiently block HIV-1 replication, while human TRIM5α was found to be less active. Surprisingly, we found that both human and rhesus TRIM5α efficiently repress human LINE-1 retrotransposition. TRIM5α interacts with LINE-1 ribonucleoprotein complexes in the cytoplasm, which is essential for restriction. In line with its postulated role as pattern recognition receptor, we show that TRIM5α also induces innate immune signaling upon interaction with LINE-1 ribonucleoprotein complexes. The signaling events activate the transcription factors AP-1 and NF-κB, leading to the down-regulation of LINE-1 promoter activity. Together, our findings identify LINE-1 as important target of human TRIM5α, which restricts and senses LINE-1 via two distinct mechanisms. Our results corroborate TRIM5α as pattern recognition receptor and shed light on its previously undescribed activity against mobile genetic elements, such as LINE-1, to protect the integrity of our genome.