HEXIM1 and NEAT1 Long Non-coding RNA Form a Multi-subunit Complex that Regulates DNA-Mediated Innate Immune Response.

The DNA-mediated innate immune response underpins anti-microbial defenses and certain autoimmune diseases. Here we used immunoprecipitation, mass spectrometry, and RNA sequencing to identify a ribonuclear complex built around HEXIM1 and the long non-coding RNA NEAT1 that we dubbed the HEXIM1-DNA-PK-paraspeckle components-ribonucleoprotein complex (HDP-RNP). The HDP-RNP contains DNA-PK subunits (DNAPKc, Ku70, and Ku80) and paraspeckle proteins (SFPQ, NONO, PSPC1, RBM14, and MATRIN3). We show that binding of HEXIM1 to NEAT1 is required for its assembly. We further demonstrate that the HDP-RNP is required for the innate immune response to foreign DNA, through the cGAS-STING-IRF3 pathway. The HDP-RNP interacts with cGAS and its partner PQBP1, and their interaction is remodeled by foreign DNA. Remodeling leads to the release of paraspeckle proteins, recruitment of STING, and activation of DNAPKc and IRF3. Our study establishes the HDP-RNP as a key nuclear regulator of DNA-mediated activation of innate immune response through the cGAS-STING pathway.

Zika virus induces massive cytoplasmic vacuolization and paraptosis-like death in infected cells.

The cytopathic effects of Zika virus (ZIKV) are poorly characterized. Innate immunity controls ZIKV infection and disease in most infected patients through mechanisms that remain to be understood. Here, we studied the morphological cellular changes induced by ZIKV and addressed the role of interferon-induced transmembrane proteins (IFITM), a family of broad-spectrum antiviral factors, during viral replication. We report that ZIKV induces massive vacuolization followed by "implosive" cell death in human epithelial cells, primary skin fibroblasts and astrocytes, a phenomenon which is exacerbated when IFITM3 levels are low. It is reminiscent of paraptosis, a caspase-independent, non-apoptotic form of cell death associated with the formation of large cytoplasmic vacuoles. We further show that ZIKV-induced vacuoles are derived from the endoplasmic reticulum (ER) and dependent on the PI3K/Akt signaling axis. Inhibiting the Sec61 ER translocon in ZIKV-infected cells blocked vacuole formation and viral production. Our results provide mechanistic insight behind the ZIKV-induced cytopathic effect and indicate that IFITM3, by acting as a gatekeeper for incoming virus, restricts virus takeover of the ER and subsequent cell death.

Atlastin Endoplasmic Reticulum-Shaping Proteins Facilitate Zika Virus Replication.

The endoplasmic reticulum (ER) is the site for Zika virus (ZIKV) replication and is central to the cytopathic effects observed in infected cells. ZIKV induces the formation of ER-derived large cytoplasmic vacuoles followed by "implosive" cell death. Little is known about the nature of the ER factors that regulate flavivirus replication. Atlastins (ATL1, -2, and -3) are dynamin-related GTPases that control the structure and the dynamics of the ER membrane. We show here that ZIKV replication is significantly decreased in the absence of ATL proteins. The appearance of infected cells is delayed, the levels of intracellular viral proteins and released virus are reduced, and the cytopathic effects are strongly impaired. We further show that ATL3 is recruited to viral replication sites and interacts with the nonstructural viral proteins NS2A and NS2B3. Thus, proteins that shape and maintain the ER tubular network ensure efficient ZIKV replication.IMPORTANCE Zika virus (ZIKV) is an emerging virus associated with Guillain-Barré syndrome, and fetal microcephaly as well as other neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We found that endoplasmic reticulum (ER)-shaping atlastin proteins (ATL1, -2, and -3), which induce ER membrane fusion, facilitate ZIKV replication. We show that ATL3 is recruited to the viral replication site and colocalize with the viral proteins NS2A and NS2B3. The results provide insights into host factors used by ZIKV to enhance its replication.

SUN2 Overexpression Deforms Nuclear Shape and Inhibits HIV.

UNLABELLED: In a previous screen of putative interferon-stimulated genes, SUN2 was shown to inhibit HIV-1 infection in an uncharacterized manner. SUN2 is an inner nuclear membrane protein belonging to the linker of nucleoskeleton and cytoskeleton complex. We have analyzed here the role of SUN2 in HIV infection. We report that in contrast to what was initially thought, SUN2 is not induced by type I interferon, and that SUN2 silencing does not modulate HIV infection. However, SUN2 overexpression in cell lines and in primary monocyte-derived dendritic cells inhibits the replication of HIV but not murine leukemia virus or chikungunya virus. We identified HIV-1 and HIV-2 strains that are unaffected by SUN2, suggesting that the effect is specific to particular viral components or cofactors. Intriguingly, SUN2 overexpression induces a multilobular flower-like nuclear shape that does not impact cell viability and is similar to that of cells isolated from patients with HTLV-I-associated adult T-cell leukemia or with progeria. Nuclear shape changes and HIV inhibition both mapped to the nucleoplasmic domain of SUN2 that interacts with the nuclear lamina. This block to HIV replication occurs between reverse transcription and nuclear entry, and passaging experiments selected for a single-amino-acid change in capsid (CA) that leads to resistance to overexpressed SUN2. Furthermore, using chemical inhibition or silencing of cyclophilin A (CypA), as well as CA mutant viruses, we implicated CypA in the SUN2-imposed block to HIV infection. Our results demonstrate that SUN2 overexpression perturbs both nuclear shape and early events of HIV infection. IMPORTANCE: Cells encode proteins that interfere with viral replication, a number of which have been identified in overexpression screens. SUN2 is a nuclear membrane protein that was shown to inhibit HIV infection in such a screen, but how it blocked HIV infection was not known. We show that SUN2 overexpression blocks the infection of certain strains of HIV before nuclear entry. Mutation of the viral capsid protein yielded SUN2-resistant HIV. Additionally, the inhibition of HIV infection by SUN2 involves cyclophilin A, a protein that binds the HIV capsid and directs subsequent steps of infection. We also found that SUN2 overexpression substantially changes the shape of the cell's nucleus, resulting in many flower-like nuclei. Both HIV inhibition and deformation of nuclear shape required the domain of SUN2 that interacts with the nuclear lamina. Our results demonstrate that SUN2 interferes with HIV infection and highlight novel links between nuclear shape and viral infection.

TLR3-responsive, XCR1+, CD141(BDCA-3)+/CD8α+-equivalent dendritic cells uncovered in healthy and simian immunodeficiency virus-infected rhesus macaques.

In mice, CD8α(+) myeloid dendritic cells (mDC) optimally cross-present Ags to CD8(+) T cells and respond strongly to TLR3 ligands. Although equivalent DC have been identified by comparative genomic analysis and functional studies in humans as XCR1(+)CD141 (BDCA-3)(+)Clec9A(+)cell adhesion molecule 1(+) mDC, and in sheep as CD26(+) mDC, these cells remained elusive in nonhuman primates. To remedy this situation, we delineated precisely DC and monocyte populations by 12-color flow cytometry and transcriptomic analyses in healthy rhesus macaques. We identified a new mDC population, with strong phenotypic and transcriptional homology to human CD141(+) and murine CD8α(+) mDC, including XCR1 membrane expression as a conserved specific marker. In contrast, high CD11c expression was not characteristic of mDC in macaques, but of CD16(+) monocytes. Like their human and murine homologs, simian XCR1(+) mDC had much stronger responses to TLR3 stimulation than other myeloid cells. The importance of this new mDC population was tested in SIV(mac251) infection, the most relevant animal model for pathogenic HIV-1 infection and vaccination. This population increased sharply and transiently during acute infection, but was reduced in blood and spleen during advanced disease. The identification of XCR1(+) mDC in rhesus macaques opens new avenues for future preclinical vaccinal studies and highlights XCR1 as a prime candidate for targeted vaccine delivery.

Pivotal role of M-DC8⁺ monocytes from viremic HIV-infected patients in TNFα overproduction in response to microbial products.

HIV infects activated CD4⁺ T cells and induces their depletion. Progressive HIV infection leading to AIDS is fueled by chronic immune hyperactivation, mediated by inflammatory cytokines like TNFα. This has been related to intestinal epithelial damage and microbial LPS translocation into the circulation. Using 11-color flow cytometry, cell sorting, and cell culture, we investigated the numbers and TNFα production of fully defined circulating dendritic cell and monocyte populations during HIV-1 infection. In 15 viremic, untreated patients, compared with 8 treated, virologically suppressed patients or to 13 healthy blood donors, circulating CD141 (BDCA-3)⁺ and CD1c (BDCA-1)⁺ dendritic cell counts were reduced. Conversely, CD14⁺ CD16⁺⁺ monocyte counts were increased, particularly those expressing M-DC8, while classical CD14⁺⁺ CD16⁻ M-DC8⁻ monocyte numbers were unchanged. Blood mononuclear cells from viremic patients produced more TNFα in response to LPS than those from virologically suppressed patients. M-DC8⁺ monocytes were mostly responsible for this overproduction. Moreover, M-DC8⁺ monocytes differentiated in vitro from classical monocytes using M-CSF and GM-CSF, which is increased in viremic patient's plasma. This M-DC8⁺ monocyte population, which is involved in the pathogenesis of chronic inflammatory diseases like Crohn disease, might thus be considered as a major actor in the immune hyperactivation fueling HIV infection progression.

Conventional Dendritic Cells and Slan+ Monocytes During HIV-2 Infection.

HIV-2 infection is characterized by low viremia and slow disease progression as compared to HIV-1 infection. Circulating CD14++CD16+ monocytes were found to accumulate and CD11c+ conventional dendritic cells (cDC) to be depleted in a Portuguese cohort of people living with HIV-2 (PLWHIV-2), compared to blood bank healthy donors (HD). We studied more precisely classical monocytes; CD16+ inflammatory (intermediate, non-classical and slan+ monocytes, known to accumulate during viremic HIV-1 infection); cDC1, important for cross-presentation, and cDC2, both depleted during HIV-1 infection. We analyzed by flow cytometry these PBMC subsets from Paris area residents: 29 asymptomatic, untreated PLWHIV-2 from the IMMUNOVIR-2 study, part of the ANRS-CO5 HIV-2 cohort: 19 long-term non-progressors (LTNP; infection ≥8 years, undetectable viral load, stable CD4 counts≥500/µL; 17 of West-African origin -WA), and 10 non-LTNP (P; progressive infection; 9 WA); and 30 age-and sex-matched controls: 16 blood bank HD with unknown geographical origin, and 10 HD of WA origin (GeoHD). We measured plasma bacterial translocation markers by ELISA. Non-classical monocyte counts were higher in GeoHD than in HD (54 vs. 32 cells/µL, p = 0.0002). Slan+ monocyte counts were twice as high in GeoHD than in HD (WA: 28 vs. 13 cells/µL, p = 0.0002). Thus cell counts were compared only between participants of WA origin. They were similar in LTNP, P and GeoHD, indicating that there were no HIV-2 related differences. cDC counts did not show major differences between the groups. Interestingly, inflammatory monocyte counts correlated with plasma sCD14 and LBP only in PLWHIV-2, especially LTNP, and not in GeoHD. In conclusion, in LTNP PLWHIV-2, inflammatory monocyte counts correlated with LBP or sCD14 plasma levels, indicating a potential innate immune response to subclinical bacterial translocation. As GeoHD had higher inflammatory monocyte counts than HD, our data also show that specific controls are important to refine innate immunity studies.

Elimination of HIV-1-infected cells by broadly neutralizing antibodies.

The Fc region of HIV-1 Env-specific broadly neutralizing antibodies (bNAbs) is required for suppressing viraemia, through mechanisms which remain poorly understood. Here, we identify bNAbs that exert antibody-dependent cellular cytotoxicity (ADCC) in cell culture and kill HIV-1-infected lymphocytes through natural killer (NK) engagement. These antibodies target the CD4-binding site, the glycans/V3 and V1/V2 loops on gp120, or the gp41 moiety. The landscape of Env epitope exposure at the surface and the sensitivity of infected cells to ADCC vary considerably between viral strains. Efficient ADCC requires sustained cell surface binding of bNAbs to Env, and combining bNAbs allows a potent killing activity. Furthermore, reactivated infected cells from HIV-positive individuals expose heterogeneous Env epitope patterns, with levels that are often but not always sufficient to trigger killing by bNAbs. Our study delineates the parameters controlling ADCC activity of bNAbs, and supports the use of the most potent antibodies to clear the viral reservoir.

Monocyte-derived dendritic cells from HLA-B27+ axial spondyloarthritis (SpA) patients display altered functional capacity and deregulated gene expression.

INTRODUCTION: This study aimed to compare the functional capacity and gene expression profile of monocyte-derived dendritic cells (MD-DCs) in HLA-B27+ axial spondyloarthritis (SpA) patients and healthy controls. METHODS: MD-DCs were differentiated with interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) for seven days, starting from purified CD14+ monocytes and stimulated with lipopolysaccharide (LPS) for six and twenty four hours. Their capacity to stimulate allogeneic CD4+ T cells from unrelated healthy donor was tested. Transcriptomic study was performed with Affymetrix HuGene 1.0 ST microarrays. Gene expression levels were compared between patients and controls using a multivariate design under a linear model (LIMMA). Real-time quantitative PCR (qRT-PCR) was performed for validation of the most striking gene expression differences. RESULTS: The stimulatory capacity of allogeneic CD4+ T cells by MD-DCs from SpA patients was decreased. Transcriptomic analysis revealed 81 genes differentially expressed in MD-DCs between SpA patients and controls (P <0.01 and fold-change <0.66 or >1.5). Four selected genes were validated by q RT-PCR: ADAMTS15, CITED2, F13A1 and SELL. Expression levels of ADAMTS15 and CITED2, encoding a metallopeptidase and a transcription factor, respectively, were inversely correlated with each other (R = 0.75, P = 0.0003). Furthermore, in silico analysis identified several genes of the Wnt signaling pathway having expression co-regulated with CITED2. CONCLUSION: This study revealed altered function and gene expression pattern in MD-DCs from HLA-B27+ axial SpA. Co-expression study showed an inverse correlation between ADAMTS15 and CITED2. Moreover, the Wnt signaling pathway appeared as deregulated in SpA MD-DCs, a finding which may be connected to Th17-driven inflammatory responses.