IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus.

Antiretroviral therapy (ART) suppresses HIV-1 viremia and prevents progression to AIDS. Nonetheless, chronic inflammation is a common problem for people living with HIV-1 on ART. One possible cause of inflammation is ongoing transcription from HIV-1 proviruses, whether or not the sequences are competent for replication. Previous work has shown that intron-containing RNA expressed from the HIV-1 provirus in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells, activates type 1 interferon. This activation required HIV-1 rev and was blocked by the XPO1 (CRM1)-inhibitor leptomycin. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with shRNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the IFN-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with non-targetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant inhibitory CARD-deletion or phosphomimetic point mutations, indicates that IFIH1 filament formation, dephosphorylation, and association with MAVS, are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1-knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 was revealed by formaldehyde crosslinking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.

AIM2 regulates anti-tumor immunity and is a viable therapeutic target for melanoma.

The STING and absent in melanoma 2 (AIM2) pathways are activated by the presence of cytosolic DNA, and STING agonists enhance immunotherapeutic responses. Here, we show that dendritic cell (DC) expression of AIM2 within human melanoma correlates with poor prognosis and, in contrast to STING, AIM2 exerts an immunosuppressive effect within the melanoma microenvironment. Vaccination with AIM2-deficient DCs improves the efficacy of both adoptive T cell therapy and anti-PD-1 immunotherapy for "cold tumors," which exhibit poor therapeutic responses. This effect did not depend on prolonged survival of vaccinated DCs, but on tumor-derived DNA that activates STING-dependent type I IFN secretion and subsequent production of CXCL10 to recruit CD8+ T cells. Additionally, loss of AIM2-dependent IL-1ß and IL-18 processing enhanced the treatment response further by limiting the recruitment of regulatory T cells. Finally, AIM2 siRNA-treated mouse DCs in vivo and human DCs in vitro enhanced similar anti-tumor immune responses. Thus, targeting AIM2 in tumor-infiltrating DCs is a promising new treatment strategy for melanoma.

USP10 Targeted Self-Deliverable siRNA to Prevent Scarring in the Cornea.

Ocular scarring after surgery, trauma, or infection leads to vision loss. The transparent cornea is an excellent model system to test anti-scarring therapies. Cholesterol-conjugated fully modified asymmetric small interfering RNAs (siRNAs) (self-deliverable siRNAs [sdRNAs]) are a novel modality for in vivo gene knockdown, transfecting cells and tissues without any additional formulations. Myofibroblasts are a main contributor to scarring and fibrosis. αv integrins play a central role in myofibroblast pathological adhesion, overcontraction, and transforming growth factor ß (TGF-ß) activation. Previously, we demonstrated that αv integrins are protected from intracellular degradation after wounding by upregulation of the deubiquitinase (DUB) ubiquitin-specific protease 10 (USP10), leading to integrin cell surface accumulation. In this study, we tested whether knockdown of USP10 with a USP10-targeting sdRNA (termed US09) will reduce scarring after wounding a rabbit cornea in vivo. The wounded corneal stroma was treated once with US09 or non-targeting control (NTC) sdRNA. At 6 weeks US09 treatment resulted in faster wound closure, limited scarring, and suppression of fibrotic markers and immune response. Specifically, fibronectin-extra domain A (EDA), collagen III, and a-smooth muscle actin (p < 0.05), CD45+ cell infiltration (p < 0.01), and apoptosis at 24 (p < 0.01) and 48 h (p < 0.05) were reduced post-wounding. Corneal thickness and cell proliferation were restored to unwounded parameters. Targeting the DUB, USP10 is a novel strategy to reduce scarring. This study indicates that ubiquitin-mediated pathways should be considered in the pathogenesis of fibrotic healing.

HIV-1-induced cytokines deplete homeostatic innate lymphoid cells and expand TCF7-dependent memory NK cells.

Human immunodeficiency virus 1 (HIV-1) infection is associated with heightened inflammation and excess risk of cardiovascular disease, cancer and other complications. These pathologies persist despite antiretroviral therapy. In two independent cohorts, we found that innate lymphoid cells (ILCs) were depleted in the blood and gut of people with HIV-1, even with effective antiretroviral therapy. ILC depletion was associated with neutrophil infiltration of the gut lamina propria, type 1 interferon activation, increased microbial translocation and natural killer (NK) cell skewing towards an inflammatory state, with chromatin structure and phenotype typical of WNT transcription factor TCF7-dependent memory T cells. Cytokines that are elevated during acute HIV-1 infection reproduced the ILC and NK cell abnormalities ex vivo. These results show that inflammatory cytokines associated with HIV-1 infection irreversibly disrupt ILCs. This results in loss of gut epithelial integrity, microbial translocation and memory NK cells with heightened inflammatory potential, and explains the chronic inflammation in people with HIV-1.

Cyclophilin A protects HIV-1 from restriction by human TRIM5α.

The HIV-1 capsid (CA) protein lattice encases viral genomic RNA and regulates steps essential to target-cell invasion1. Cyclophilin A (CypA) has interacted with the CA of lentiviruses related to HIV-1 for millions of years2-7. Disruption of the CA-CypA interaction decreases HIV-1 infectivity in human cells8-12 but stimulates infectivity in non-human primate cells13-15. Genetic and biochemical data suggest that CypA protects HIV-1 from a CA-specific restriction factor in human cells16-20. Discovery of the CA-specific restriction factor tripartite-containing motif 5α (TRIM5α)21 and multiple, independently derived, TRIM5-CypA fusion genes4,5,15,22-26 pointed to human TRIM5α being the CypA-sensitive restriction factor. However, HIV-1 restriction by human TRIM5α in tumour cell lines is minimal21 and inhibition of such activity by CypA has not been detected27. Here, by exploiting reverse genetic tools optimized for primary human blood cells, we demonstrate that disruption of the CA-CypA interaction renders HIV-1 susceptible to potent restriction by human TRIM5α, with the block occurring before reverse transcription. Endogenous TRIM5α associated with virion cores as they entered the cytoplasm, but only when the CA-CypA interaction was disrupted. These experiments resolve the long-standing mystery of the role of CypA in HIV-1 replication by demonstrating that this ubiquitous cellular protein shields HIV-1 from previously inapparent restriction by human TRIM5α.

Intron-containing RNA from the HIV-1 provirus activates type I interferon and inflammatory cytokines.

HIV-1-infected people who take drugs that suppress viremia to undetectable levels are protected from developing AIDS. Nonetheless, HIV-1 establishes proviruses in long-lived CD4+ memory T cells, and perhaps other cell types, that preclude elimination of the virus even after years of continuous antiviral therapy. Here we show that the HIV-1 provirus activates innate immune signaling in isolated dendritic cells, macrophages, and CD4+ T cells. Immune activation requires transcription from the HIV-1 provirus and expression of CRM1-dependent, Rev-dependent, RRE-containing, unspliced HIV-1 RNA. If rev is provided in trans, all HIV-1 coding sequences are dispensable for activation except those cis-acting sequences required for replication or splicing. Our results indicate that the complex, post-transcriptional regulation intrinsic to HIV-1 RNA is detected by the innate immune system as a danger signal, and that drugs which disrupt HIV-1 transcription or HIV-1 RNA metabolism would add qualitative benefit to current antiviral drug regimens.

Primate immunodeficiency virus proteins Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex.

Host factors that silence provirus transcription in CD4+ memory T cells help HIV-1 escape eradication by the host immune system and by antiviral drugs1. These same factors, however, must be overcome for HIV-1 to propagate. Here we show that Vpx and Vpr encoded by diverse primate immunodeficiency viruses activate provirus transcription. Vpx and Vpr are adaptor proteins for the DCAF1-CUL4A/B E3 ubiquitin ligase that degrade SAMHD1 and increase reverse transcription2-4. Nonetheless, Vpx and Vpr have effects on reporter gene expression that are not explained by SAMHD1 degradation5-8. A screen for factors that mimic these effects identified the human silencing hub (HUSH) complex, FAM208A (TASOR/RAP140), MPHOSPH8 (MPP8), PPHLN1 (PERIPHILIN) and MORC29-13. Vpx associated with the HUSH complex and decreased steady-state level of these proteins in a DCAF1/CUL4A/B/proteasome-dependent manner14,15. Replication kinetics of HIV-1 and SIVMAC was accelerated to a similar extent by vpx or FAM208A knockdown. Finally, vpx increased steady-state levels of LINE-1 ORF1p, as previously described for FAM208A disruption11. These results demonstrate that the HUSH complex represses primate immunodeficiency virus transcription, and that, to counteract this restriction, viral Vpx or Vpr proteins degrade the HUSH complex.

Comparative Analysis of Immune Cells Reveals a Conserved Regulatory Lexicon.

Most well-characterized enhancers are deeply conserved. In contrast, genome-wide comparative studies of steady-state systems showed that only a small fraction of active enhancers are conserved. To better understand conservation of enhancer activity, we used a comparative genomics approach that integrates temporal expression and epigenetic profiles in an innate immune system. We found that gene expression programs diverge among mildly induced genes, while being highly conserved for strongly induced genes. The fraction of conserved enhancers varies greatly across gene expression programs, with induced genes and early-response genes, in particular, being regulated by a higher fraction of conserved enhancers. Clustering of conserved accessible DNA sequences within enhancers resulted in over 60 sequence motifs including motifs for known factors, as well as many with unknown function. We further show that the number of instances of these motifs is a strong predictor of the responsiveness of a gene to pathogen detection.

HIV-1 R5 Macrophage-Tropic Envelope Glycoprotein Trimers Bind CD4 with High Affinity, while the CD4 Binding Site on Non-macrophage-tropic, T-Tropic R5 Envelopes Is Occluded.

HIV-1 R5 variants exploit CCR5 as a coreceptor to infect both T cells and macrophages. R5 viruses that are transmitted or derived from immune tissue and peripheral blood are mainly inefficient at mediating infection of macrophages. In contrast, highly macrophage-tropic (mac-tropic) R5 viruses predominate in brain tissue and can be detected in cerebrospinal fluid but are infrequent in immune tissue or blood even in late disease. These mac-tropic R5 variants carry envelope glycoproteins (Envs) adapted to exploit low levels of CD4 on macrophages to induce infection. However, it is unclear whether this adaptation is conferred by an increased affinity of the Env trimer for CD4 or is mediated by postbinding structural rearrangements in the trimer that enhance the exposure of the coreceptor binding site and facilitate events leading to fusion and virus entry. In this study, we investigated CD4 binding to mac-tropic and non-mac-tropic Env trimers and showed that CD4-IgG binds efficiently to mac-tropic R5 Env trimers, while binding to non-mac-tropic trimers was undetectable. Our data indicated that the CD4 binding site (CD4bs) is highly occluded on Env trimers of non-mac-tropic R5 viruses. Such viruses may therefore infect T cells via viral synapses where Env and CD4 become highly concentrated. This environment will enable high-avidity interactions that overcome extremely low Env-CD4 affinities.IMPORTANCE HIV R5 variants bind to CD4 and CCR5 receptors on T cells and macrophages to initiate infection. Transmitted HIV variants infect T cells but not macrophages, and these viral strains persist in immune tissue even in late disease. Here we show that the binding site for CD4 present on HIV's envelope protein is occluded on viruses replicating in immune tissue. This occlusion likely prevents antibody binding to this site and neutralization of the virus, but it makes it difficult for virus-CD4 interactions to occur. Such viruses probably pass from T cell to T cell via cell contacts where CD4 is highly concentrated and allows infection via inefficient envelope-CD4 binding. Our data are highly relevant for vaccines that aim to induce antibodies targeting the CD4 binding site on the envelope protein.

Ebola Virus Glycoprotein with Increased Infectivity Dominated the 2013-2016 Epidemic.

The magnitude of the 2013-2016 Ebola virus disease (EVD) epidemic enabled an unprecedented number of viral mutations to occur over successive human-to-human transmission events, increasing the probability that adaptation to the human host occurred during the outbreak. We investigated one nonsynonymous mutation, Ebola virus (EBOV) glycoprotein (GP) mutant A82V, for its effect on viral infectivity. This mutation, located at the NPC1-binding site on EBOV GP, occurred early in the 2013-2016 outbreak and rose to high frequency. We found that GP-A82V had heightened ability to infect primate cells, including human dendritic cells. The increased infectivity was restricted to cells that have primate-specific NPC1 sequences at the EBOV interface, suggesting that this mutation was indeed an adaptation to the human host. GP-A82V was associated with increased mortality, consistent with the hypothesis that the heightened intrinsic infectivity of GP-A82V contributed to disease severity during the EVD epidemic.

HIV-1 Nef promotes infection by excluding SERINC5 from virion incorporation.

HIV-1 Nef, a protein important for the development of AIDS, has well-characterized effects on host membrane trafficking and receptor downregulation. By an unidentified mechanism, Nef increases the intrinsic infectivity of HIV-1 virions in a host-cell-dependent manner. Here we identify the host transmembrane protein SERINC5, and to a lesser extent SERINC3, as a potent inhibitor of HIV-1 particle infectivity that is counteracted by Nef. SERINC5 localizes to the plasma membrane, where it is efficiently incorporated into budding HIV-1 virions and impairs subsequent virion penetration of susceptible target cells. Nef redirects SERINC5 to a Rab7-positive endosomal compartment and thereby excludes it from HIV-1 particles. The ability to counteract SERINC5 was conserved in Nef encoded by diverse primate immunodeficiency viruses, as well as in the structurally unrelated glycosylated Gag from murine leukaemia virus. These examples of functional conservation and convergent evolution emphasize the fundamental importance of SERINC5 as a potent anti-retroviral factor.

Lv4 Is a Capsid-Specific Antiviral Activity in Human Blood Cells That Restricts Viruses of the SIVMAC/SIVSM/HIV-2 Lineage Prior to Integration.

HIV-2 and SIVMAC are AIDS-causing, zoonotic lentiviruses that jumped to humans and rhesus macaques, respectively, from SIVSM-bearing sooty mangabey monkeys. Cross-species transmission events such as these sometimes necessitate virus adaptation to species-specific, host restriction factors such as TRIM5. Here, a new human restriction activity is described that blocks viruses of the SIVSM/SIVMAC/HIV-2 lineage. Human T, B, and myeloid cell lines, peripheral blood mononuclear cells and dendritic cells were 4 to >100-fold less transducible by VSV G-pseudotyped SIVMAC, HIV-2, or SIVSM than by HIV-1. In contrast, transduction of six epithelial cell lines was equivalent to that by HIV-1. Substitution of HIV-1 CA with the SIVMAC or HIV-2 CA was sufficient to reduce HIV-1 transduction to the level of the respective vectors. Among such CA chimeras there was a general trend such that CAs from epidemic HIV-2 Group A and B isolates were the most infectious on human T cells, CA from a 1° sooty mangabey isolate was the least infectious, and non-epidemic HIV-2 Group D, E, F, and G CAs were in the middle. The CA-specific decrease in infectivity was observed with either HIV-1, HIV-2, ecotropic MLV, or ALV Env pseudotypes, indicating that it was independent of the virus entry pathway. As2O3, a drug that suppresses TRIM5-mediated restriction, increased human blood cell transduction by SIVMAC but not by HIV-1. Nonetheless, elimination of TRIM5 restriction activity did not rescue SIVMAC transduction. Also, in contrast to TRIM5-mediated restriction, the SIVMAC CA-specific block occurred after completion of reverse transcription and the formation of 2-LTR circles, but before establishment of the provirus. Transduction efficiency in heterokaryons generated by fusing epithelial cells with T cells resembled that in the T cells, indicative of a dominant-acting SIVMAC restriction activity in the latter. These results suggest that the nucleus of human blood cells possesses a restriction factor specific for the CA of HIV-2/SIVMAC/SIVSM and that cross-species transmission of SIVSM to human T cells necessitated adaptation of HIV-2 to this putative restriction factor.