Premature activation of the SLX4 complex by Vpr promotes G2/M arrest and escape from innate immune sensing.

The HIV auxiliary protein Vpr potently blocks the cell cycle at the G2/M transition. Here, we show that G2/M arrest results from untimely activation of the structure-specific endonuclease (SSE) regulator SLX4 complex (SLX4com) by Vpr, a process that requires VPRBP-DDB1-CUL4 E3-ligase complex. Direct interaction of Vpr with SLX4 induced the recruitment of VPRBP and kinase-active PLK1, enhancing the cleavage of DNA by SLX4-associated MUS81-EME1 endonucleases. G2/M arrest-deficient Vpr alleles failed to interact with SLX4 or to induce recruitment of MUS81 and PLK1. Furthermore, knockdown of SLX4, MUS81, or EME1 inhibited Vpr-induced G2/M arrest. In addition, we show that the SLX4com is involved in suppressing spontaneous and HIV-1-mediated induction of type 1 interferon and establishment of antiviral responses. Thus, our work not only reveals the identity of the cellular factors required for Vpr-mediated G2/M arrest but also identifies the SLX4com as a regulator of innate immunity.

DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Harnessing the cooperation between DNA-PK and cGAS in cancer therapies: The cooperation between DNA-PK and cGAS shapes tumour immunogenicity.

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is central for the initiation of anti-tumoural immune responses. Enormous effort has been made to optimise the design and administration of STING agonists to stimulate tumour immunogenicity. However, in certain contexts the cGAS-STING axis fuels tumourigenesis. Here, we review recent findings on the regulation of cGAS expression and activity. We particularly focus our attention on the DNA-dependent protein kinase (DNA-PK) complex, that recently emerged as an activator of inflammatory responses in tumour cells. We propose that stratification analyses on cGAS and DNA-PK expression/activation status should be carried out to predict treatment efficacy. We herein also provide insights into non-canonical functions borne by cGAS and cGAMP, highlighting how they may influence tumourigenesis. All these parameters should be taken into consideration concertedly to choose strategies aiming to effectively boost tumour immunogenicity.

SAMHD1 restricts the replication of human immunodeficiency virus type 1 by depleting the intracellular pool of deoxynucleoside triphosphates.

SAMHD1 restricts the infection of dendritic and other myeloid cells by human immunodeficiency virus type 1 (HIV-1), but in lentiviruses of the simian immunodeficiency virus of sooty mangabey (SIVsm)-HIV-2 lineage, SAMHD1 is counteracted by the virion-packaged accessory protein Vpx. Here we found that SAMHD1 restricted infection by hydrolyzing intracellular deoxynucleoside triphosphates (dNTPs), lowering their concentrations to below those required for the synthesis of the viral DNA by reverse transcriptase (RT). SAMHD1-mediated restriction was alleviated by the addition of exogenous deoxynucleosides. An HIV-1 with a mutant RT with low affinity for dNTPs was particularly sensitive to SAMHD1-mediated restriction. Vpx prevented the SAMHD1-mediated decrease in dNTP concentration and induced the degradation of human and rhesus macaque SAMHD1 but had no effect on mouse SAMHD1. Nucleotide-pool depletion could be a general mechanism for protecting cells from infectious agents that replicate through a DNA intermediate.

V1b vasopressin receptor trafficking and signaling: Role of arrestins, G proteins and Src kinase.

The signaling pathway of G protein-coupled receptors is strongly linked to their trafficking profile. Little is known about the molecular mechanisms involved in the vasopressin receptor V1b subtype (V1b R) trafficking and its impact on receptor signaling and regulation. For this purpose, we investigated the role of ß-arrestins in receptor desensitization, internalization and recycling and attempted to dissect the V1b R-mediated MAP kinase pathway. Using MEF cells Knocked-out for ß-arrestins 1 and 2, we demonstrated that both ß-arrestins 1 and 2 play a fundamental role in internalization and recycling of V1b R with a rapid and transient V1b R-ß-arrestin interaction in contrast to a slow and long-lasting ß-arrestin recruitment of the V2 vasopressin receptor subtype (V2 R). Using V1b R-V2 R chimeras and V1b R C-terminus truncations, we demonstrated the critical role of the V1b R C-terminus in its interaction with ß-arrestins thereby regulating the receptor internalization and recycling kinetics in a phosphorylation-independent manner. In parallel, V1b R MAP kinase activation was dependent on arrestins and Src-kinase but independent on G proteins. Interestingly, Src interacted with hV1b R at basal state and dissociated when receptor internalization occurred. Altogether, our data describe for the first time the trafficking profile and MAP kinase pathway of V1b R involving both arrestins and Src kinase family.

HIV-1 Tat assembles a multifunctional transcription elongation complex and stably associates with the 7SK snRNP.

HIV-1 transactivator Tat has greatly contributed to our understanding of transcription elongation by RNAPII. We purified HIV-1 Tat-associated factors from HeLa nuclear extract and show that Tat forms two distinct and stable complexes. Tatcom1 consists of the core active P-TEFb, MLL-fusion partners involved in leukemia (AF9, AFF4, AFF1, ENL, and ELL), and PAF1 complex. Importantly, Tatcom1 formation relies on P-TEFb while optimal CDK9 CTD-kinase activity is AF9 dependent. MLL-fusion partners and PAF1 are required for Tat transactivation. Tatcom2 is composed of CDK9, CycT1, and 7SK snRNP lacking HEXIM. Tat remodels 7SK snRNP by interacting directly with 7SK RNA, leading to the formation of a stress-resistant 7SK snRNP particle. Besides the identification of factors required for Tat transactivation and important for P-TEFb function, our data show a coordinated control of RNAPII elongation by different classes of transcription elongation factors associated in a single complex and acting at the same promoter.

SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx.

The primate lentivirus auxiliary protein Vpx counteracts an unknown restriction factor that renders human dendritic and myeloid cells largely refractory to HIV-1 infection. Here we identify SAMHD1 as this restriction factor. SAMHD1 is a protein involved in Aicardi-Goutières syndrome, a genetic encephalopathy with symptoms mimicking congenital viral infection, that has been proposed to act as a negative regulator of the interferon response. We show that Vpx induces proteasomal degradation of SAMHD1. Silencing of SAMHD1 in non-permissive cell lines alleviates HIV-1 restriction and is associated with a significant accumulation of viral DNA in infected cells. Concurrently, overexpression of SAMHD1 in sensitive cells inhibits HIV-1 infection. The putative phosphohydrolase activity of SAMHD1 is probably required for HIV-1 restriction. Vpx-mediated relief of restriction is abolished in SAMHD1-negative cells. Finally, silencing of SAMHD1 markedly increases the susceptibility of monocytic-derived dendritic cells to infection. Our results demonstrate that SAMHD1 is an antiretroviral protein expressed in cells of the myeloid lineage that inhibits an early step of the viral life cycle.

How SAMHD1 changes our view of viral restriction.

Recent studies have uncovered sterile alpha motif and HD domain 1 (SAMHD1) as the restriction factor that blocks HIV-1 replication in myeloid cells. In contrast to previously identified HIV-1 restriction factors, SAMHD1 does not meet a countermeasure developed by HIV-1. However, HIV-2 and certain simian immunodeficiency virus (SIV) strains express the auxiliary protein Vpx that potently blocks SAMHD1. It is therefore perplexing why this function has been lost or not acquired during the course of lentiviral evolution. This article summarizes the similarities and differences between SAMHD1 and other HIV-1 restriction factors, while highlighting the new questions that are emerging about the crosstalk between restriction factors and innate immune responses.

The unexpected role of the STING protein in lipid metabolism.

Detection of cytosolic pathological nucleic acids is a key step for the initiation of innate immune responses. In the past decade, the stimulator of interferon genes (STING) adaptor protein has emerged as a central platform enabling the activation of inflammatory responses in the presence of cytosolic DNAs. This has prompted a plethora of approaches aiming at modulating STING activation in order to boost or inhibit inflammatory responses. However, recent work has revealed that STING is also a direct regulator of metabolic homeostasis. In particular, STING regulates lipid metabolism directly, a function that is conserved throughout evolution. This indicates that STING targeting strategies must take into consideration potential metabolic side effects that may alter disease course, but also suggests that targeting STING may open the route to novel treatments for metabolic disorders. Here we discuss recent work describing the metabolic function of STING and the implications of these findings.

La détection des acides nucléiques pathologiques cytosoliques est une étape clé pour le déclenchement des réponses immunitaires innées. Au cours de la dernière décennie, la protéine adaptatrice STING (stimulator of interferon genes) est apparue comme une plateforme centrale permettant l'activation des réponses inflammatoires en présence d'ADN cytosolique. Cela a donné lieu à une multitude d'approches visant à moduler l'activation de STING afin de stimuler ou d'inhiber les réponses inflammatoires. Cependant, des travaux récents ont révélé que STING est également un régulateur direct de l'homéostasie métabolique. En particulier, STING régule directement le métabolisme des lipides, une fonction qui est conservée au cours de l'évolution. Cela indique que les stratégies de ciblage de STING doivent prendre en compte les effets secondaires métaboliques potentiels qui peuvent modifier l'évolution de la maladie, mais suggère également la possibilité que le ciblage de STING puisse ouvrir la voie à de nouvelles façons de traiter les pathologies présentant une composante métabolique. Nous discutons ici les travaux récents décrivant la fonction métabolique de STING et les implications de ces résultats.

Multiplexed targeted analysis of polyunsaturated fatty acids and oxylipins using liquid chromatography-tandem mass spectrometry.

Polyunsaturated fatty acids (PUFAs) and their oxidized products (oxylipins) are important mediators in intra- and extra-cellular signaling. We describe here the simultaneous quantification of 163 PUFAs and oxylipins using liquid chromatography-mass spectrometry (LC-MS). The protocol details steps for PUFA purification from various biological materials, the conditions for LC-MS analysis, as well as quantitative approaches for data evaluation. We provide an example of PUFA quantification in animal tissue along with the bioinformatic protocol, enabling efficient inter-sample comparison and statistical analysis. For complete details on the use and execution of this protocol, please refer to Vila et al.,1 Costanza et al.,2 Blomme et al.,3 and Blomme et al.4.

Dietary restriction induces a sexually dimorphic type I interferon response in mice with gene-environment interactions.

Intermittent fasting (IF) is an established intervention to treat the growing obesity epidemic. However, the interaction between dietary interventions and sex remains a significant knowledge gap. In this study, we use unbiased proteome analysis to identify diet-sex interactions. We report sexual dimorphism in response to intermittent fasting within lipid and cholesterol metabolism and, unexpectedly, in type I interferon signaling, which was strongly induced in females. We verify that secretion of type I interferon is required for the IF response in females. Gonadectomy differentially alters the every-other-day fasting (EODF) response and demonstrates that sex hormone signaling can either suppress or enhance the interferon response to IF. IF fails to potentiate a stronger innate immune response when IF-treated animals were challenged with a viral mimetic. Lastly, the IF response changes with genotype and environment. These data reveal an interesting interaction between diet, sex, and the innate immune system.

SAMHD1 restricts HIV-1 reverse transcription in quiescent CD4(+) T-cells.

BACKGROUND: Quiescent CD4+ T lymphocytes are highly refractory to HIV-1 infection due to a block at reverse transcription. RESULTS: Examination of SAMHD1 expression in peripheral blood lymphocytes shows that SAMHD1 is expressed in both CD4+ and CD8+ T cells at levels comparable to those found in myeloid cells. Treatment of CD4+ T cells with Virus-Like Particles (VLP) containing Vpx results in the loss of SAMHD1 expression that correlates with an increased permissiveness to HIV-1 infection and accumulation of reverse transcribed viral DNA without promoting transcription from the viral LTR. Importantly, CD4+ T-cells from patients with Aicardi-Goutières Syndrome harboring mutation in the SAMHD1 gene display an increased susceptibility to HIV-1 infection that is not further enhanced by VLP-Vpx-treatment. CONCLUSION: Here, we identified SAMHD1 as the restriction factor preventing efficient viral DNA synthesis in non-cycling resting CD4+ T-cells. These results highlight the crucial role of SAMHD1 in mediating restriction of HIV-1 infection in quiescent CD4+ T-cells and could impact our understanding of HIV-1 mediated CD4+ T-cell depletion and establishment of the viral reservoir, two of the HIV/AIDS hallmarks.

Role of SAMHD1 nuclear localization in restriction of HIV-1 and SIVmac.

BACKGROUND: SAMHD1 is a nuclear protein that blocks lentiviral infection before reverse transcription in macrophages and dendritic cells. The viral accessory protein Vpx overcomes the SAMHD1-mediated lentiviral block by inducing its proteasomal degradation. RESULTS: Here, we identified the nuclear localization signal (NLS) of SAMHD1, and studied its contribution to restriction of HIV-1 and SIVmac. By studying the cellular distribution of different SAMHD1 variants, we mapped the nuclear localization of SAMHD1 to residues 11KRPR14. Mutagenesis of these residues changed the cellular distribution of SAMHD1 from the nucleus to the cytoplasm. SAMHD1 mutants that lost nuclear localization restricted HIV-1 and SIV as potently as the wild type protein. Interestingly, SAMHD1 mutants that localized to the cytoplasm were not degraded by nuclear Vpx alleles. Therefore, nuclear Vpx alleles require nuclear localization of SAMHD1 in order to induce its degradation. In agreement, SIVmac viruses encoding Vpx did not overcome the restriction imposed by the cytoplasmic variants of SAMHD1. CONCLUSIONS: We mapped the NLS of SAMHD1 to residues 11KRPR14 and studied the contribution of SAMHD1 nuclear localization to restriction of HIV-1 and SIV. These experiments demonstrate that cytoplasmic variants of SAMHD1 potently block lentiviral infection and are resistant to Vpx-mediated degradation. The nuclear Vpx alleles studied here are only capable of degrading a nuclearly localized SAMHD1 suggesting that Vpx-mediated degradation of SAMHD1 is initiated in the nucleus.

Protocol to induce and assess cGAS-STING pathway activation in vitro.

The cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway plays a pivotal role in several cellular processes including pathogen recognition and inflammatory responses. We describe a protocol to activate the cGAS-STING pathway in murine cells using nucleic acids transfection. We describe how to prepare the nucleic acid probes and validate activation of the pathway by western blot and gene expression analysis. The protocol can be applied to investigate cGAS-STING signaling in both murine and human cell lines. For complete details on the use and execution of this protocol, please refer to Vila et al. (2022).

Activation of STING in the pancreatic tumor microenvironment: A novel therapeutic opportunity.

Pancreatic ductal adenocarcinoma (PDAC) is a cancer of poor prognosis that presents with a dense desmoplastic stroma that contributes to therapeutic failure. PDAC patients are mostly unresponsive to immunotherapy. However, hopes to elicit response to immunotherapy have emerged with novel strategies targeting the Stimulator of Interferon Genes (STING) protein, which is a major regulator of tumor-associated inflammation. Combination of STING agonists with conventional immunotherapy approaches has proven to potentiate therapeutic benefits in several cancers. However, recent data underscore that the output of STING activation varies depending on the cellular and tissue context. This suggests that tumor heterogeneity, and in particular the heterogeneity of the tumor microenvironment (TME), is a key factor determining whether STING activation would bear benefits for patients. In this review, we discuss the potential benefits of STING activation in PDAC. To this aim, we describe the major components of the PDAC TME, and the expected consequences of STING activation.

Alternative pathways driven by STING: From innate immunity to lipid metabolism.

The Stimulator of Interferon Genes (STING) is a major adaptor protein that is central to the initiation of type I interferon responses and proinflammatory signalling. STING-dependent signalling is triggered by the presence of cytosolic nucleic acids that are generated following pathogen infection or cellular stress. Beyond this central role in controlling immune responses through the production of cytokines and chemokines, recent reports have uncovered inflammation-independent STING functions. Amongst these, a rapidly growing body of evidence demonstrates a key role of STING in controlling metabolic pathways at several levels. Since immunity and metabolic homeostasis are tightly interconnected, these findings deepen our understanding of the involvement of STING in human pathologies. Here, we discuss these findings and reflect on their impact on our current understanding of how nucleic acid immunity controls homeostasis and promotes pathological outcomes.

STING orchestrates the crosstalk between polyunsaturated fatty acid metabolism and inflammatory responses.

Concerted alteration of immune and metabolic homeostasis underlies several inflammation-related pathologies, ranging from metabolic syndrome to infectious diseases. Here, we explored the coordination of nucleic acid-dependent inflammatory responses and metabolic homeostasis. We reveal that the STING (stimulator of interferon genes) protein regulates metabolic homeostasis through inhibition of the fatty acid desaturase 2 (FADS2) rate-limiting enzyme in polyunsaturated fatty acid (PUFA) desaturation. STING ablation and agonist-mediated degradation increased FADS2-associated desaturase activity and led to accumulation of PUFA derivatives that drive thermogenesis. STING agonists directly activated FADS2-dependent desaturation, promoting metabolic alterations. PUFAs in turn inhibited STING, thereby regulating antiviral responses and contributing to resolving STING-associated inflammation. Thus, we have unveiled a negative regulatory feedback loop between STING and FADS2 that fine-tunes inflammatory responses. Our results highlight the role of metabolic alterations in human pathologies associated with aberrant STING activation and STING-targeting therapies.

Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes.

The maintenance of genomic stability in multicellular organisms relies on the DNA damage response (DDR). The DDR encompasses several interconnected pathways that cooperate to ensure the repair of genomic lesions. Besides their repair functions, several DDR proteins have emerged as involved in the onset of inflammatory responses. In particular, several actors of the DDR have been reported to elicit innate immune activation upon detection of cytosolic pathological nucleic acids. Conversely, pattern recognition receptors (PRRs), initially described as dedicated to the detection of cytosolic immune-stimulatory nucleic acids, have been found to regulate DDR. Thus, although initially described as operating in specific subcellular localizations, actors of the DDR and nucleic acid immune sensors may be involved in interconnected pathways, likely influencing the efficiency of one another. Within this mini review, we discuss evidences for the crosstalk between PRRs and actors of the DDR. For this purpose, we mainly focus on cyclic GMP-AMP (cGAMP) synthetase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16), as major PRRs involved in the detection of aberrant nucleic acid species, and components of the DNA-dependent protein kinase (DNA-PK) complex, involved in the repair of double strand breaks that were recently described to qualify as potential PRRs. Finally, we discuss how the crosstalk between DDR and nucleic acid-associated Interferon responses cooperate for the fine-tuning of innate immune activation, and therefore dictate pathological outcomes. Understanding the molecular determinants of such cooperation will be paramount to the design of future therapeutic approaches.

Human immunodeficiency virus type 1 Nef incorporation into virions does not increase infectivity.

The viral protein Nef contributes to the optimal infectivity of human and simian immunodeficiency viruses. The requirement for Nef during viral biogenesis particles suggests that Nef might play a role in this process. Alternatively, because Nef is incorporated into viruses, it might play a role when progeny virions reach target cells. We challenged these hypotheses by manipulating the amounts of Nef incorporated in viruses while keeping its expression level constant in producer cells. This was achieved by forcing the incorporation of Nef into viral particles by fusing a Vpr sequence to the C-terminal end of Nef. A cleavage site for the viral protease was introduced between Nef and Vpr to allow the release of Nef fragments from the fusion protein during virus maturation. We show that the resulting Nef-CS-Vpr fusion partially retains the ability of Nef to downregulate cell surface CD4 and that high amounts of Nef-CS-Vpr are incorporated into viral particles compared with what is seen for wild-type Nef. The fusion protein is processed during virion maturation and releases Nef fragments similar to those found in viruses produced in the presence of wild-type Nef. Unlike viruses produced in the presence of wild-type Nef, viruses produced in the presence of Nef-CS-Vpr do not have an increase in infectivity and are as poorly infectious as viruses produced in the absence of Nef. These findings demonstrate that the presence of Nef in viral particles is not sufficient to increase human immunodeficiency virus type 1 infectivity and suggest that Nef plays a role during the biogenesis of viral particles.

Nef-induced CD4 endocytosis in human immunodeficiency virus type 1 host cells: role of p56lck kinase.

Human immunodeficiency virus type 1 (HIV-1) Nef interferes with the endocytic machinery to modulate the cell surface expression of CD4. However, the basal trafficking of CD4 is governed by different rules in the target cells of HIV-1: whereas CD4 is rapidly internalized from the cell surface in myeloid cells, CD4 is stabilized at the plasma membrane through its interaction with the p56(lck) kinase in lymphoid cells. In this study, we showed that Nef was able to downregulate CD4 in both lymphoid and myeloid cell lines but that an increase in the internalization rate of CD4 could be observed only in lymphoid cells. Expression of p56(lck) in nonlymphoid CD4-expressing cells restores the ability of Nef in order to increase the internalization rate of CD4. Concurrent with this observation, the expression of a p56(lck)-binding-deficient mutant of CD4 in lymphoid cells abrogates the Nef-induced acceleration of CD4 internalization. We also show that the expression of Nef causes a decrease in the association of p56(lck) with cell surface-expressed CD4. Regardless of the presence of p56(lck), the downregulation of CD4 by Nef was followed by CD4 degradation. Our results imply that Nef uses distinct mechanisms to downregulate the cell surface expression levels of CD4 in either lymphoid or myeloid target cells of HIV-1.