Measuring the subcellular compartmentalization of viral infections by protein complementation assay.

The recent emergence and reemergence of viruses in the human population has highlighted the need to develop broader panels of therapeutic molecules. High-throughput screening assays opening access to untargeted steps of the viral replication cycle will provide powerful leverage to identify innovative antiviral molecules. We report here the development of an innovative protein complementation assay, termed αCentauri, to measure viral translocation between subcellular compartments. As a proof of concept, the Centauri fragment was either tethered to the nuclear pore complex or sequestered in the nucleus, while the complementary α fragment (<16 amino acids) was attached to the integrase proteins of infectious HIV-1. The translocation of viral ribonucleoproteins from the cytoplasm to the nuclear envelope or to the nucleoplasm efficiently reconstituted superfolder green fluorescent protein or NanoLuc αCentauri reporters. These fluorescence- or bioluminescence-based assays offer a robust readout of specific steps of viral infection in a multiwell format that is compatible for high-throughput screening and is validated by a short hairpin RNA-based prototype screen.

[Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy]. / Apport de la microscopie de fluorescence super-résolue : dans l'intimité des virus !

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

Illuminating the nanoscopic world of viruses by fluorescence super-resolution microscopy.

The recent revolution in optical fluorescence microscopy, supported by the optimization of both spatial resolution and acquisition speed, led to the ability to visualize nano-scaled objects. Currently, the use of a new generation of super-resolution fluorescence microscopes coupled to improved fluorescent probes gives the possibility to study the replicative cycle of viruses in living cells, at the single-virus and molecule level. In this review, after a brief chronological description of these new approaches, we highlight several examples of super-resolution microscopies that have allowed to revisit our understanding of several human viruses and of host-pathogen interactions.

[HIV capsid and productive infection: taking steps in the right direction]. / Capside du VIH et infection productive : comment arriver à bon pore ?

The Human Immunodeficiency Virus Type-1, the causative agent of AIDS, reaches its site of replication by trafficking through the cytoplasm towards the nucleus, benefiting from an active nuclear import through the nuclear pore in order to integrate in the genome of the host cell. Although it is generally accepted that the viral genome remains within the viral capsid for some time after cell entry, the mechanisms responsible for controlled uncoating, which is essential for productive infection, remain poorly understood. Numerous studies now show that the integrity of the viral capsid is essential for transport towards the nucleus, for reverse transcription and nuclear import, and to prevent sensing by innate immune receptors. This review aims to report recent developments in our understanding of the early stages of HIV infection, from entry into the cell to integration, highlighting the cellular partners of the HIV-1 capsid that promote or antagonize infection, as well as the different techniques that are developed for fundamental research and the identification of potential therapeutic targets.

PML/TRIM19-Dependent Inhibition of Retroviral Reverse-Transcription by Daxx.

PML (Promyelocytic Leukemia protein), also known as TRIM19, belongs to the family of tripartite motif (TRIM) proteins. PML is mainly expressed in the nucleus, where it forms dynamic structures known as PML nuclear bodies that recruit many other proteins, such as Sp100 and Daxx. While the role of PML/TRIM19 in antiviral defense is well documented, its effect on HIV-1 infection remains unclear. Here we show that infection by HIV-1 and other retroviruses triggers the formation of PML cytoplasmic bodies, as early as 30 minutes post-infection. Quantification of the number and size of PML cytoplasmic bodies revealed that they last approximately 8 h, with a peak at 2 h post-infection. PML re-localization is blocked by reverse-transcription inhibitors and is not observed following infection with unrelated viruses, suggesting it is specifically triggered by retroviral reverse-transcription. Furthermore, we show that PML interferes with an early step of retroviral infection since PML knockdown dramatically increases reverse-transcription efficiency. We demonstrate that PML does not inhibit directly retroviral infection but acts through the stabilization of one of its well-characterized partners, Daxx. In the presence of PML, cytoplasmic Daxx is found in the vicinity of incoming HIV-1 capsids and inhibits reverse-transcription. Interestingly, Daxx not only interferes with exogenous retroviral infections but can also inhibit retrotransposition of endogenous retroviruses, thus identifying Daxx as a broad cellular inhibitor of reverse-transcription. Altogether, these findings unravel a novel antiviral function for PML and PML nuclear body-associated protein Daxx.

Microtubule-associated proteins 1 (MAP1) promote human immunodeficiency virus type I (HIV-1) intracytoplasmic routing to the nucleus.

After cell entry, HIV undergoes rapid transport toward the nucleus using microtubules and microfilaments. Neither the cellular cytoplasmic components nor the viral proteins that interact to mediate transport have yet been identified. Using a yeast two-hybrid screen, we identified four cytoskeletal components as putative interaction partners for HIV-1 p24 capsid protein: MAP1A, MAP1S, CKAP1, and WIRE. Depletion of MAP1A/MAP1S in indicator cell lines and primary human macrophages led to a profound reduction in HIV-1 infectivity as a result of impaired retrograde trafficking, demonstrated by a characteristic accumulation of capsids away from the nuclear membrane, and an overall defect in nuclear import. MAP1A/MAP1S did not impact microtubule network integrity or cell morphology but contributed to microtubule stabilization, which was shown previously to facilitate infection. In addition, we found that MAP1 proteins interact with HIV-1 cores both in vitro and in infected cells and that interaction involves MAP1 light chain LC2. Depletion of MAP1 proteins reduced the association of HIV-1 capsids with both dynamic and stable microtubules, suggesting that MAP1 proteins help tether incoming viral capsids to the microtubular network, thus promoting cytoplasmic trafficking. This work shows for the first time that following entry into target cells, HIV-1 interacts with the cytoskeleton via its p24 capsid protein. Moreover, our results support a role for MAP1 proteins in promoting efficient retrograde trafficking of HIV-1 by stimulating the formation of stable microtubules and mediating the association of HIV-1 cores with microtubules.

TRIM5α is a SUMO substrate.

BACKGROUND: The TRIM5α restriction factor interferes with retroviral infections by inhibiting an early step of viral replication. TRIM5α activity was recently proposed to be regulated by the SUMO machinery and one SUMO consensus conjugation site as well as three putative SUMO interacting motifs (SIMs) were identified within TRIM5α sequence. Whereas mutation of the SIM sequences was found to abolish TRIM5α antiviral activity, mutation of the consensus SUMO conjugation site did not affect its restriction capacity, although this putative site has never been shown to be actually a SUMO substrate. FINDINGS: Here we further demonstrate that TRIM5α relies on the SUMO machinery to promote restriction, since SUMO1 overexpression enhances TRIM5α-mediated retroviral inhibition whereas knockdown of SUMO1 or E2 SUMO conjugating enzyme Ubc9 prevents restriction. Furthermore, we show for the first time that TRIM5α is SUMOylated both in vitro and in cellulo and that Lysine 10 is the main SUMOylation site. Mutation of the consensus SUMO conjugation motif in position 10 abrogated SUMOylation at this position, but did not disrupt TRIM5α antiviral activity. CONCLUSIONS: Altogether, our results confirm that the SUMO machinery is involved in TRIM5α-mediated retroviral restriction, and demonstrate that TRIM5α is a SUMO 1 and SUMO 2 substrate. The inability to abrogate TRIM5α antiviral activity by mutating its main SUMO conjugation motif supports the notion that non-covalent interaction with SUMO or SUMOylated proteins rather than TRIM5α direct SUMOylation is required.

RANBP2 evolution and human disease.

Ran-binding protein 2 (RANBP2)/Nup358 is a nucleoporin and a key component of the nuclear pore complex. Through its multiple functions (e.g., SUMOylation, regulation of nucleocytoplasmic transport) and subcellular localizations (e.g., at the nuclear envelope, kinetochores, annulate lamellae), it is involved in many cellular processes. RANBP2 dysregulation or mutation leads to the development of human pathologies, such as acute necrotizing encephalopathy 1, cancer, neurodegenerative diseases, and it is also involved in viral infections. The chromosomal region containing the RANBP2 gene is highly dynamic, with high structural variation and recombination events that led to the appearance of a gene family called RANBP2 and GCC2 Protein Domains (RGPD), with multiple gene loss/duplication events during ape evolution. Although RGPD homoplasy and maintenance during evolution suggest they might confer an advantage to their hosts, their functions are still unknown and understudied. In this review, we discuss the appearance and importance of RANBP2 in metazoans and its function-related pathologies, caused by an alteration of its expression levels (through promotor activity, post-transcriptional, or post-translational modifications), its localization, or genetic mutations.

Assessing the Impact of Persistent HIV Infection on Innate Lymphoid Cells Using In Vitro Models.

Pathogens that persist in their host induce immune dysfunctions even in the absence of detectable replication. To better understand the phenotypic and functional changes that persistent infections induce in sentinel innate immune cells, we developed human PBMC-based HIV models of persistent infection. Autologous nonactivated PBMCs were cocultured with chronically infected, acutely infected, or uninfected cells and were then analyzed by unsupervised high-dimensional flow cytometry. Using this approach, we identified prevalent patterns of innate immune dysfunctions associated with persistent HIV infections that at least in part mirror immune dysfunctions observed in patients. In one or more models of chronic infection, bystander CD16+ NK cells expressing markers of activation, such as CD94, CD45RO, CD62L, CD69, CD25, and immune checkpoints PD1, Tim3, TIGIT, NKG2A and Lag3, were significantly reduced. Conversely, helper ILC subsets expressing PDL1/PDL2 were significantly enriched in chronic infection compared with either uninfected or acute infection, suggesting that chronic HIV-1 infection was associated with an inhibitory environment for bystander ILC and NK subsets. The cell-based models of persistent infection that we describe here provide versatile tools to explore the molecular mechanisms of these immune dysfunctions and unveil the contribution of innate immunity in sustaining pathogen persistence.

Rapid and inexpensive bedside diagnosis of RAN binding protein 2-associated acute necrotizing encephalopathy.

Acute necrotizing encephalopathy 1 (ANE1) is a very rare disorder associated with a dominant heterozygous mutation in the RANBP2 (RAN binding protein 2) gene. ANE1 is frequently triggered by a febrile infection and characterized by serious and irreversible neurological damage. Although only a few hundred cases have been reported, mutations in RANBP2 are only partially penetrant and can occur de novo, suggesting that their frequency may be higher in some populations. Genetic diagnosis is a lengthy process, potentially delaying definitive diagnosis. We therefore developed a rapid bedside qPCR-based tool for early diagnosis and screening of ANE1 mutations. Primers were designed to specifically assess RANBP2 and not RGPD (RANBP2 and GCC2 protein domains) and discriminate between wild-type or mutant RANBP2. Nasal epithelial cells were obtained from two individuals with known RANBP2 mutations and two healthy control individuals. RANBP2-specific reverse transcription followed by allele-specific primer qPCR amplification confirmed the specific detection of heterozygously expressed mutant RANBP2 in the ANE1 samples. This study demonstrates that allele-specific qPCR can be used as a rapid and inexpensive diagnostic tool for ANE1 using preexisting equipment at local hospitals. It can also be used to screen non-hospitalized family members and at risk-population to better establish the frequency of non-ANE-associated RANBP2 mutations, as well as possible tissue-dependent expression patterns. Systematic review registration: The protocol was registered in the international prospective register of systematic reviews (PROSPERO- CRD42023443257).

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.

Usutu Virus escapes langerin-induced restriction to productively infect human Langerhans cells, unlike West Nile virus.

Usutu virus (USUV) and West Nile virus (WNV) are phylogenetically close emerging arboviruses and constitute a global public health threat. Since USUV and WNV are transmitted by mosquitoes, the first immune cells they encounter are skin-resident dendritic cells, the most peripheral outpost of immune defense. This unique network is composed of Langerhans cells (LCs) and dermal DCs, which reside in the epidermis and the dermis, respectively. Using human skin explants, we show that while both viruses can replicate in keratinocytes, they can also infect resident DCs with distinct tropism: WNV preferentially infects DCs in the dermis, whereas USUV has a greater propensity to infect LCs. Using both purified human epidermal LCs (eLCs) and monocyte derived LCs (MoLCs), we confirm that LCs sustain a faster and more efficient replication of USUV than WNV and that this correlates with a more intense innate immune response to USUV compared with WNV. Next, we show that ectopic expression of the LC-specific C-type lectin receptor (CLR), langerin, in HEK293T cells allows WNV and USUV to bind and enter, but supports the subsequent replication of USUV only. Conversely, blocking or silencing langerin in MoLCs or eLCs made them resistant to USUV infection, thus demonstrating that USUV uses langerin to enter and replicate in LCs. Altogether, our results demonstrate that LCs constitute privileged target cells for USUV in human skin, because langerin favours its entry and replication. Intriguingly, this suggests that USUV efficiently escapes the antiviral functions of langerin, which normally safeguards LCs from most viral infections.

Regulation of Viral Restriction by Post-Translational Modifications.

Intrinsic immunity is orchestrated by a wide range of host cellular proteins called restriction factors. They have the capacity to interfere with viral replication, and most of them are tightly regulated by interferons (IFNs). In addition, their regulation through post-translational modifications (PTMs) constitutes a major mechanism to shape their action positively or negatively. Following viral infection, restriction factor modification can be decisive. Palmitoylation of IFITM3, SUMOylation of MxA, SAMHD1 and TRIM5α or glycosylation of BST2 are some of those PTMs required for their antiviral activity. Nonetheless, for their benefit and by manipulating the PTMs machinery, viruses have evolved sophisticated mechanisms to counteract restriction factors. Indeed, many viral proteins evade restriction activity by inducing their ubiquitination and subsequent degradation. Studies on PTMs and their substrates are essential for the understanding of the antiviral defense mechanisms and provide a global vision of all possible regulations of the immune response at a given time and under specific infection conditions. Our aim was to provide an overview of current knowledge regarding the role of PTMs on restriction factors with an emphasis on their impact on viral replication.

Lack of endogenous TRIM5alpha-mediated restriction in rhesus macaque dendritic cells.

Rhesus macaques are resistant to infection by HIV-1 as a result of an innate cellular restriction mechanism attributable to the expression of rhTRIM5alpha, a member of the large tripartite motif (TRIM) protein family. TRIM5alpha-mediated restriction, which occurs before reverse transcription through targeting of the HIV-1 capsid, has been identified in a number of macaque primary cells and cell lines and is thought to occur in all macaque cell types. We report, however, that rhesus macaque dendritic cells (DCs) lack TRIM5alpha-mediated restriction and are equally permissive to HIV-1 infection as human DCs. Evidence suggests that, although TRIM5alpha RNA levels are normal in these cells, the protein may be dysfunctional. We propose that abrogation of TRIM5alpha-mediated restriction in DCs, although still operative in cells that replicate HIV-1 (macrophages, T lymphocytes), illustrates the need for innate mechanisms to not inhibit adaptive immune responses to ensure an optimal fight against pathogens.

Implications of Nef: host cell interactions in viral persistence and progression to AIDS.

The HIV and SIV Nef accessory proteins are potent enhancers of viral persistence and accelerate progression to AIDS in HIV-1-infected patients and non-human primate models. Although relatively small (27-35 kD), Nef can interact with a multitude of cellular factors and induce complex changes in trafficking, signal transduction, and gene expression that together converge to promote viral replication and immune evasion. In particular, Nef recruits several immunologically relevant cellular receptors to the endocytic machinery to reduce the recognition and elimination of virally infected cells by the host immune system, while simultaneously interacting with various kinases to promote T cell activation and viral replication. This review provides an overview on selected Nef interactions with host cell proteins, and discusses their possible relevance for viral spread and pathogenicity.

Daxx Inhibits HIV-1 Reverse Transcription and Uncoating in a SUMO-Dependent Manner.

Death domain-associated protein 6 (Daxx) is a multifunctional, ubiquitously expressed and highly conserved chaperone protein involved in numerous cellular processes, including apoptosis, transcriptional repression, and carcinogenesis. In 2015, we identified Daxx as an antiretroviral factor that interfered with HIV-1 replication by inhibiting the reverse transcription step. In the present study, we sought to unravel the molecular mechanism of Daxx-mediated restriction and, in particular, to identify the protein(s) that Daxx targets in order to achieve its antiviral activity. First, we show that the SUMO-interacting motif (SIM) located at the C-terminus of the protein is strictly required for Daxx to inhibit HIV-1 reverse transcription. By performing a quantitative proteomic screen combined with classical biochemical analyses, we found that Daxx associated with incoming HIV-1 cores through a SIM-dependent interaction with cyclophilin A (CypA) and capsid (CA). Daxx was found to reside within a multiprotein complex associated with viral capsids, also containing TNPO3, TRIM5α, and TRIM34. Given the well-known influence of these cellular factors on the stability of HIV-1 cores, we investigated the effect of Daxx on the cytoplasmic fate of incoming cores and found that Daxx prevented HIV-1 uncoating in a SIM-dependent manner. Altogether, our findings suggest that, by recruiting TNPO3, TRIM5α, and TRIM34 and possibly other proteins onto incoming HIV-1 cores through a SIM-dependent interaction with CA-bound CypA, Daxx increases their stability, thus preventing uncoating and reverse transcription. Our study uncovers a previously unknown function of Daxx in the early steps of HIV-1 infection and further illustrates how reverse transcription and uncoating are two tightly interdependent processes.

Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.

Bisarsenical labeling of HIV-1 for real-time fluorescence microscopy.

Imaging studies have benefited from the development of a novel technique for non-destructive labeling of proteins within living cells, based on the use of a reagent called FlAsH-EDT2, a bisarsenical derivative of fluorescein capable of binding with high affinity and specificity to a tetracysteine motif in the protein of interest. This technique has been adapted for the stable, sensitive and specific molecular tagging of HIV-1 IN enabling the tracking of incoming viral particles inside infected living cells. Here we present the experimental steps required for the efficient labeling of HIV-1 IN, namely, molecular insertion of a tetracysteine tag, production of viruses, labeling in vitro of tagged viruses, infection of target cells and visualization of particles by fluorescence microscopy.

TRIM8 is required for virus-induced IFN response in human plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) play a crucial role in antiviral innate immunity through their unique capacity to produce large amounts of type I interferons (IFNs) upon viral detection. Tripartite motif (TRIM) proteins have recently come forth as important modulators of innate signaling, but their involvement in pDCs has not been investigated. Here, we performed a rationally streamlined small interfering RNA (siRNA)-based screen of TRIM proteins in human primary pDCs to identify those that are critical for the IFN response. Among candidate hits, TRIM8 emerged as an essential regulator of IFN regulatory factor 7 (IRF7) function. Mechanistically, TRIM8 protects phosphorylated IRF7 (pIRF7) from proteasomal degradation in an E3 ubiquitin ligase-independent manner by preventing its recognition by the peptidyl-prolyl isomerase Pin1. Our findings uncover a previously unknown regulatory mechanism of type I IFN production in pDCs by which TRIM8 and Pin1 oppositely regulate the stability of pIRF7.

Transportin-1 binds to the HIV-1 capsid via a nuclear localization signal and triggers uncoating.

The initial steps of HIV replication in host cells prime the virus for passage through the nuclear pore and drive the establishment of a productive and irreparable infection1,2. The timely release of the viral genome from the capsid-referred to as uncoating-is emerging as a critical parameter for nuclear import, but the triggers and mechanisms that orchestrate these steps are unknown. Here, we identify ß-karyopherin Transportin-1 (TRN-1) as a cellular co-factor of HIV-1 infection, which binds to incoming capsids, triggers their uncoating and promotes viral nuclear import. Depletion of TRN-1, which we characterized by mass spectrometry, significantly reduced the early steps of HIV-1 infection in target cells, including primary CD4+ T cells. TRN-1 bound directly to capsid nanotubes and induced dramatic structural damage, indicating that TRN-1 is necessary and sufficient for uncoating in vitro. Glycine 89 on the capsid protein, which is positioned within a nuclear localization signal in the cyclophilin A-binding loop, is critical for engaging the hydrophobic pocket of TRN-1 at position W730. In addition, TRN-1 promotes the efficient nuclear import of both viral DNA and capsid protein. Our study suggests that TRN-1 mediates the timely release of the HIV-1 genome from the capsid protein shell and efficient viral nuclear import.