Spinal cord injury dysregulates fibro-adipogenic progenitors miRNAs signaling to promote neurogenic heterotopic ossifications.

Neurogenic heterotopic ossifications are intramuscular bone formations developing following central nervous system injury. The pathophysiology is poorly understood and current treatments for this debilitating condition remain unsatisfying. Here we explored the role of miRNAs in a clinically relevant mouse model that combines muscle and spinal cord injury, and in patients' cells. We found an osteo-suppressive miRNAs response in injured muscle that was hindered when the spinal cord injury was associated. In isolated fibro-adipogenic progenitors from damaged muscle (cells at the origin of ossification), spinal cord injury induced a downregulation of osteo-suppressive miRNAs while osteogenic markers were overexpressed. The overexpression of selected miRNAs in patient's fibro-adipogenic progenitors inhibited mineralization and osteo-chondrogenic markers in vitro. Altogether, we highlighted an osteo-suppressive mechanism involving multiple miRNAs in response to muscle injury that prevents osteogenic commitment which is ablated by the neurologic lesion in heterotopic ossification pathogenesis. This provides new research hypotheses for preventive treatments.

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.

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.

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.

[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.

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.

RanBP2 regulates the anti-retroviral activity of TRIM5α by SUMOylation at a predicted phosphorylated SUMOylation motif.

TRIM5α is a cytoplasmic restriction factor that blocks post-entry retroviral infection. Evidence suggests that its antiviral activity can be regulated by SUMO, but how this is achieved remains unknown. Here, we show that TRIM5α forms a complex with RanGAP1, Ubc9, and RanBP2 at the nuclear pore, and that RanBP2 E3 SUMO ligase promotes the SUMOylation of endogenous TRIM5α in the cytoplasm. Loss of RanBP2 blocked SUMOylation of TRIM5α, altered its localization in primary cells, and suppressed the antiviral activity of both rhesus and human orthologs. In cells, human TRIM5α is modified on K84 within a predicted phosphorylated SUMOylation motif (pSUM) and not on K10 as found in vitro. Non-modified TRIM5α lacked antiviral activity, indicating that only SUMOylated TRIM5α acts as a restriction factor. This work illustrates the importance of the nuclear pore in intrinsic antiviral immunity, acting as a hub where virus, SUMO machinery, and restriction factors can meet.

Endogenous TRIM5α Function Is Regulated by SUMOylation and Nuclear Sequestration for Efficient Innate Sensing in Dendritic Cells.

During retroviral infection, viral capsids are subject to restriction by the cellular factor TRIM5α. Here, we show that dendritic cells (DCs) derived from human and non-human primate species lack efficient TRIM5α-mediated retroviral restriction. In DCs, endogenous TRIM5α accumulates in nuclear bodies (NB) that partly co-localize with Cajal bodies in a SUMOylation-dependent manner. Nuclear sequestration of TRIM5α allowed potent induction of type I interferon (IFN) responses during infection, mediated by sensing of reverse transcribed DNA by cGAS. Overexpression of TRIM5α or treatment with the SUMOylation inhibitor ginkgolic acid (GA) resulted in enforced cytoplasmic TRIM5α expression and restored efficient viral restriction but abrogated type I IFN production following infection. Our results suggest that there is an evolutionary trade-off specific to DCs in which restriction is minimized to maximize sensing. TRIM5α regulation via SUMOylation-dependent nuclear sequestration adds to our understanding of how restriction factors are regulated.

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.

Human nucleoporins promote HIV-1 docking at the nuclear pore, nuclear import and integration.

The nuclear pore complex (NPC) mediates nucleo-cytoplasmic transport of macromolecules and is an obligatory point of passage and functional bottleneck in the replication of some viruses. The Human Immunodeficiency Virus (HIV) has evolved the required mechanisms for active nuclear import of its genome through the NPC. However the mechanisms by which the NPC allows or even assists HIV translocation are still unknown. We investigated the involvement of four key nucleoporins in HIV-1 docking, translocation, and integration: Nup358/RanBP2, Nup214/CAN, Nup98 and Nup153. Although all induce defects in infectivity when depleted, only Nup153 actually showed any evidence of participating in HIV-1 translocation through the nuclear pore. We show that Nup358/RanBP2 mediates docking of HIV-1 cores on NPC cytoplasmic filaments by interacting with the cores and that the C-terminus of Nup358/RanBP2 comprising a cyclophilin-homology domain contributes to binding. We also show that Nup214/CAN and Nup98 play no role in HIV-1 nuclear import per se: Nup214/CAN plays an indirect role in infectivity read-outs through its effect on mRNA export, while the reduction of expression of Nup98 shows a slight reduction in proviral integration. Our work shows the involvement of nucleoporins in diverse and functionally separable steps of HIV infection and nuclear import.

Residual HIV-1 DNA Flap-independent nuclear import of cPPT/CTS double mutant viruses does not support spreading infection.

BACKGROUND: The human immunodeficiency virus type 1 (HIV-1) central DNA Flap is generated during reverse transcription as a result of (+) strand initiation at the central polypurine tract (cPPT) and termination after a ca. 100 bp strand displacement at the central termination sequence (CTS). The central DNA Flap is a determinant of HIV-1 nuclear import, however, neither cPPT nor CTS mutations entirely abolish nuclear import and infection. Therefore, to determine whether or not the DNA Flap is essential for HIV-1 nuclear import, we generated double mutant (DM) viruses, combining cPPT and CTS mutations to abolish DNA Flap formation. RESULTS: The combination of cPPT and CTS mutations reduced the proportion of viruses forming the central DNA Flap at the end of reverse transcription and further decreased virus infectivity in one-cycle titration assays. The most affected DM viruses were unable to establish a spreading infection in the highly permissive MT4 cell line, nor in human primary peripheral blood mononuclear cells (PBMCs), indicating that the DNA Flap is required for virus replication. Surprisingly, we found that DM viruses still maintained residual nuclear import levels, amounting to 5-15% of wild-type virus, as assessed by viral DNA circle quantification. Alu-PCR quantification of integrated viral genome also indicated 5-10% residual integration levels compared to wild-type virus. CONCLUSION: This work establishes that the central DNA Flap is required for HIV-1 spreading infection but points to a residual DNA Flap independent nuclear import, whose functional significance remains unclear since it is not sufficient to support viral replication.

Cross-species amplification of 41 microsatellites in European cyprinids: A tool for evolutionary, population genetics and hybridization studies.

BACKGROUND: Cyprinids display the most abundant and widespread species among the European freshwater Teleostei and are known to hybridize quite commonly. Nevertheless, a limited number of markers for conducting comparative differentiation, evolutionary and hybridization dynamics studies are available to date. FINDINGS: Five multiplex PCR sets were optimized in order to assay 41 cyprinid-specific polymorphic microsatellite loci (including 10 novel loci isolated from Chondrostoma nasus nasus, Chondrostoma toxostoma toxostoma and Leuciscus leuciscus) for 503 individuals (440 purebred specimens and 63 hybrids) from 15 European cyprinid species. The level of genetic diversity was assessed in Alburnus alburnus, Alburnoides bipunctatus, C. genei, C. n. nasus, C. soetta, C. t. toxostoma, L. idus, L. leuciscus, Pachychilon pictum, Rutilus rutilus, Squalius cephalus and Telestes souffia. The applicability of the markers was also tested on Abramis brama, Blicca bjoerkna and Scardinius erythrophtalmus specimens. Overall, between 24 and 37 of these markers revealed polymorphic for the investigated species and 23 markers amplified for all the 15 European cyprinid species. CONCLUSIONS: The developed set of markers demonstrated its performance in discriminating European cyprinid species. Furthermore, it allowed detecting and characterizing hybrid individuals. These microsatellites will therefore be useful to perform comparative evolutionary and population genetics studies dealing with European cyprinids, what is of particular interest in conservation issues and constitutes a tool of choice to conduct hybridization studies.