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.

Correlation between low FAT1 expression and early affected muscle in facioscapulohumeral muscular dystrophy.

OBJECTIVE: Facioscapulohumeral muscular dystrophy (FSHD) is linked to either contraction of D4Z4 repeats on chromosome 4 or to mutations in the SMCHD1 gene, both of which result in the aberrant expression of the transcription factor DUX4. However, it is still difficult to correlate these genotypes with the phenotypes observed in patients. Because we have recently shown that mice with disrupted Fat1 functions exhibit FSHD-like phenotypes, we have investigated the expression of the human FAT1 gene in FSHD. METHODS: We first analyzed FAT1 expression in FSHD adult muscles and determined whether FAT1 expression was driven by DUX4. We next determined FAT1 expression levels in 64 muscles isolated from 16 control fetuses. These data were further complemented with analysis of Fat1 expression in developing mouse embryos. RESULTS: We demonstrated that FAT1 expression is independent of DUX4. Moreover, we observed that (1) in control fetal human biopsies or in developing mouse embryos, FAT1 is expressed at lower levels in muscles that are affected at early stages of FSHD progression than in muscles that are affected later or are nonaffected; and (2) in adult muscle biopsies, FAT1 expression is lower in FSHD muscles compared to control muscles. INTERPRETATION: We propose a revised model for FSHD in which FAT1 levels might play a role in determining which muscles will exhibit early and late disease onset, whereas DUX4 may worsen the muscle phenotype.

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.

α-Cyclodextrin enhances myoblast fusion and muscle differentiation by the release of IL-4.

Muscle fibers are formed during embryonic development by the fusion of mononucleated myoblasts. The spatial structure and molecular composition of the sarcolemma are crucial for the myoblast recognition and fusion steps. Cyclodextrins are a group of substances that have the ability to solubilize lipids through the formation of molecular inclusion complexes. Previously, we have shown that methyl-ß-cyclodextrin (MbCD) enhances muscle differentiation. Here, we analyzed the effects of α-cyclodextrin (aCD) during myogenesis. Myogenic cultures treated with aCD showed an increase in myoblast fusion and in the expression of myogenin, sarcomeric tropomyosin and desmin. aCD-conditioned media accelerates myogenesis in a similar way as aCD does, and increased levels of IL-4 were found in aCD-conditioned media. aCD-induced effects on myogenesis were inhibited by an anti-IL4 antibody. These results show that α-cyclodextrin induces myogenic differentiation by the release of IL-4.

Membrane cholesterol depletion by methyl-beta-cyclodextrin enhances the expression of cardiac differentiation markers.

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-beta-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/beta-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. beta-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac beta-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

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.

Activated dendritic cells modulate proliferation and differentiation of human myoblasts.

Idiopathic Inflammatory Myopathies (IIMs) are a heterogeneous group of autoimmune diseases affecting skeletal muscle tissue homeostasis. They are characterized by muscle weakness and inflammatory infiltration with tissue damage. Amongst the cells in the muscle inflammatory infiltration, dendritic cells (DCs) are potent antigen-presenting and key components in autoimmunity exhibiting an increased activation in inflamed tissues. Since, the IIMs are characterized by the focal necrosis/regeneration and muscle atrophy, we hypothesized that DCs may play a role in these processes. Due to the absence of a reliable in vivo model for IIMs, we first performed co-culture experiments with immature DCs (iDC) or LPS-activated DCs (actDC) and proliferating myoblasts or differentiating myotubes. We demonstrated that both iDC or actDCs tightly interact with myoblasts and myotubes, increased myoblast proliferation and migration, but inhibited myotube differentiation. We also observed that actDCs increased HLA-ABC, HLA-DR, VLA-5, and VLA-6 expression and induced cytokine secretion on myoblasts. In an in vivo regeneration model, the co-injection of human myoblasts and DCs enhanced human myoblast migration, whereas the absolute number of human myofibres was unchanged. In conclusion, we suggest that in the early stages of myositis, DCs may play a crucial role in inducing muscle-damage through cell-cell contact and inflammatory cytokine secretion, leading to muscle regeneration impairment.

A soluble and active form of Wnt-3a protein is involved in myogenic differentiation after cholesterol depletion.

Cholesterol is one of the major lipids of plasma membranes. Recently, we have shown that cholesterol depletion by methyl-beta-cyclodextrin (M beta CD) induces the activation of the Wnt/beta-catenin pathway and enhances myogenic differentiation. Here, we show that M beta CD-conditioned media accelerates myogenesis in a similar way as M beta CD does, suggesting that the effects induced by M beta CD could be caused by soluble factors present in the culture medium. Soluble Wnt-3 protein is significantly enhanced in M beta CD-conditioned medium. Wnt-3a-enriched media induces myogenesis as much as M beta CD does, whereas Wnt-5a-enriched media inhibits. We suggest that Wnt-3a is involved in the myogenic induction observed after cholesterol depletion.

Role of non-motile microtubule-associated proteins in virus trafficking.

Viruses are entirely dependent on their ability to infect a host cell in order to replicate. To reach their site of replication as rapidly and efficiently as possible following cell entry, many have evolved elaborate mechanisms to hijack the cellular transport machinery to propel themselves across the cytoplasm. Long-range movements have been shown to involve motor proteins along microtubules (MTs) and direct interactions between viral proteins and dynein and/or kinesin motors have been well described. Although less well-characterized, it is also becoming increasingly clear that non-motile microtubule-associated proteins (MAPs), including structural MAPs of the MAP1 and MAP2 families, and microtubule plus-end tracking proteins (+TIPs), can also promote viral trafficking in infected cells, by mediating interaction of viruses with filaments and/or motor proteins, and modulating filament stability. Here we review our current knowledge on non-motile MAPs, their role in the regulation of cytoskeletal dynamics and in viral trafficking during the early steps of infection.

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.

miRNA expression in control and FSHD fetal human muscle biopsies.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is an autosomal-dominant disorder and is one of the most common forms of muscular dystrophy. We have recently shown that some hallmarks of FSHD are already expressed in fetal FSHD biopsies, thus opening a new field of investigation for mechanisms leading to FSHD. As microRNAs (miRNAs) play an important role in myogenesis and muscle disorders, in this study we compared miRNAs expression levels during normal and FSHD muscle development. METHODS: Muscle biopsies were obtained from quadriceps of both healthy control and FSHD1 fetuses with ages ranging from 14 to 33 weeks of development. miRNA expression profiles were analyzed using TaqMan Human MicroRNA Arrays. RESULTS: During human skeletal muscle development, in control muscle biopsies we observed changes for 4 miRNAs potentially involved in secondary muscle fiber formation and 5 miRNAs potentially involved in fiber maturation. When we compared the miRNA profiles obtained from control and FSHD biopsies, we did not observe any differences in the muscle specific miRNAs. However, we identified 8 miRNAs exclusively expressed in FSHD1 samples (miR-330, miR-331-5p, miR-34a, miR-380-3p, miR-516b, miR-582-5p, miR-517* and miR-625) which could represent new biomarkers for this disease. Their putative targets are mainly involved in muscle development and morphogenesis. Interestingly, these FSHD1 specific miRNAs do not target the genes previously described to be involved in FSHD. CONCLUSIONS: This work provides new candidate mechanisms potentially involved in the onset of FSHD pathology. Whether these FSHD specific miRNAs cause deregulations during fetal development, or protect against the appearance of the FSHD phenotype until the second decade of life still needs to be investigated.

Differences in the expression and distribution of flotillin-2 in chick, mice and human muscle cells.

Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.

Cholesterol depletion by methyl-ß-cyclodextrin enhances cell proliferation and increases the number of desmin-positive cells in myoblast cultures.

Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-ß-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/ß-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3ß, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/ß-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/ß-catenin signaling pathway.

Regenerative potential of human muscle stem cells in chronic inflammation.

INTRODUCTION: Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle. METHODS: As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation. RESULTS: After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group. CONCLUSIONS: In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.

Wnt/beta-catenin pathway activation and myogenic differentiation are induced by cholesterol depletion.

Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.

Association between the muscle-specific proteins desmin and caveolin-3 in muscle cells.

The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts.

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.