Hepatitis C virus replication requires integrity of mitochondria-associated ER membranes.

Background & Aims: Chronic HCV infection causes cellular stress, fibrosis and predisposes to hepatocarcinogenesis. Mitochondria play key roles in orchestrating stress responses by regulating bioenergetics, inflammation and apoptosis. To better understand the role of mitochondria in the viral life cycle and disease progression of chronic hepatitis C, we studied morphological and functional mitochondrial alterations induced by HCV using productively infected hepatoma cells and patient livers. Methods: Biochemical and imaging assays were used to assess localization of cellular and viral proteins and mitochondrial functions in cell cultures and liver biopsies. Cyclophilin D (CypD) knockout was performed using CRISPR/Cas9 technology. Viral replication was quantified by quantitative reverse-transcription PCR and western blotting. Results: Several HCV proteins were found to associate with mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), the points of contact between the ER and mitochondria. Downregulation of CypD, which is known to disrupt MAM integrity, reduced viral replication, suggesting that MAMs play an important role in the viral life cycle. This process was rescued by ectopic CypD expression. Furthermore, HCV proteins were found to associate with voltage dependent anion channel 1 (VDAC1) at MAMs and to reduce VDAC1 protein levels at MAMs in vitro and in patient biopsies. This association did not affect MAM-associated functions in glucose homeostasis and Ca2+ signaling. Conclusions: HCV proteins associate specifically with MAMs and MAMs play an important role in viral replication. The association between viral proteins and MAMs did not impact Ca2+ signaling between the ER and mitochondria or glucose homeostasis. Whether additional functions of MAMs and/or VDAC are impacted by HCV and contribute to the associated pathology remains to be assessed. Impact and implications: Hepatitis C virus infects the liver, where it causes inflammation, cell damage and increases the long-term risk of liver cancer. We show that several HCV proteins interact with mitochondria in liver cells and alter the composition of mitochondrial subdomains. Importantly, HCV requires the architecture of these mitochondrial subdomains to remain intact for efficient viral replication.

Heparanase-1: From Cancer Biology to a Future Antiviral Target.

Heparan sulfate proteoglycans (HSPGs) are a major constituent of the extracellular matrix (ECM) and are found to be implicated in viral infections, where they play a role in both cell entry and release for many viruses. The enzyme heparanase-1 is the only known endo-beta-D-glucuronidase capable of degrading heparan sulphate (HS) chains of HSPGs and is thus important for regulating ECM homeostasis. Heparanase-1 expression is tightly regulated as the uncontrolled cleavage of HS may result in abnormal cell activation and significant tissue damage. The overexpression of heparanase-1 correlates with pathological scenarios and is observed in different human malignancies, such as lymphoma, breast, colon, lung, and hepatocellular carcinomas. Interestingly, heparanase-1 has also been documented to be involved in numerous viral infections, e.g., HSV-1, HPV, DENV. Moreover, very recent reports have demonstrated a role of heparanase-1 in HCV and SARS-CoV-2 infections. Due to the undenied pro-carcinogenic role of heparanase-1, multiple inhibitors have been developed, some reaching phase II and III in clinical studies. However, the use of heparanase inhibitors as antivirals has not yet been proposed. If it can be assumed that heparanase-1 is implicated in numerous viral life cycles, its inhibition by specific heparanase-acting compounds should result in a blockage of viral infection. This review addresses the perspectives of using heparanase inhibitors, not only for cancer treatment, but also as antivirals. Eventually, the development of a novel class antivirals targeting a cellular protein could help to alleviate the resistance problems seen with some current antiretroviral therapies.

Heparanase-1 is upregulated by hepatitis C virus and favors its replication.

BACKGROUND & AIMS: Over time, chronic HCV infection can lead to hepatocellular carcinoma (HCC), a process that involves changes to the liver extracellular matrix (ECM). However, the exact mechanisms by which HCV induces HCC remain unclear. Therefore, we sought to investigate the impact of HCV on the liver ECM, with a focus on heparanase-1 (HPSE). METHODS: HPSE expression was assessed by quantitative reverse-transcription PCR, immunoblotting and immunofluorescence in liver biopsies infected or not with HCV, and in 10-day-infected hepatoma Huh7.5 cells. Cell lines deficient for or overexpressing HPSE were established to study its role during infection. RESULTS: HCV propagation led to significant HPSE induction, in vivo and in vitro. HPSE enhanced infection when exogenously expressed or supplemented as a recombinant protein. Conversely, when HPSE expression was downregulated or its activity blocked, HCV infection dropped, suggesting a role of HPSE in the HCV life cycle. We further studied the underlying mechanisms of such observations and found that HPSE favored HCV release by enhancing CD63 synthesis and exosome secretion, but not by stimulating HCV entry or genome replication. We also showed that virus-induced oxidative stress was involved in HPSE induction, most likely through NF-κB activation. CONCLUSIONS: We report for the first time that HCV infection is favored by HPSE, and upregulates HPSE expression and secretion, which may result in pathogenic alterations of the ECM. LAY SUMMARY: Chronic hepatitis C virus (HCV) infection can lead to hepatocellular carcinoma development in a process that involves derangement of the extracellular matrix (ECM). Herein, we show that heparanase-1, a protein involved in ECM degradation and remodeling, favors HCV infection and is upregulated by HCV infection; this upregulation may result in pathogenic alterations of the ECM.

Molecular Crosstalk between the Hepatitis C Virus and the Extracellular Matrix in Liver Fibrogenesis and Early Carcinogenesis.

Chronic infection by the hepatitis C virus (HCV) is a major cause of liver diseases, predisposing to fibrosis and hepatocellular carcinoma. Liver fibrosis is characterized by an overly abundant accumulation of components of the hepatic extracellular matrix, such as collagen and elastin, with consequences on the properties of this microenvironment and cancer initiation and growth. This review will provide an update on mechanistic concepts of HCV-related liver fibrosis/cirrhosis and early stages of carcinogenesis, with a dissection of the molecular details of the crosstalk during disease progression between hepatocytes, the extracellular matrix, and hepatic stellate cells.

Innovative particle standards and long-lived imaging for 2D and 3D dSTORM.

Direct stochastic optical reconstruction microscopy (dSTORM), developed in the last decade, has revolutionised optical microscopy by enabling scientists to visualise objects beyond the resolution provided by conventional microscopy (200 nm). We developed an innovative method based on blinking particle standards and conditions for long-lived imaging over several weeks. Stable localisation precisions within the 10 nm-range were achieved for single virions and in cellulo 2D imaging of centrosomes, as well as their reliable reconstruction in 3D dSTORM.

CD81 large extracellular loop-containing fusion proteins with a dominant negative effect on HCV cell spread and replication.

The roles of CD81 in the hepatitis C virus (HCV) life cycle are multiple but remain ill characterized. CD81 is known to interact with the HCV glycoproteins as an attachment factor. It also has an important role in the post-attachment entry process. Its interaction with claudin-1, for example, is vital for viral uptake and trafficking. Furthermore, CD81 and its role in membrane organization and trafficking are thought to play a pivotal role in HCV replication. Some of these functions are particularly limited to human CD81; others can be substituted with CD81 molecules from other species. However, with the exception of the large extracellular loop sequence, the structure-function analysis of CD81 in the HCV infectious cycle remains ill characterized. We describe here the fusion molecules between the large extracellular loops of human or mouse CD81 and lipid-raft-associated or unassociated GPI anchors. These fusion molecules have strong antiviral activity in a dominant negative fashion, independent of membrane raft association. Their expression in the hepatoma cell line Huh7.5 blocks HCV uptake, transmission and replication. These molecules will be useful to decipher the various roles of CD81 in the HCV life cycle and transmission in more detail.

Hepatitis C virus infection propagates through interactions between Syndecan-1 and CD81 and impacts the hepatocyte glycocalyx.

The hepatitis C virus (HCV) infects hepatocytes after binding to heparan sulfate proteoglycans, in particular Syndecan-1, followed by recognition of the tetraspanin CD81 and other receptors. Heparan sulfate proteoglycans are found in a specific microenvironment coating the hepatocyte surface called the glycocalyx and are receptors for extracellular matrix proteins, cytokines, growth factors, lipoproteins, and infectious agents. We investigated the mutual influence of HCV infection on the glycocalyx and revealed new links between Syndecan-1 and CD81. Hepatocyte infection by HCV was inhibited after knocking down Syndecan-1 or Xylosyltransferase 2, a key enzyme of Syndecan-1 biosynthesis. Simultaneous knockdown of Syndecan-1 and CD81 strongly inhibited infection, suggesting their cooperative action. At early infection stages, Syndecan-1 and virions colocalized at the plasma membrane and were internalized in endosomes. Direct interactions between Syndecan-1 and CD81 were revealed in primary and transformed hepatocytes by immunoprecipitation and proximity ligation assays. Expression of Syndecan-1 and Xylosyltransferase 2 was altered within days post-infection, and the remaining Syndecan-1 pool colocalized poorly with CD81. The data indicate a profound reshuffling of the hepatocyte glycocalyx during HCV infection, possibly required for establishing optimal conditions of viral propagation.

HSP90 Chaperoning in Addition to Phosphoprotein Required for Folding but Not for Supporting Enzymatic Activities of Measles and Nipah Virus L Polymerases.

UNLABELLED: Nonsegmented negative-stranded RNA viruses, or members of the order Mononegavirales, share a conserved gene order and the use of elaborate transcription and replication machinery made up of at least four molecular partners. These partners have coevolved with the acquisition of the permanent encapsidation of the entire genome by the nucleoprotein (N) and the use of this N-RNA complex as a template for the viral polymerase composed of the phosphoprotein (P) and the large enzymatic protein (L). Not only is P required for polymerase function, but it also stabilizes the L protein through an unknown underlying molecular mechanism. By using NVP-AUY922 and/or 17-dimethylaminoethylamino-17-demethoxygeldanamycin as specific inhibitors of cellular heat shock protein 90 (HSP90), we found that efficient chaperoning of L by HSP90 requires P in the measles, Nipah, and vesicular stomatitis viruses. While the production of P remains unchanged in the presence of HSP90 inhibitors, the production of soluble and functional L requires both P and HSP90 activity. Measles virus P can bind the N terminus of L in the absence of HSP90 activity. Both HSP90 and P are required for the folding of L, as evidenced by a luciferase reporter insert fused within measles virus L. HSP90 acts as a true chaperon; its activity is transient and dispensable for the activity of measles and Nipah virus polymerases of virion origin. That the cellular chaperoning of a viral polymerase into a soluble functional enzyme requires the assistance of another viral protein constitutes a new paradigm that seems to be conserved within the Mononegavirales order. IMPORTANCE: Viruses are obligate intracellular parasites that require a cellular environment for their replication. Some viruses particularly depend on the cellular chaperoning apparatus. We report here that for measles virus, successful chaperoning of the viral L polymerase mediated by heat shock protein 90 (HSP90) requires the presence of the viral phosphoprotein (P). Indeed, while P protein binds to the N terminus of L independently of HSP90 activity, both HSP90 and P are required to produce stable, soluble, folded, and functional L proteins. Once formed, the mature P+L complex no longer requires HSP90 to exert its polymerase functions. Such a new paradigm for the maturation of a viral polymerase appears to be conserved in several members of the Mononegavirales order, including the Nipah and vesicular stomatitis viruses.

Structural basis for the activation of innate immune pattern-recognition receptor RIG-I by viral RNA.

RIG-I is a key innate immune pattern-recognition receptor that triggers interferon expression upon detection of intracellular 5'triphosphate double-stranded RNA (5'ppp-dsRNA) of viral origin. RIG-I comprises N-terminal caspase activation and recruitment domains (CARDs), a DECH helicase, and a C-terminal domain (CTD). We present crystal structures of the ligand-free, autorepressed, and RNA-bound, activated states of RIG-I. Inactive RIG-I has an open conformation with the CARDs sequestered by a helical domain inserted between the two helicase moieties. ATP and dsRNA binding induce a major rearrangement to a closed conformation in which the helicase and CTD bind the blunt end 5'ppp-dsRNA with perfect complementarity but incompatibly with continued CARD binding. We propose that after initial binding of 5'ppp-dsRNA to the flexibly linked CTD, co-operative tight binding of ATP and RNA to the helicase domain liberates the CARDs for downstream signaling. These findings significantly advance our molecular understanding of the activation of innate immune signaling helicases.

Rapid titration of measles and other viruses: optimization with determination of replication cycle length.

BACKGROUND: Measles virus (MV) is a member of the Paramyxoviridae family and an important human pathogen causing strong immunosuppression in affected individuals and a considerable number of deaths worldwide. Currently, measles is a re-emerging disease in developed countries. MV is usually quantified in infectious units as determined by limiting dilution and counting of plaque forming unit either directly (PFU method) or indirectly from random distribution in microwells (TCID50 method). Both methods are time-consuming (up to several days), cumbersome and, in the case of the PFU assay, possibly operator dependent. METHODS/FINDINGS: A rapid, optimized, accurate, and reliable technique for titration of measles virus was developed based on the detection of virus infected cells by flow cytometry, single round of infection and titer calculation according to the Poisson's law. The kinetics follow up of the number of infected cells after infection with serial dilutions of a virus allowed estimation of the duration of the replication cycle, and consequently, the optimal infection time. The assay was set up to quantify measles virus, vesicular stomatitis virus (VSV), and human immunodeficiency virus type 1 (HIV-1) using antibody labeling of viral glycoprotein, virus encoded fluorescent reporter protein and an inducible fluorescent-reporter cell line, respectively. CONCLUSION: Overall, performing the assay takes only 24-30 hours for MV strains, 12 hours for VSV, and 52 hours for HIV-1. The step-by-step procedure we have set up can be, in principle, applicable to accurately quantify any virus including lentiviral vectors, provided that a virus encoded gene product can be detected by flow cytometry.

Identification of a methylated oligoribonucleotide as a potent inhibitor of HIV-1 reverse transcription complex.

Upon HIV-1 infection of a target cell, the viral reverse transcriptase (RT) copies the genomic RNA to synthesize the viral DNA. The genomic RNA is within the incoming HIV-1 core where it is coated by molecules of nucleocapsid (NC) protein that chaperones the reverse transcription process. Indeed, the RT chaperoning properties of NC extend from the initiation of cDNA synthesis to completion of the viral DNA. New and effective drugs against HIV-1 continue to be required, which prompted us to search for compounds aimed at inhibiting NC protein. Here, we report that the NC chaperoning activity is extensively inhibited in vitro by small methylated oligoribonucleotides (mODN). These mODNs were delivered intracellularly using a cell-penetrating-peptide and found to impede HIV-1 replication in primary human cells at nanomolar concentrations. Extensive analysis showed that viral cDNA synthesis was severely impaired by mODNs. Partially resistant viruses with mutations in NC and RT emerged after months of passaging in cell culture. A HIV-1 molecular clone (NL4.3) bearing these mutations was found to replicate at high concentrations of mODN, albeit with a reduced fitness. Small, methylated ODNs such as mODN-11 appear to be a new type of highly potent inhibitor of HIV-1.

[Cellular receptors and in vivo measles virus propagation]. / Récepteurs cellulaires du virus de la rougeole et dissémination in vivo.

Since the nineties, the analysis of the molecular mechanisms gover- ning the entry of measles virus has strongly impacted our knowledge of viral propagation into tissues during the natural infection. The identification of cellular receptors and the 3D structure of the viral glycoprotein mediating virus attachment have revealed how an RNA virus with error-prone poly- merase has remained immunologically monomorphous despite of more than twenty circulating genotypes around the world. In addition, the neo-targeting towards another cellular receptor suggests its involvement in the attenuation phenotype of the vaccine strains. Likewise, from this virus, the first class of an envelope glycoprotein supporting any retargeting has been developed.

A role for CD81 on the late steps of HIV-1 replication in a chronically infected T cell line.

BACKGROUND: HIV-1 uses cellular co-factors for virion formation and release. The virus is able to incorporate into the viral particles host cellular proteins, such as tetraspanins which could serve to facilitate HIV-1 egress. Here, we investigated the implication of several tetraspanins on HIV-1 formation and release in chronically infected T-lymphoblastic cells, a model that permits the study of the late steps of HIV-1 replication. RESULTS: Our data revealed that HIV-1 Gag and Env structural proteins co-localized with tetraspanins in the form of clusters. Co-immunoprecipitation experiments showed that Gag proteins interact, directly or indirectly, with CD81, and less with CD82, in tetraspanin-enriched microdomains composed of CD81/CD82/CD63. In addition, when HIV-1 producing cells were treated with anti-CD81 antibodies, or upon CD81 silencing by RNA interference, HIV-1 release was significantly impaired, and its infectivity was modulated. Finally, CD81 downregulation resulted in Gag redistribution at the cell surface. CONCLUSION: Our findings not only extend the notion that HIV-1 assembly can occur on tetraspanin-enriched microdomains in T cells, but also highlight a critical role for the tetraspanin CD81 on the late steps of HIV replication.

A clathrin-dynamin-dependent endocytic pathway for the uptake of HIV-1 by direct T cell-T cell transmission.

Cellular contacts between HIV-1-infected cells and target primary T CD4+ lymphocytes trigger the formation of a structure known as the virological synapse. As a consequence, viral production in HIV-1-infected cells is polarized towards the virological synapse and nascent viral particles are directly transferred to target T CD4+ lymphocytes. In this study, we performed short time cocultures of target primary T CD4+ lymphocytes with effector T cells infected by either HIV-1 NL4-3 or BaL. Using flow cytometry and immuno-confocal analyses, we investigated the transfer of HIV-1 virion antigens. We found that after 3 h of coculture, unstimulated T CD4+ lymphocytes captured complete HIV-1 virions from infected T cells during cell-cell contacts. Virus transfer occurred through a dynamin-dependent pathway and could be inhibited by chlorpromazine, an inhibitor of clathrin-dependent endocytosis. Transferred HIV-1 virions were located in compartments close to the surface of the target cell in a polarized manner. These compartments were positive for clathrin and the early endosomal marker EEA1 but were negative for caveolin-1. Furthermore, the great majority of internalized HIV-1 particles did not colocalize with Lamp1, a well-known marker for the lysosomal-degradative pathway. Similar results were observed when stimulated primary T CD4+ lymphocytes were the target cells. Our results suggest a mechanism of cell to cell HIV-1 transfer through a clathrin- and dynamin-dependent early endocytic pathway where internalized HIV-1 particles would not reach Lamp1 positive compartments, suggesting that during HIV-1 transfer by cell-cell contacts, virions can be taken up by endocytosis but not be degraded in lysosomes.

[Revisiting HIV-1 assembly]. / Une nouvelle vision de l'assemblage du VIH-1.

During the late stage of virus replication, incorporation of the envelope glycoproteins (Env) by Gag cores takes place together with the proteolytic maturation of Gag and Gag-Pol precursors. Assembly is initially driven by Gag oligomerisation, which requires two platorms. The first one is formed by specific membrane subdomains with which Gag molecules interact via the N-terminal MA domain, and the second by the viral genomic RNA undergoing specific interactions with the NC domain of Gag. To complete viral budding, the Gag "late domain" subsequently associates with members of the ESCRT complexes involved in the budding of vesicles in late endosomes (LE). While the cellular trafficking of the viral components is still poorly understood, there is an ongoing debate on the site of HIV-1 assembly, because this process might take place either at the plasma membrane or in intracellular compartments such as the LE, depending on the virus/cell system studied. This site may depend on the interplay of multiple overlapping trafficking signals bear by Gag and Env. Our recent results indicate that it may rely on the chronic or acute nature of the viral infection more than on the cell type. In chronically infected cells, virions probably assemble and accumulate in intracellular compartments hidden from the immune system. Release of virions in the form of bursts would be triggered during cell-cell interactions, through a specialized structure called the virological synapse.

Intracellular HIV-1 Gag localization is impaired by mutations in the nucleocapsid zinc fingers.

BACKGROUND: The HIV-1 nucleocapsid protein (NC) is formed of two CCHC zinc fingers flanked by highly basic regions. HIV-1 NC plays key roles in virus structure and replication via its nucleic acid binding and chaperoning properties. In fact, NC controls proviral DNA synthesis by reverse transcriptase (RT), gRNA dimerization and packaging, and virion assembly. RESULTS: We previously reported a role for the first NC zinc finger in virion structure and replication 1. To investigate the role of both NC zinc fingers in intracellular Gag trafficking, and in virion assembly, we generated series of NC zinc fingers mutations. Results show that all Zinc finger mutations have a negative impact on virion biogenesis and maturation and rendered defective the mutant viruses. The NC zinc finger mutations caused an intracellular accumulation of Gag, which was found either diffuse in the cytoplasm or at the plasma membrane but not associated with endosomal membranes as for wild type Gag. Evidences are also provided showing that the intracellular interactions between NC-mutated Gag and the gRNA were impaired. CONCLUSION: These results show that Gag oligomerization mediated by gRNA-NC interactions is required for correct Gag trafficking, and assembly in HIV-1 producing cells and the release of infectious viruses.

Assembly of infectious HIV-1 in human epithelial and T-lymphoblastic cell lines.

The canonical view of the ultimate steps of HIV-1 replication is that virus assembly and budding are taking place at the plasma membrane of infected cells. Surprisingly, recent studies revealed that these steps also occur on endosomal membranes in the interior of infected cells, such as macrophages. This prompted us to revisit the site of HIV-1 assembly in human epithelial-like cells and in infected human T-lymphoblastic cells. To address this question, we investigated the intracellular location of the major viral structural components of HIV-1, namely Gag, Env and the genomic RNA. Using a sub-cellular fractionation method, as well as immuno-confocal and electron microscopy, we show that Gag, the Env glycoproteins and the genomic RNA accumulate in late endosomes that contain infectious HIV-1 particles. In epithelial-like 293T cells, HIV-1 assembles and buds both at the plasma membrane and in endosomes, while in chronically infected human T lymphocytes, viral assembly mostly occurs within the cell where large amounts of infectious virions accumulate in endosomal compartments. In addition, HIV-1 release could be enhanced by ionomycin, a drug stimulating calcium-dependent exocytosis. These results favour the view that newly made Gag molecules associate with the genomic RNA in the cytosol, then viral core complexes can be targeted to late endosomes together with Env, where infectious HIV-1 are made and subsequently released by exocytosis.

Inhibition of coreceptor-independent cell-to-cell human immunodeficiency virus type 1 transmission by a CD4-immunoglobulin G2 fusion protein.

We have previously shown (J. Blanco et al., J. Biol. Chem. 279:51305-51314, 2004) that the contact between HIV producing cells and primary CD4(+) T cells may induce the uptake of human immunodeficiency virus (HIV) particles by target cells in the absence of HIV envelope-mediated membrane fusion or productive HIV replication. HIV uptake by CD4(+) T cells was dependent on cellular contacts mediated by the binding of gp120 to CD4 but was independent of the expression of the appropriate HIV coreceptor, CCR5 or CXCR4. Here, we have characterized the effect of agents blocking gp120 binding to CD4 on cell-to-cell HIV transmission. A recombinant CD4-based protein (CD4-immunoglobulin G2 [IgG2]), that is currently being evaluated in clinical trials, completely inhibited the uptake of HIV particles by CD4(+) T cells from persistently infected cells expressing R5, X4, or X4/T-20-resistant HIV-1 envelope glycoproteins. Consequently, both the release of viral particles from endocytic vesicles and the infection of reporter U87-CD4 cells were also prevented. The polyanionic anti-HIV agent dextran sulfate failed to prevent the intracellular uptake of virions by CD4(+) T cells. Indeed, it increased HIV uptake in a dose-dependent manner, suggesting functional differences between the specific gp120-targeting CD4-IgG2 agent and nonspecific HIV binding inhibitors. Thus, the inhibition of the specific interaction between gp120 and CD4 protein could be an effective strategy to inhibit HIV binding to CD4(+) T cells, and the mechanism by which CD4(+) T cells lacking the appropriate coreceptor may be converted in HIV carriers.