HIV fusion: Catch me if you can

International audienc

Maturation extracellulaire du virus de l’hépatite C: Une cape d’invisibilité plasmatique

International audienc

Overview of HCV Life Cycle with a Special Focus on Current and Possible Future Antiviral Targets

Hepatitis C infection is the leading cause of liver diseases worldwide and a major health concern that affects an estimated 3% of the global population. Novel therapies available since 2014 and 2017 are very efficient and the WHO considers HCV eradication possible by the year 2030. These treatments are based on the so-called direct acting antivirals (DAAs) that have been developed through research efforts by academia and industry since the 1990s. After a brief overview of the HCV life cycle, we describe here the functions of the different targets of current DAAs, the mode of action of these DAAs and potential future inhibitors

HCV Interplay with Lipoproteins: Inside or Outside the Cells?

International audienceHepatitis C virus (HCV) infection is a major public health issue leading to chronic liver diseases. HCV particles are unique owing to their particular lipid composition, namely the incorporation of neutral lipids and apolipoproteins. The mechanism of association between HCV virion components and these lipoproteins factors remains poorly understood as well as its impact in subsequent steps of the viral life cycle, such as entry into cells. It was proposed that the lipoprotein biogenesis pathway is involved in HCV morphogenesis; yet, recent evidence indicated that HCV particles can mature and evolve biochemically in the extracellular medium after egress. In addition, several viral, cellular and blood components have been shown to influence and regulate this specific association. Finally, this specific structure and composition of HCV particles was found to influence entry into cells as well as their stability and sensitivity to neutralizing antibodies. Due to its specific particle composition, studying the association of HCV particles with lipoproteins remains an important goal towards the rational design of a protective vaccine

Le virus de l’hépatite Delta : Un agent pathogènequi retourne facilement sa veste ?

International audienceLe virus de l'hépatite Delta Un agent pathogène qui retourne facilement sa veste ? Anaïs Vignon, Solène Denolly, Jimena Perez-Vargas, François-Loïc Cosset > Le virus de l'hépatite Delta (VHD) a été découvert il y a 40 ans dans le foie de personnes infectées par le virus de l'hépatite B (VHB, de la famille des Hepadnaviridae). Parmi les 250 millions de personnes chroniquement infectées par VHB, 15 à 20 millions sont coinfectées par VHD [1]. La surinfection par VHD aggrave l'hépatite déjà causée par VHB, et augmente le risque de développer cirrhose et hépatocarcinome [2]. Du fait de son association avec VHB, VHD est connu en tant que virus « satellite » de VHB, ce qui signifie qu'il en est dépendant (Figure 1) [3]. La particule virale de VHD est formée d'un complexe ribonucléoprotéique constitué du génome viral à ARN associé aux deux formes structurelles de la protéine codée par VHD, S-HDAg et L-HDAg, et repose sur les glycoprotéines de surface de VHB pour l'assemblage des virions, leur enveloppement et leur transmission cellulaire (Figure 1A). Ainsi, pour que VHD puisse former sa particule virale et se disséminer, VHB doit être présent dans la même cellule. Lorsque VHB est absent, VHD reste capable de se répliquer et reste plusieurs semaines dans la cellule, mais il est incapable de se disséminer (Figure 1B) [2]. Cependant, l'idée que VHB est l'unique virus « assistant » utilisé par VHD a récemment été remise en cause. En effet, le génome et les protéines de VHD ont été détectés dans les glandes salivaires de patients atteints du syndrome de Sjögren (une maladie auto-immune systémique), alors que ni les séquences génomiques de VHB ni des anticorp

Daclatasvir Prevents Hepatitis C Virus Infectivity by Blocking Transfer of Viral Genome to Assembly Sites.

International audienceDaclatasvir is a direct-acting antiviral agent and potent inhibitor of NS5A, which is involved in replication of the hepatitis C virus (HCV) genome, presumably via membranous web shaping, and assembly of new virions, likely via transfer of the HCV RNA genome to viral particle assembly sites. Daclatasvir inhibits the formation of new membranous web structures and, ultimately, of replication complex vesicles, but also inhibits an early assembly step. We investigated the relationship between daclatasvir-induced clustering of HCV proteins, intracellular localization of viral RNAs and inhibition of viral particle assembly.HCVcc particles were produced from Huh7.5 hepatocarcinoma cells in presence of daclatasvir for short time periods. Infectivity as well as production of physical particles were quantified and producer cells were subjected to subcellular fractionation. Intracellular colocalization between core, E2, NS5A, NS4B proteins, and viral RNAs was quantitatively analyzed by confocal microscopy and by structured illumination microscopy.Short exposure of HCV-infected cells to daclatasvir reduced viral assembly and induced clustering of structural proteins with non-structural HCV proteins, including core, E2, NS4B and NS5A. These clustered structures appeared to be inactive assembly platforms, likely owing to loss of functional connection with replication complexes. Daclatasvir greatly reduced delivery of viral genomes to these core clusters without altering HCV RNA colocalization with NS5A. In contrast, daclatasvir neither induced clustered structures nor inhibited HCV assembly in cells infected with a daclatasvir-resistant mutant (NS5A-Y93H), indicating that daclatasvir targets a mutual, specific function of NS5A inhibiting both processes.In addition to inhibiting replication complex biogenesis, daclatasvir prevents viral assembly by blocking transfer of the viral genome to assembly sites. This leads to clustering of HCV proteins, because viral particles and replication complex vesicles cannot form and/or egress. This dual mode of action of daclatasvir could explain its efficacy in blocking HCV replication in cultured cells and in treatment of patients with HCV infection

Unlike for cellular mRNAs and other viral internal ribosome entry sites (IRESs), the eIF3 subunit e is not required for the translational activity of the HCV IRES

International audienceViruses depend on the host cell translation machinery for their replication, and one common strategy is the presence of internal ribosome entry sites (IRESs) in the viral RNAs, using different sets of host translation initiation factors. The hepatitis C virus (HCV) IRES binds eukaryotic translation initiation factor 3 (eIF3), but the exact functional role of the eIF3 complex and of its subunits remains to be precisely defined. Toward this goal, here we focused on eIF3 subunit e. We used an in vitro assay combining a ribosome-depleted rabbit reticulocyte lysate and ribosomes prepared from HeLa or Huh-7.5 cells transfected with either control or eIF3e siRNAs. eIF3e silencing reduced translation mediated by the 5'UTR of various cellular genes and HCV-like IRESs. However, this effect was not observed with the bona fide HCV IRES. Silencing of eIF3e reduced the intracellular levels of the c, d, and l subunits of eIF3 and their association with the eIF3 core subunit a. A pulldown analysis of eIF3 subunits associated with the HCV IRES disclosed similar effects and that the a subunit is critical for binding to the HCV IRES. Carrying out HCV infections of control and eIF3e-silenced Huh-7.5 cells, we found that in agreement with the in vitro findings, eIF3e silencing does not reduce HCV replication and viral protein expression. We conclude that unlike for host cellular mRNAs, the entire eIF3 is not required for HCV RNA translation, favoring viral expression under conditions of low eIF3e levels

The interplays between Crimean-Congo hemorrhagic fever virus (CCHFV) M segment-encoded accessory proteins and structural proteins promote virus assembly and infectivity

International audienceCrimean-Congo hemorrhagic fever virus (CCHFV) is a tick-borne orthonairovirus that has become a serious threat to the public health. CCHFV has a single-stranded, tripartite RNA genome composed of L, M, and S segments. Cleavage of the M polyprotein precursor generates the two envelope glycoproteins (GPs) as well as three secreted nonstructural proteins GP38 and GP85 or GP160, representing GP38 only or GP38 linked to a mucin-like protein (MLD), and a double-membrane-spanning protein called NSm. Here, we examined the relevance of each M-segment non-structural proteins in virus assembly, egress and infectivity using a well-established CCHFV virus-like-particle system (tc-VLP). Deletion of MLD protein had no impact on infectivity although it reduced by 60% incorporation of GPs into particles. Additional deletion of GP38 abolished production of infectious tc-VLPs. The loss of infectivity was associated with impaired Gc maturation and exclusion from the Golgi, showing that Gn is not sufficient to target CCHFV GPs to the site of assembly. Consistent with this, efficient complementation was achieved in cells expressing MLD-GP38 in trans with increased levels of preGc to Gc conversion, co-targeting to the Golgi, resulting in particle incorporation and restored infectivity. Contrastingly, a MLD-GP38 variant retained in the ER allowed preGc cleavage but failed to rescue miss-localization or infectivity. NSm deletion, conversely, did not affect trafficking of Gc but interfered with Gc processing, particle formation and secretion. NSm expression affected N-glycosylation of different viral proteins most likely due to increased speed of trafficking through the secretory pathway. This highlights a potential role of NSm in overcoming Golgi retention and facilitating CCHFV egress. Thus, deletions of GP38 or NSm demonstrate their important role on CCHFV particle production and infectivity. GP85 is an essential viral factor for preGc cleavage, trafficking and Gc incorporation into particles, whereas NSm protein is involved in CCHFV assembly and virion secretion

The SARS-CoV-2 Envelope and Membrane proteins modulate maturation and retention of the Spike protein, allowing optimal formation of VLPs in presence of Nucleoprotein

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. Like for other coronaviruses, its particles are composed of four structural proteins, namely Spike S, Envelope E, Membrane M and Nucleoprotein N proteins. The involvement of each of these proteins and their interplays during the assembly process of this new virus are poorly-defined and are likely β-coronavirus-type different. Therefore, we sought to investigate how SARS-CoV-2 behaves for its assembly by expression assays of S, in combination with E, M and/or N. By combining biochemical and imaging assays, we showed that E and M regulate intracellular trafficking of S and hence its furin-mediated processing. Indeed, our imaging data revealed that S remains at ERGIC or Golgi compartments upon expression of E or M, like for SARS-CoV-2 infected cells. By studying a mutant of S, we showed that its cytoplasmic tail, and more specifically, its C-terminal retrieval motif, is required for the M-mediated retention in the ERGIC, whereas E induces S retention by modulating the cell secretory pathway. We also highlighted that E and M induce a specific maturation of S N-glycosylation, which is observed on particles and lysates from infected cells independently of its mechanisms of intracellular retention. Finally, we showed that both M, E and N are required for optimal production of virus-like-proteins. Altogether, our results indicated that E and M proteins influence the properties of S proteins to promote assembly of viral particles. Our results therefore highlight both similarities and dissimilarities in these events, as compared to other β-coronaviruses