Ion-channel function and cross-species determinants in viral assembly of nonprimate hepacivirus p7

Nonprimate hepacivirus (NPHV), the closest homolog of hepatitis C virus (HCV) described to date, has recently been discovered in horses. Even though the two viruses share a similar genomic organization, conservation of the encoded hepaciviral proteins remains undetermined. The HCV p7 protein is localized within endoplasmic reticulum (ER) membranes and is important for the production of infectious particles. In this study, we analyzed the structural and functional features of NPHV p7 in addition to its role during virus assembly. Three-dimensional homology models for NPHV p7 using various nuclear magnetic resonance spectroscopy (NMR) structures were generated, highlighting the conserved residues important for ion channel function. By applying a liposome permeability assay, we observed that NPHV p7 exhibited liposome permeability features similar to those of HCV p7, indicative of similar ion channel activity. Next, we characterized the viral protein using a p7-based trans-complementation approach. A similar subcellular localization pattern at the ER membrane was observed, although production of infectious particles was likely hindered by genetic incompatibilities with HCV proteins. To further characterize these cross-species constraints, chimeric viruses were constructed by substituting different regions of HCV p7 with NPHV p7. The N terminus and transmembrane domains were nonexchangeable and therefore constitute a cross-species barrier in hepaciviral assembly. In contrast, the basic loop and the C terminus of NPHV p7 were readily exchangeable, allowing production of infectious trans-complemented viral particles. In conclusion, comparison of NPHV and HCV p7 revealed structural and functional homology of these proteins, including liposome permeability, and broadly acting determinants that modulate hepaciviral virion assembly and contribute to the host-species barrier were identified

PicPreview and PicSummary: Two Timesaving Plugins for the Fluorescence Microscopist

This article targets cell biologists who use fluorescence microscopy but lack automatic tools to summarize and manage their image datasets. When using microscopy to document a phenotype, multiple and random pictures are required to reflect the biological diversity of each imaged sample. Managing, integrating and summarizing the acquired data can be a daunting task that becomes extremely time-consuming unless one automatizes it. Unfortunately, if many biologists use microscopy, only a few have automatized procedures to cope with the data generated. For the majority of microscope users, the two developed complementary ImageJ plugins, PicPreview and PicSummary, will allow, in a few clicks and in an instant, to obtain an overview of all pictures taken for each sample of an experiment and a summary with one user-defined representative picture per sample. The plugins and a video tutorial, as well as demonstration pictures, are available as supplementary data at the journal website. PicPreview and PicSummary should save precious time in managing microscopy datasets and in preparing figures for publications

Entry and replication of recombinant hepatitis C viruses in cell culture.

Hepatitis C virus (HCV) is a positive-strand enveloped RNA virus and belongs to the Flaviviridae family. The heavy health burden associated with the virus infection in humans and the intriguing peculiarities of the interaction between the HCV replication cycle and the hepatocyte host cell have stimulated a flourishing research field. The present review aims at recapitulating the different viral and cellular systems modelling HCV entry and replication, and in particular at gathering the tools available to dissect the HCV entry pathway

HCV Pit Stop at the Lipid Droplet: Refuel Lipids and Put on a Lipoprotein Coat before Exit

The replication cycle of the liver-tropic hepatitis C virus (HCV) is tightly connected to the host lipid metabolism, during the virus entry, replication, assembly and egress stages, but also while the virus circulates in the bloodstream. This interplay coins viral particle properties, governs viral cell tropism, and facilitates immune evasion. This review summarizes our knowledge of these interactions focusing on the late steps of the virus replication cycle. It builds on our understanding of the cell biology of lipid droplets and the biosynthesis of liver lipoproteins and attempts to explain how HCV hijacks these organelles and pathways to assemble its lipo-viro-particles. In particular, this review describes (i) the mechanisms of viral protein translocation to and from the lipid droplet surface and the orchestration of an interface between replication and assembly complexes, (ii) the importance of the triglyceride mobilization from the lipid droplets for HCV assembly, (iii) the interplay between HCV and the lipoprotein synthesis pathway including the role played by apolipoproteins in virion assembly, and finally (iv) the consequences of these complex virus–host interactions on the virion composition and its biophysical properties. The wealth of data accumulated in the past years on the role of the lipid metabolism in HCV assembly and its imprint on the virion properties will guide vaccine design efforts and reinforce our understanding of the hepatic lipid metabolism in health and disease

The Railmap of Type I Interferon Induction: Subcellular Network Plan and How Viruses Can Change Tracks

The innate immune response constitutes the cell’s first line of defense against viruses and culminates in the expression of type I interferon (IFN) and IFN-stimulated genes, inducing an antiviral state in infected and neighboring cells. Efficient signal transduction is a key factor for strong but controlled type I IFN expression and depends on the compartmentalization of different steps of the signaling cascade and dynamic events between the involved compartments or organelles. This compartmentalization of the innate immune players not only relies on their association with membranous organelles but also includes the formation of supramolecular organizing centers (SMOCs) and effector concentration by liquid–liquid phase separation. For their successful replication, viruses need to evade innate defenses and evolve a multitude of strategies to impair type I IFN induction, one of which is the disruption of spatial immune signaling dynamics. This review focuses on the role of compartmentalization in ensuring an adequate innate immune response to viral pathogens, drawing attention to crucial translocation events occurring downstream of pattern recognition and leading to the expression of type I IFN. Furthermore, it intends to highlight concise examples of viral countermeasures interfering with this spatial organization to alleviate the innate immune response

Incorporation of Hepatitis C Virus E1 and E2 Glycoproteins: The Keystones on a Peculiar Virion

Hepatitis C virus (HCV) encodes two envelope glycoproteins, E1 and E2. Their structure and mode of fusion remain unknown, and so does the virion architecture. The organization of the HCV envelope shell in particular is subject to discussion as it incorporates or associates with host-derived lipoproteins, to an extent that the biophysical properties of the virion resemble more very-low-density lipoproteins than of any virus known so far. The recent development of novel cell culture systems for HCV has provided new insights on the assembly of this atypical viral particle. Hence, the extensive E1E2 characterization accomplished for the last two decades in heterologous expression systems can now be brought into the context of a productive HCV infection. This review describes the biogenesis and maturation of HCV envelope glycoproteins, as well as the interplay between viral and host factors required for their incorporation in the viral envelope, in a way that allows efficient entry into target cells and evasion of the host immune response

Biochemical and functional characterisation of virion-associated Hepatitis C virus envelope glycoproteins

Hepatitis C is a major global health burden with unsatisfactory treatments and no vaccine available. It is caused by a small positive-strand RNA enveloped virus, namely hepatitis C virus (HCV). HCV encodes 10 proteins, including three structural proteins, the capsid protein (Core) and two envelope proteins (E1 and E2), the focus of this thesis. My thesis first concentrated on E2 hypervariable region 1 (HVR1). HVR1 constitutes an immunological decoy as it elicits isolate-specific antibodies but continuously escapes their neutralisation by adapting its aminoacid sequence. Using a set of new anti-HVR1 antibodies, we found that the C-terminus of HVR1 carries virus neutralising determinants and defined key residues in their epitope. Furthermore, anti-HVR1 antibodies did not inhibit E2 interaction with CD81 or heparin but still inhibited cell-cultured HCV (HCVcc) infection after virus attachment had occurred, confirming a role for HVR1 in the active steps of entry. In addition, we developed a new method to synchronise HCV infection, based on magnetic adsorption. Briefly, HCV was combined with magnetic beads and pulled down onto the cell surface under a magnetic field. This proved to be both very simple and highly efficient as HCV particles were found to be adsorbed in only two minutes onto the cell surface, more efficiently than after a standard, on-synchronised infection. This new method is amenable to study early steps of virus infection, while optimising the infection efficiency. Lastly, the characteristics of HCV glycoproteins in the HCVcc system were analysed. Importantly, the secreted envelope glycoproteins were mostly associated with infectious virions, despite the low specific infectivity inherent to the HCVcc system. Importantly, while intracellular glycoproteins were competent for interaction with entry factors (glycosaminoglycans and CD81) they were found to undergo drastic changes during virion morphogenesis and secretion. Indeed, some E2 glycans were modified and most importantly, virion-associated glycoproteins formed high molecular mass covalent complexes that retained a functional conformation. This study is the first one to describe envelope glycoproteins incorporated onto infectious HCV particle and uncovers unexpected features in HCV assembly. Altogether, this work permitted to delineate more precisely a neutralising epitope in E2 HVR1 and its function in entry. It also gave further insights into the incorporation of HCV glycoproteins in the virion envelope. Together, these results will contribute significantly to future studies aimed at understanding the processes involved in HCV assembly and entry.LILLE2-BU Santé-Recherche (593502101) / SudocSudocFranceF

The ATGL lipase cooperates with ABHD5 to mobilize lipids for hepatitis C virus assembly.

Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/β hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis

Characterization of the Envelope Glycoproteins Associated with Infectious Hepatitis C Virus ▿

Hepatitis C is caused by an enveloped virus whose entry is mediated by two glycoproteins, namely, E1 and E2, which have been shown to assemble as a noncovalent heterodimer. Despite extensive research in the field of such an important human pathogen, hepatitis C virus (HCV) glycoproteins have only been studied so far in heterologous expression systems, and their organization at the surfaces of infectious virions has not yet been described. Here, we characterized the envelope glycoproteins associated with cell-cultured infectious virions and compared them with their prebudding counterparts. Viral particles were analyzed by ultracentrifugation, and the envelope glycoproteins were characterized by coimmunoprecipitation and receptor pulldown assays. Furthermore, their oligomeric state was determined by sedimentation through sucrose gradients and by separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. In sucrose gradient analyses, HCV envelope glycoproteins were associated with fractions containing the most infectious viral particles. Importantly, besides maturation of some of their glycans, HCV envelope glycoproteins showed a dramatic change in their oligomeric state after incorporation into the viral particle. Indeed, virion-associated E1 and E2 envelope glycoproteins formed large covalent complexes stabilized by disulfide bridges, whereas the intracellular forms of these proteins assembled as noncovalent heterodimers. Furthermore, the virion-associated glycoprotein complexes were recognized by the large extracellular loop of CD81 as well as conformation-sensitive antibodies, indicating that these proteins are in a functional conformation. Overall, our study fills a gap in the description of HCV outer morphology and should guide further investigations into virus entry and assembly