Ultrastructural and biochemical analyses of hepatitis C virus-associated host cell membranes.

International audienceLike most other positive-strand RNA viruses, hepatitis C virus (HCV) induces changes in the host cell's membranes, resulting in a membranous web. The non-structural proteins of the viral replication complex are thought to be associated with these newly synthesized membranes. We studied this phenomenon, using a Huh7.5 cell clone displaying high levels of replication of a subgenomic replicon of the JFH-1 strain. Electron microscopy of ultrathin sections of these cells revealed the presence of numerous double membrane vesicles (DMVs), resembling those observed for other RNA viruses such as poliovirus and coronavirus. Some sections had more discrete multivesicular units consisting of circular concentric membranes organized into clusters surrounded by a wrapping membrane. These structures were highly specific to HCV as they were not detected in naive Huh7.5 cells. Preparations enriched in these structures were separated from other endoplasmic reticulum-derived membranes by cell cytoplasm homogenization and ultracentrifugation on a sucrose gradient. They were found to contain the non-structural NS3 and NS5A HCV proteins, HCV RNA and LC3-II, a specific marker of autophagic membranes. By analogy to other viral models, HCV may induce DMVs by activating the autophagy pathway. This could represent a strategy to conceal the viral RNA and help the virus to evade double-stranded RNA-triggered host antiviral responses. More detailed characterization of these virus-cell interactions may facilitate the development of new treatments active against HCV and other RNA viruses that are dependent on newly synthesized cellular membranes for replication

Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy.

The original publication is available at www.springerlink.comInternational audienceThe mechanisms underlying hepatitis C virus (HCV) morphogenesis remain elusive, but lipid droplets have recently been shown to be important organelles for virus production. We investigated the interaction between HCV-like particles and lipid droplets by three-dimensional reconstructions of serial ultrathin electron microscopy sections of cells producing the HCV core protein. The budding of HCV-like particles was mostly initiated at membranes close to the lipid droplets rather than at membranes directly apposed to the lipid droplets. This may have important implications for our understanding of the complex relationship between HCV and lipids and may make easier to dissect out the HCV life cycle

Sequential biogenesis of host cell membrane rearrangements induced by hepatitis C virus infection.: HCV-induced membrane rearrangements

International audienceLike most positive-strand RNA viruses, hepatitis C virus (HCV) forms a membrane-associated replication complex consisting of replicating RNA, viral and host proteins anchored to altered cell membranes. We used a combination of qualitative and quantitative electron microscopy (EM), immuno-EM, and the 3D reconstruction of serial EM sections to analyze the host cell membrane alterations induced by HCV. Three different types of membrane alteration were observed: vesicles in clusters (ViCs), contiguous vesicles (CVs), and double-membrane vesicles (DMVs). The main ultrastructural change observed early in infection was the formation of a network of CVs surrounding the lipid droplets. Later stages in the infectious cycle were characterized by a large increase in the number of DMVs, which may be derived from the CVs. These DMVs are thought to constitute the membranous structures harboring the viral replication complexes in which viral replication is firmly and permanently established and to protect the virus against double-stranded RNA-triggered host antiviral responses

Ultrastructural and quantitative analysis of the lipid droplet clustering induced by hepatitis C virus core protein.: HCV-induced lipid droplet clustering

International audienceHepatitis C virus (HCV) release is linked to the formation of lipid droplet (LD) clusters in the perinuclear area of infected cells, induced by the core protein. We used electron microscopy (EM) to monitor and compare the number and size of LD in cells producing the mature and immature forms of the HCV core protein, and 3D EM to reconstruct whole cells producing the mature core protein. Only the mature protein coated the LD and induced their clustering and emergence from endoplasmic reticulum membranes enriched in this protein. We found no particular association between LD clusters and the centrosome in reconstructed cells. The LD clustering induced by the mature core protein was associated with an increase in LD synthesis potentially due, at least in part, to the ability of this protein to coat the LD. These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis

The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model.: HCV genotype 3-specific steatosis

International audienceBackground and aims: The prevalence and severity of liver steatosis are higher in patients infected with genotype 3 hepatitis C virus (HCV) than in patients infected with other genotypes. HCV core protein is known to affect lipid metabolism, inducing lipid droplet accumulation both in vitro and in vivo. We used an in vitro cellular model to investigate whether an HCV core protein with residues specific to genotype 3 increased this phenomenon. Methods: Sequence comparisons for HCV core protein domain II, which is known to interact with lipid droplets, identified the phenylalanine (F) residue at position 164 as the only residue specific to genotype 3. We compared the area covered by lipid droplets in sections of cells producing a wild-type genotype 1a HCV core protein with that in cells producing a Y164F mutant protein. Results: Cumulative lipid droplet area was significantly greater in sections of cells producing the Y164F mutant HCV core protein than in cells producing the wild-type protein (p<0.001). The frequency of cell sections containing more than 3 μm2 of lipid droplets, in particular, was higher for the mutant than for the wild-type protein. Conclusion: Our data provide a molecular explanation for HCV genotype 3-specific lipid accumulation. This difference between genotypes may be due to phenylalanine having a higher affinity for lipids than tyrosine (Y). These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis

LIF-Dependent Signaling: New Pieces in the Lego

LIF, a member of the IL6 family of cytokine, displays pleiotropic effects on various cell types and organs. Its critical role in stem cell models (e.g.: murine ES, human mesenchymal cells) and its essential non redundant function during the implantation process of embryos, in eutherian mammals, put this cytokine at the core of many studies aiming to understand its mechanisms of action, which could benefit to medical applications. In addition, its conservation upon evolution raised the challenging question concerning the function of LIF in species in which there is no implantation. We present the recent knowledge about the established and potential functions of LIF in different stem cell models, (embryonic, hematopoietic, mesenchymal, muscle, neural stem cells and iPSC). We will also discuss EVO-DEVO aspects of this multifaceted cytokine

Clinical spectrum of MTOR-related hypomelanosis of Ito with neurodevelopmental abnormalities.

PURPOSE: Hypomelanosis of Ito (HI) is a skin marker of somatic mosaicism. Mosaic MTOR pathogenic variants have been reported in HI with brain overgrowth. We sought to delineate further the pigmentary skin phenotype and clinical spectrum of neurodevelopmental manifestations of MTOR-related HI. METHODS: From two cohorts totaling 71 patients with pigmentary mosaicism, we identified 14 patients with Blaschko-linear and one with flag-like pigmentation abnormalities, psychomotor impairment or seizures, and a postzygotic MTOR variant in skin. Patient records, including brain magnetic resonance image (MRI) were reviewed. Immunostaining (n = 3) for melanocyte markers and ultrastructural studies (n = 2) were performed on skin biopsies. RESULTS: MTOR variants were present in skin, but absent from blood in half of cases. In a patient (p.[Glu2419Lys] variant), phosphorylation of p70S6K was constitutively increased. In hypopigmented skin of two patients, we found a decrease in stage 4 melanosomes in melanocytes and keratinocytes. Most patients (80%) had macrocephaly or (hemi)megalencephaly on MRI. CONCLUSION: MTOR-related HI is a recognizable neurocutaneous phenotype of patterned dyspigmentation, epilepsy, intellectual deficiency, and brain overgrowth, and a distinct subtype of hypomelanosis related to somatic mosaicism. Hypopigmentation may be due to a defect in melanogenesis, through mTORC1 activation, similar to hypochromic patches in tuberous sclerosis complex