Differentiated Cells in Prolonged Hypoxia Produce Highly Infectious Native-Like Hepatitis C Virus Particles.

BACKGROUND AND AIMS: Standard hepatitis C virus (HCV) cell-culture models present an altered lipid metabolism and thus produce lipid-poor lipoviral particles (LVPs). These models are thereby weakly adapted to explore the complete natural viral life cycle. APPROACH AND RESULTS: To overcome these limitations, we used an HCV cell-culture model based on both cellular differentiation and sustained hypoxia to better mimic the host-cell environment. The long-term exposure of Huh7.5 cells to DMSO and hypoxia (1% O2 ) significantly enhanced the expression of major differentiation markers and the cellular hypoxia adaptive response by contrast with undifferentiated and normoxic (21% O2 ) standard conditions. Because hepatocyte-like differentiation and hypoxia are key regulators of intracellular lipid metabolism, we characterized the distribution of lipid droplets (LDs) and demonstrated that experimental cells significantly accumulate larger and more numerous LDs relative to standard cell-culture conditions. An immunocapture (IC) and transmission electron microscopy (TEM) method showed that differentiated and hypoxic Huh7.5 cells produced lipoproteins significantly larger than those produced by standard Huh7.5 cell cultures. The experimental cell culture model is permissive to HCV-Japanese fulminant hepatitis (JFH1) infection and produces very-low-buoyant-density LVPs that are 6-fold more infectious than LVPs formed by standard JFH1-infected Huh7.5 cells. Finally, the IC-TEM approach and antibody-neutralization experiments revealed that LVPs were highly lipidated, had a global ultrastructure and a conformation of the envelope glycoprotein complex E1E2 close to that of the ones circulating in infected individuals. CONCLUSIONS: This relevant HCV cell culture model thus mimics the complete native intracellular HCV life cycle and, by extension, can be proposed as a model of choice for studies of other hepatotropic viruses.

The envelope protein of Zika virus interacts with apolipoprotein E early in the infectious cycle and this interaction is conserved on the secreted viral particles.

BACKGROUND: Zika virus (ZIKV), a member of the Flaviviridae family, has caused massive outbreaks of infection in tropical areas over the last decade and has now begun spreading to temperate countries. Little is currently known about the specific host factors involved in the intracellular life cycle of ZIKV. Flaviviridae viruses interact closely with host-cell lipid metabolism and associated secretory pathways. Another Flaviviridae, hepatitis C virus, is highly dependent on apolipoprotein E (ApoE) for the completion of its infectious cycle. We therefore investigated whether ZIKV also interacted with this protein. METHODS: ZIKV infections were performed on both liver and microglia derived cell lines in order to proceed to colocalization analysis and immunoprecipitation assays of ApoE and Zika envelope glycoprotein (Zika E). Transmission electron microscopy combined to immunogold labeling was also performed on the infected cells and related supernatant to study the association of ApoE and Zika E protein in the virus-induced membrane rearrangements and secreted particles, respectively. Finally, the potential of neutralization of anti-ApoE antibodies on ZIKV particles was studied. RESULT: We demonstrated an interaction between ApoE and the Zika E protein. This specific interaction was observed in virus-induced host-cell membrane rearrangements, but also on newly formed intracellular particles. The partial neutralizing effect of anti-ApoE antibody and the immunogold labeling of the two proteins on secreted virions indicates that this interaction is conserved during ZIKV intracellular trafficking and release. CONCLUSIONS: These data suggest that another member of the Flaviviridae also interacts with ApoE, indicating that this could be a common mechanism for the viruses from this family.

Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity.

OBJECTIVE: Infection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene expression. Given that miRNAs were indicated to regulate between 30% and 75% of all human genes, we aimed to investigate the functional and regulatory role of miRNAs for the HCV life cycle. DESIGN: To systematically reveal human miRNAs affecting the HCV life cycle, we performed a two-step functional high-throughput miRNA mimic screen in Huh7.5.1 cells infected with recombinant cell culture-derived HCV. miRNA targeting was then assessed using a combination of computational and functional approaches. RESULTS: We uncovered miR-501-3p and miR-619-3p as novel modulators of HCV assembly/release. We discovered that these miRNAs regulate O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) protein expression and identified OGT and O-GlcNAcylation as regulators of HCV morphogenesis and infectivity. Furthermore, increased OGT expression in patient-derived liver tissue was associated with HCV-induced liver disease and cancer. CONCLUSION: miR-501-3p and miR-619-3p and their target OGT are previously undiscovered regulatory host factors for HCV assembly and infectivity. In addition to its effect on HCV morphogenesis, OGT may play a role in HCV-induced liver disease and hepatocarcinogenesis.

Hepatitis E Virus Lifecycle and Identification of 3 Forms of the ORF2 Capsid Protein.

BACKGROUND & AIMS: Hepatitis E virus (HEV) infection is a major cause of acute hepatitis worldwide. Approximately 2 billion people live in areas endemic for HEV and are at risk of infection. The HEV genome encodes 3 proteins, including the ORF2 capsid protein. Detailed analyses of the HEV life cycle has been hampered by the lack of an efficient viral culture system. METHODS: We performed studies with gt3 HEV cell culture-produced particles and patient blood and stool samples. Samples were fractionated on iodixanol gradients and cushions. Infectivity assays were performed in vitro and in human liver chimeric mice. Proteins were analyzed by biochemical and proteomic approaches. Infectious particles were analyzed by transmission electron microscopy. HEV antigen levels were measured with the Wantaï enzyme-linked immunosorbent assay. RESULTS: We developed an efficient cell culture system and isolated HEV particles that were infectious in vitro and in vivo. Using transmission electron microscopy, we defined the ultrastructure of HEV cell culture-produced particles and particles from patient sera and stool samples. We also identified the precise sequence of the infectious particle-associated ORF2 capsid protein. In cultured cells and in samples from patients, HEV produced 3 forms of the ORF2 capsid protein: infectious/intracellular ORF2 (ORF2i), glycosylated ORF2 (ORF2g), and cleaved ORF2 (ORF2c). The ORF2i protein associated with infectious particles, whereas the ORF2g and ORF2c proteins were massively secreted glycoproteins not associated with infectious particles. ORF2g and ORF2c were the most abundant antigens detected in sera from patients. CONCLUSIONS: We developed a cell culture system and characterized HEV particles; we identified 3 ORF2 capsid proteins (ORF2i, ORF2g, and ORFc). These findings will advance our understanding of the HEV life cycle and improve diagnosis.

Ultrastructural organisation of HCV from the bloodstream of infected patients revealed by electron microscopy after specific immunocapture.

OBJECTIVE: HCV particles are associated with very low-density lipoprotein components in chronically infected patients. These hybrid particles, or 'lipo-viro particles' (LVPs), are rich in triglycerides, and contain the viral RNA, the capsid protein, E1E2 envelope glycoproteins and apolipoproteins B and E. However, their specific ultrastructural organisation has yet to be determined. We developed a strategy for the preparation of any viral sample that preserves the native structure of the LVPs, facilitating their precise morphological characterisation. DESIGN: Using a strategy based on the direct specific immunocapture of particles on transmission electron microscopy (TEM) grids, we characterised the precise morphology of the viral particle by TEM. RESULTS: The LVP consists of a broad nucleocapsid surrounding an electron-dense centre, presumably containing the HCV genome. The nucleocapsid is surrounded by an irregular, detergent-sensitive crescent probably composed of lipids. Lipid content may determine particle size. These particles carry HCV E1E2, ApoB and ApoE, as shown in our immuno-EM analysis. Our results also suggest that these putative LVPs circulate in the serum of patients as part of a mixed population, including lipoprotein-like particles and complete viral particles. CONCLUSIONS: Twenty-five years after the discovery of HCV, this study finally provides information about the precise morphological organisation of viral particles. It is truly remarkable that our TEM images fully confirm the ultrastructure of LVPs predicted by several authors, almost exclusively from the results of molecular biology studies.

The association of hepatitis C virus glycoproteins with apolipoproteins E and B early in assembly is conserved in lipoviral particles.

In patients chronically infected with hepatitis C virus and in the HCV cell culture system (HCVcc), it is known that highly infectious virus particles have low to very low buoyant densities. These low densities have been attributed to the association of HCV with lipoprotein components, which occur during the viral morphogenesis. The resulting hybrid particles are known as lipoviral particles (LVP); however, very little is known about how these particles are created. In our study, we used Huh7.5 cells to investigate the intracellular association between envelope proteins and apolipoproteins B and E (ApoB and ApoE, respectively). In particular, we were interested in the role of this association in initiating LVP morphogenesis. Co-immunoprecipitation assays revealed that ApoB, ApoE, and HCV glycoproteins formed a protein complex early in the HCV lifecycle. Confocal analyses of naïve, E1E2-transduced and HCVcc-infected cells showed that HCV glycoproteins, ApoB and ApoE were found strongly colocalized only in the endoplasmic reticulum. We also found that HCV glycoproteins, ApoB and ApoE were already associated with intracellular infectious viral particles and, furthermore, that the protein complex was conserved in the infectious viral particles present in the supernatant of infected Huh7.5 cells. The association of HCV glycoproteins with ApoE was also evidenced in the HCVpp system, using the non-hepatic HEK293T cell line. We suggest that the complex formed by HCV E1E2, ApoB, and ApoE may initiate lipoviral particle morphogenesis.

The development of children's inhibition: does parenting matter?

Whereas a large body of research has investigated the maturation of inhibition in relation to the prefrontal cortex, far less research has been devoted to environmental factors that could contribute to inhibition improvement. The aim of the current study was to test whether and to what extent parenting matters for inhibition development from 2 to 8years of age. Data were collected from 421 families, with 348 mother-child dyads and 342 father-child dyads participating. Children's inhibition capacities and parenting behaviors were assessed in a three-wave longitudinal data collection. The main analyses examined the impact of parenting on the development of children's inhibition capacities. They were conducted using a multilevel modeling (MLM) framework. The results lead to the conclusion that both mothers and fathers contribute through their child-rearing behavior to their children's executive functioning, even when controlling for age-related improvement (maturation) and important covariates such as gender, verbal IQ, and place of enrollment. More significant relations between children's inhibition development and parenting were displayed for mothers than for fathers. More precisely, parenting behaviors that involve higher monitoring, lower discipline, inconsistency and negative controlling, and a positive parenting style are associated with good development of inhibition capacities in children.

Multilevel mediation: cumulative contextual risk, maternal differential treatment, and children's behavior within families.

This study tests the hypothesis that links between contextual risk and children's outcomes are partially explained by differential parenting. Using multi-informant measurement and including up to four children per family (Mage  = 3.51, SD = 2.38) in a sample of 397 families, indirect effects (through maternal differential parenting: self-reported and observed) of cumulative contextual risk on four child outcomes were investigated. Cumulative risk was associated with higher levels of differential parenting and, in turn, with higher levels of behavioral problems. Indirect effects were strongest for attentional and social problems but also evident for aggression. The link between differential parenting and outcome was moderated by favoritism, but this was only evident for maternal report and strongest for aggression.

Vaccine-induced cross-genotype reactive neutralizing antibodies against hepatitis C virus.

We detected cross-reactive neutralizing antibodies (NtAb) against hepatitis C virus (HCV) in chimpanzees vaccinated with HCV-1 (genotype 1a) recombinant E1/E2 envelope glycoproteins. Five vaccinated chimpanzees, protected following HCV-1 challenge, were initially studied using the heterologous H77 (genotype 1a) HCVpp assay. All animals had developed NtAb after the second vaccination; 4 animals had reciprocal titers of ≥200 at the time of challenge. Using genotypes 1a-6a HCV pseudoparticles (HCVpp) and cell culture-derived HCV (HCVcc) assays, cross-reactive NtAb were detected against 1a, 4a, 5a, and 6a, with limited reactivity against 2a and 3a. Our study provides encouragement for the development of a recombinant envelope-based vaccine against hepatitis C.

Neutralizing antibodies to hepatitis C virus in perinatally infected children followed up prospectively.

Little is known about the presence and role of neutralizing antibodies (NtAbs) in perinatal hepatitis C virus (HCV) infection. Using HCV pseudoparticles, NtAbs were studied longitudinally in 12 HCV-infected children with or without evidence of acute hepatitis during the first year of life. Broadly reactive NtAbs of maternal origin did not prevent vertical HCV transmission or progression to chronicity. NtAbs against homologous genotype or subtype appeared during the chronic phase and were more abundant and sustained in children with acute hepatitis. Cross-reactive NtAbs were present in both groups of children, but their appearance did not correlate with better control of viremia or HCV clearance.

Advantages of a single-cycle production assay to study cell culture-adaptive mutations of hepatitis C virus.

The JFH1 strain of hepatitis C virus (HCV) is unique among HCV isolates, in that the wild-type virus can traverse the entire replication cycle in cultured cells. However, without adaptive mutations, only low levels of infectious virus are produced. In the present study, the effects of five mutations that were selected during serial passage in Huh-7.5 cells were studied. Recombinant genomes containing all five mutations produced 3-4 logs more infectious virions than did wild type. Neither a coding mutation in NS5A nor a silent mutation in E2 was adaptive, whereas coding mutations in E2, p7, and NS2 all increased virus production. A single-cycle replication assay in CD81-deficient cells was developed to study more precisely the effect of the adaptive mutations. The E2 mutation had minimal effect on the amount of infectious virus released but probably enhanced entry into cells. In contrast, both the p7 and NS2 mutations independently increased the amount of virus released.

Apolipoprotein c1 association with hepatitis C virus.

Accumulating evidence suggests that cellular lipoprotein components are involved in hepatitis C virus (HCV) morphogenesis, but the precise contribution of these components remains unclear. We investigated the involvement of apolipoprotein C1 (ApoC1) in HCV infection in the HCV pseudotyped particle system (HCVpp), in the recently developed cell culture infection model (HCVcc), and in authentic HCV isolated from viremic chimpanzees. Viral genomes associated with HCVcc or authentic HCV were efficiently immunoprecipitated by anti-ApoC1, demonstrating that ApoC1 was a normal component of HCV. The infectivities of HCVpp that had been mixed with ApoC1 and, more importantly, untreated HCVcc collected from lysates or media of infected Huh7.5 cells were directly neutralized by anti-ApoC1. Indeed, convalescent anti-HCV immunoglobulin G and anti-ApoC1 each neutralized over 75% of infectious HCVcc particles, indicating that many, if not all, infectious particles were recognized by both antibodies. Moreover, peptides corresponding to the C-terminal region of ApoC1 blocked infectivity of both HCVpp and HCVcc. Altogether, these results suggest that ApoC1 associates intracellularly via its C-terminal region with surface components of virions during viral morphogenesis and may play a major role in the replication cycle of HCV.

Previously infected chimpanzees are not consistently protected against reinfection or persistent infection after reexposure to the identical hepatitis C virus strain.

Protective immunity after resolved hepatitis C virus (HCV) infection has been reported. However, the breadth of this immunity has remained controversial, and the role of neutralizing antibodies has not been well-defined. In the present study, two chimpanzees (CH96A008 and CH1494) with resolved monoclonal H77C (genotype 1a) infection were rechallenged with low-dose homologous H77C virus about 12 months after viral clearance; CH96A008 became persistently infected, and CH1494 had transient viremia lasting 2 weeks. CH1494 was subsequently either partially or completely protected following five homologous rechallenges with monoclonal H77C or polyclonal H77 and after six heterologous rechallenges with HC-J4 (genotype 1b) or HC-J6 (genotype 2a) viruses. Subsequently, a final challenge with H77C resulted in persistent HCV infection. In both chimpanzees, serum neutralizing antibodies against retroviral pseudoparticles bearing the H77C envelope proteins were not detected during the initial infection or during rechallenge. However, anamnestic cellular immune responses developed during the initial homologous rechallenge, in particular in CH96A008, which developed a persistent infection. Polyprotein sequences of viruses recovered from CH1494 after the two homologous rechallenges that resulted in transient viremia were identical with the H77C virus. In contrast, the polyprotein sequences of viruses recovered from both chimpanzees after homologous rechallenge resulting in persistent infection had numerous changes. These findings have important implications for our understanding of immunity against HCV; even in the best-case scenario with autologous rechallenge, low-level viral persistence was seen in the presence of primed T-cell responses.

Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus.

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a. Results from this study suggest that these MAbs may have the potential to prevent HCV infection.

Polyclonal immunoglobulins from a chronic hepatitis C virus patient protect human liver-chimeric mice from infection with a homologous hepatitis C virus strain.

UNLABELLED: The role of the humoral immune response in the natural course of hepatitis C virus (HCV) infection is widely debated. Most chronically infected patients have immunoglobulin G (IgG) antibodies capable of neutralizing HCV pseudoparticles (HCVpp) in vitro. It is, however, not clear whether these IgG can prevent a de novo HCV infection in vivo and contribute to the control of viremia in infected individuals. We addressed this question with homologous in vivo protection studies in human liver-urokinase-type plasminogen activator (uPA)(+/+) severe combined immune deficient (SCID) mice. Chimeric mice were loaded with chronic phase polyclonal IgG and challenged 3 days later with a 100% infectious dose of the acute phase H77C virus, both originating from patient H. Passive immunization induced sterilizing immunity in five of eight challenged animals. In the three nonprotected animals, the HCV infection was attenuated, as evidenced by altered viral kinetics in comparison with five control IgG-treated animals. Plasma samples obtained from the mice at viral challenge neutralized H77C-HCVpp at dilutions as high as 1/400. Infection was completely prevented when, before administration to naïve chimeric mice, the inoculum was pre-incubated in vitro at an IgG concentration normally observed in humans. CONCLUSION: Polyclonal IgG from a patient with a long-standing HCV infection not only displays neutralizing activity in vitro using the HCVpp system, but also conveys sterilizing immunity toward the ancestral HCV strain in vivo, using the human liver-chimeric mouse model. Both experimental systems will be useful tools to identify neutralizing antibodies for future clinical use.

Validation of the preschool and primary school form of a questionnaire assessing parents' childrearing behavior.

This study presents a validation of a scale that assesses parents' childrearing behavior toward young children. The scale was validated on 565 parents of 2- to 7-year-old children. The current results replicated the factor solution of the original scale designed for parents of school-aged children. The scale demonstrated good psychometric properties: moderate to high internal consistency, the expected relations with criterion variables (parental self-efficacy beliefs, child's behavior and personality), and discriminative properties according to the parents' gender and educational level, the child's age and gender, and the difference between referred and nonreferred children.

Endoplasmic Reticulum Detergent-Resistant Membranes Accommodate Hepatitis C Virus Proteins for Viral Assembly.

During Hepatitis C virus (HCV) morphogenesis, the non-structural protein 2 (NS2) brings the envelope proteins 1 and 2 (E1, E2), NS3, and NS5A together to form a complex at the endoplasmic reticulum (ER) membrane, initiating HCV assembly. The nature of the interactions in this complex is unclear, but replication complex and structural proteins have been shown to be associated with cellular membrane structures called detergent-resistant membranes (DRMs). We investigated the role of DRMs in NS2 complex formation, using a lysis buffer combining Triton and n-octyl glucoside, which solubilized both cell membranes and DRMs. When this lysis buffer was used on HCV-infected cells and the resulting lysates were subjected to flotation gradient centrifugation, all viral proteins and DRM-resident proteins were found in soluble protein fractions. Immunoprecipitation assays demonstrated direct protein-protein interactions between NS2 and E2 and E1 proteins, and an association of NS2 with NS3 through DRMs. The well-folded E1E2 complex and NS5A were not associated, instead interacting separately with the NS2-E1-E2-NS3 complex through less stable DRMs. Core was also associated with NS2 and the E1E2 complex through these unstable DRMs. We suggest that DRMs carrying this NS2-E1-E2-NS3-4A-NS5A-core complex may play a central role in HCV assembly initiation, potentially as an assembly platform.

LARP1 binding to hepatitis C virus particles is correlated with intracellular retention of viral infectivity.

Hepatitis C virus (HCV) virions contain a subset of host liver cells proteome often composed of interesting virus-interacting factors. A proteomic analysis performed on double gradient-purified clinical HCV highlighted the translation regulator LARP1 on these virions. This finding was validated using post-virion capture and immunoelectron microscopy, as well as immunoprecipitation applied to in vitro (Huh7.5 liver cells) grown (Gt2a, JFH1 strain) and patient-derived (Gt1a) HCV particles. Upon HCV infection of Huh7.5 cells, we observed a drastic transfer of LARP1 to lipid droplets, inducing colocalization with core proteins. RNAi-mediated depletion of LARP1 using the C911 control approach decreased extracellular infectivity of HCV Gt1a (H77), Gt2a (JFH1), and Gt3a (S52 chimeric strain), yet increased their intracellular infectivity. This latter effect was unrelated to changes in the hepatocyte secretory pathway, as evidenced using a functional RUSH assay. These results indicate that LARP1 binds to HCV, an event associated with retention of intracellular infectivity.