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.
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.
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.
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.
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Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 capsid protein. Recently, we demonstrated that HEV produces three different forms of ORF2: (i) the ORF2i form (infectious ...ORF2) which is the component of infectious particles, (ii) the secreted ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are not associated with infectious particles, but are the major antigens in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). The status and biological relevance of ORF2 N-glycosylation in HEV lifecycle remain to be elucidated. Here, we generated and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. We also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. Hence, our study led to new insights into the molecular mechanisms of ORF2 expression.
Hepatitis C Virus (HCV) infects over 150 million people worldwide. In most cases HCV infection becomes chronic, causing liver disease ranging from fibrosis to cirrhosis and hepatocellular carcinoma. ...HCV affects the cholesterol homeostasis and at the molecular level, every step of the virus life cycle is intimately connected to lipid metabolism. In this review, we present an update on the lipids and apolipoproteins that are involved in the HCV infectious cycle steps: entry, replication and assembly. Moreover, the result of the assembly process is a lipoviroparticle, which represents a peculiarity of hepatitis C virion. This review illustrates an example of an intricate virus-host interaction governed by lipid metabolism.
Summary
GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G‐proteins of the Arf family, which regulate membrane ...dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf‐guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA‐mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.
Core plays a critical role during hepatitis C virus (HCV) assembly, not only as a structural component of the virion, but also as a regulator of the formation of assembly sites. In this study, we ...observed that core is expressed later than other HCV proteins in a single viral cycle assay, resulting in a relative increase of core expression during a late step of the viral life cycle. This delayed core expression results from an increase of core half-life, indicating that core is initially degraded and is stabilized at a late step of the HCV life cycle. Stabilization-mediated delayed kinetics of core expression were also observed using heterologous expression systems. Core stabilization did not depend on its interaction with non-structural proteins or lipid droplets but was correlated with its expression levels and its oligomerization status. Therefore in the course of a HCV infection, core stabilization is likely to occur when the prior amplification of the viral genome during an initial replication step allows core to be synthesized at higher levels as a stable protein, during the assembly step of the viral life cycle.
HDV superinfection of chronically HBV-infected patients is the most aggressive form of chronic viral hepatitis, with an accelerated progression towards fibrosis/cirrhosis and increased risk of liver ...failure, hepatocellular carcinoma, and death. While HDV infection is not susceptible to available direct anti-HBV drugs, suboptimal responses are obtained with interferon-α-based therapies, and the number of investigational drugs remains limited. We therefore analyzed the effect of several innate immune stimulators on HDV replication in infected hepatocytes.
We used in vitro models of HDV and HBV infection based on primary human hepatocytes (PHHs) and the non-transformed HepaRG cell line that are relevant to explore new innate immune therapies.
We describe here, for the first time, anti-HDV effects of Pam3CSK4 and BS1, agonists of Toll-like receptor (TLR)-1/2, and the lymphotoxin-β receptor (LTβR), respectively. Both types of agonists induced dose-dependent reductions of total intracellular HDV genome and antigenome RNA and of HDV protein levels, without toxicity in cells monoinfected with HDV or co/superinfected with HBV. Moreover, both molecules negatively affected HDV progeny release and strongly decreased their specific infectivity. The latter effect is particularly important since HDV is thought to persist in humans through constant propagation.
Immune-modulators inducing NF-κB pathways in hepatocytes can inhibit HDV replication and should be further evaluated as a possible therapeutic approach in chronically HBV/HDV-infected patients.
Hepatitis delta virus causes the most severe form of viral hepatitis. Despite positive recent developments, effective treatments remain a major clinical need. Herein, we show that immune-modulators that trigger the NF-κB pathways could be effective for the treatment of hepatitis delta infections.
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•HDV leads to the most severe form of chronic viral hepatitis and treatment options are limited.•We demonstrate, for the first time, that immune-modulators inducing the NF-κB pathways can inhibit HDV in hepatocytes.•Pam3CSK4 (TLR1/2 agonist) and BS1 (LTBR agonist) reduced the intracellular and extracellular HDV RNAs and proteins.•Pam3CSK4 and BS1 also strongly reduced specific infectivity of remaining HDV virion progeny.•Pam3CSK4 and BS1 will thereby probably counteract viral spreading in vivo.
Recent reports indicate that the replication of hepatitis C virus (HCV) depends on the GBF1-Arf1-COP-I pathway. We generated Huh-7-derived cell lines resistant to brefeldin A (BFA), which is an ...inhibitor of this pathway. The resistant cell lines could be sorted into two phenotypes regarding BFA-induced toxicity, inhibition of albumin secretion, and inhibition of HCV infection. Two cell lines were more than 100 times more resistant to BFA than the parental Huh-7 cells in these 3 assays. This resistant phenotype was correlated with the presence of a point mutation in the Sec7 domain of GBF1, which is known to impair the binding of BFA. Surprisingly, the morphology of the cis-Golgi of these cells remained sensitive to BFA at concentrations of the drug that allowed albumin secretion, indicating a dichotomy between the phenotypes of secretion and Golgi morphology. Cells of the second group were about 10 times more resistant than parental Huh-7 cells to the BFA-induced toxicity. The EC50 for albumin secretion was only 1.5-1.8 fold higher in these cells than in Huh-7 cells. However their level of secretion in the presence of inhibitory doses of BFA was 5 to 15 times higher. Despite this partially effective secretory pathway in the presence of BFA, the HCV infection was almost as sensitive to BFA as in Huh-7 cells. This suggests that the function of GBF1 in HCV replication does not simply reflect its role of regulator of the secretory pathway of the host cell. Thus, our results confirm the involvement of GBF1 in HCV replication, and suggest that GBF1 might fulfill another function, in addition to the regulation of the secretory pathway, during HCV replication.
Immune-mediated induction of cytidine deaminase APOBEC3B (A3B) expression leads to HBV covalently closed circular DNA (cccDNA) decay. Here, we aimed to decipher the signalling pathway(s) and ...regulatory mechanism(s) involved in A3B induction and related HBV control.
Differentiated HepaRG cells (dHepaRG) knocked-down for NF-κB signalling components, transfected with siRNA or micro RNAs (miRNA), and primary human hepatocytes ± HBV or HBVΔX or HBV-RFP, were treated with lymphotoxin beta receptor (LTβR)-agonist (BS1). The biological outcomes were analysed by reverse transcriptase-qPCR, immunoblotting, luciferase activity, chromatin immune precipitation, electrophoretic mobility-shift assay, targeted-bisulfite-, miRNA-, RNA-, genome-sequencing, and mass-spectrometry.
We found that canonical and non-canonical NF-κB signalling pathways are mandatory for A3B induction and anti-HBV effects. The degree of immune-mediated A3B production is independent of A3B promoter demethylation but is controlled post-transcriptionally by the miRNA 138-5p expression (hsa-miR-138-5p), promoting A3B mRNA decay. Hsa-miR-138-5p over-expression reduced A3B levels and its antiviral effects. Of note, established infection inhibited BS1-induced A3B expression through epigenetic modulation of A3B promoter. Twelve days of treatment with a LTβR-specific agonist BS1 is sufficient to reduce the cccDNA pool by 80% without inducing significant damages to a subset of cancer-related host genes. Interestingly, the A3B-mediated effect on HBV is independent of the transcriptional activity of cccDNA as well as on rcDNA synthesis.
Altogether, A3B represents the only described enzyme to target both transcriptionally active and inactive cccDNA. Thus, inhibiting hsa-miR-138-5p expression should be considered in the combinatorial design of new therapies against HBV, especially in the context of immune-mediated A3B induction.
Immune-mediated induction of cytidine deaminase APOBEC3B is transcriptionally regulated by NF-κB signalling and post-transcriptionally downregulated by hsa-miR-138-5p expression, leading to cccDNA decay. Timely controlled APOBEC3B-mediated cccDNA decay occurs independently of cccDNA transcriptional activity and without damage to a subset of cancer-related genes. Thus, APOBEC3B-mediated cccDNA decay could offer an efficient therapeutic alternative to target hepatitis B virus chronic infection.
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•Impairment of NF-κB signalling prevents APOBEC3B induction and cccDNA decay.•APOBEC3B is post-transcriptionally regulated by the hsa-miR-138-5p.•Over-expression of the hsa-miR-138-5p inhibits APOBEC3B expression and cccDNA decay.•A3B timely induces cccDNA decay without damage to cancer-related genes.•APOBEC3B-mediated cccDNA decay is independent of cccDNA transcriptional activity.
GBF1 has emerged as a host factor required for the replication of positive-sense single-stranded RNA viruses of different families, but its mechanism of action is still unknown. GBF1 is a guanine ...nucleotide exchange factor for Arf family members. Recently, we identified Arf4 and Arf5 (class II Arfs) as host factors required for the replication of hepatitis C virus (HCV), a GBF1-dependent virus. To assess whether a GBF1/class II Arf pathway is conserved among positive-sense single-stranded RNA viruses, we investigated yellow fever virus (YFV), Sindbis virus (SINV), coxsackievirus B4 (CVB4) and human coronavirus 229E (HCoV-229E). We found that GBF1 is involved in the replication of these viruses. However, using siRNA or CRISPR-Cas9 technologies, it was seen that the depletion of Arf1, Arf3, Arf4 or Arf5 had no impact on viral replication. In contrast, the depletion of Arf pairs suggested that class II Arfs could be involved in HCoV-229E, YFV and SINV infection, as for HCV, but not in CVB4 infection. In addition, another Arf pair, Arf1 and Arf4, appears to be essential for YFV and SINV infection, but not for infection by other viruses. Finally, CVB4 infection was not inhibited by any combination of Arf depletion. We conclude that the mechanism of action of GBF1 in viral replication appears not to be conserved, and that a subset of positive-sense single-stranded RNA viruses from different families might require class II Arfs for their replication.
GBF1 has emerged as a host factor required for the genome replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the ...onset of genome replication but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, coimmunoprecipitation, and proximity ligation assays and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A reverse yeast two-hybrid screen to identify mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is nonreplicative despite conserved protease activity and does not interact with GBF1. The mutated residue is exposed at the surface of NS3, suggesting it is part of the domain of NS3 that interacts with GBF1. The corresponding mutation in strain JFH-1 (S77D) produces a similar phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV genome replication.
Single-stranded, positive-sense RNA viruses rely to a significant extent on host factors to achieve the replication of their genome. GBF1 is such a cellular protein that is required for the replication of several RNA viruses, but its mechanism of action during viral infections is not yet defined. In this study, we investigated potential interactions that GBF1 might engage in with proteins of HCV, a GBF1-dependent virus. We found that GBF1 interacts with NS3, a nonstructural protein involved in HCV genome replication, and our results suggest that this interaction is important for GBF1 function during HCV replication. Interestingly, GBF1 interaction with HCV appears different from its interaction with enteroviruses, another group of GBF1-dependent RNA viruses, in keeping with the fact that HCV and enteroviruses use different functions of GBF1.
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