Glycometabolism regulates hepatitis C virus release.

HCV cell-culture system uses hepatoma-derived cell lines for efficient virus propagation. Tumor cells cultured in glucose undergo active aerobic glycolysis, but switch to oxidative phosphorylation for energy production when cultured in galactose. Here, we investigated whether modulation of glycolysis in hepatocytes affects HCV infection. We showed HCV release, but not entry, genome replication or virion assembly, is significantly blocked when cells are cultured in galactose, leading to accumulation of intracellular infectious virions within multivesicular body (MVB). Blockade of the MVB-lysosome fusion or treatment with pro-inflammatory cytokines promotes HCV release in galactose. Furthermore, we found this glycometabolic regulation of HCV release is mediated by MAPK-p38 phosphorylation. Finally, we showed HCV cell-to-cell transmission is not affected by glycometabolism, suggesting that HCV cell-to-supernatant release and cell-to-cell transmission are two mechanistically distinct pathways. In summary, we demonstrated glycometabolism regulates the efficiency and route of HCV release. We proposed HCV may exploit the metabolic state in hepatocytes to favor its spread through the cell-to-cell transmission in vivo to evade immune response.

A Nanoparticle-Based Hepatitis C Virus Vaccine With Enhanced Potency.

Despite the emergence of new direct-acting antivirals, hepatitis C virus (HCV) chronic infection and its consequent fibrosis and hepatocarcinoma remain a significant burden for public health, thus requiring an effective preventive vaccine. Our group previously showed that a subunit vaccine based on recombinant soluble E2 (sE2) can induce broadly neutralizing antibodies. To improve the immunogenicity of sE2, we designed and produced a fusion protein (sE2-ferritin) comprising sE2 and a ferritin unit in Drosophila S2 cells, which self-assembled into a nanoparticle with sE2 displayed on the surface. The sE2 moiety on the sE2-ferritin nanoparticle not only had nearly natural conformation but also had better affinities than the unfused sE2 to neutralizing antibodies, receptor, and patient serum. Mouse immunization studies showed that sE2-ferritin was more potent than sE2 in inducing anti-HCV broadly neutralizing antibodies. Our results demonstrate that sE2-ferritin is a vaccine candidate superior to previously developed sE2, providing a new possibility for controlling HCV.

Functional expression and characterization of the envelope glycoprotein E1E2 heterodimer of hepatitis C virus.

Hepatitis C virus (HCV) is a member of Hepacivirus and belongs to the family of Flaviviridae. HCV infects millions of people worldwide and may lead to cirrhosis and hepatocellular carcinoma. HCV envelope proteins, E1 and E2, play critical roles in viral cell entry and act as major epitopes for neutralizing antibodies. However, unlike other known flaviviruses, it has been challenging to study HCV envelope proteins E1E2 in the past decades as the in vitro expressed E1E2 heterodimers are usually of poor quality, making the structural and functional characterization difficult. Here we express the ectodomains of HCV E1E2 heterodimer with either an Fc-tag or a de novo designed heterodimeric tag and are able to isolate soluble E1E2 heterodimer suitable for functional and structural studies. Then we characterize the E1E2 heterodimer by electron microscopy and model the structure by the coevolution based modeling strategy with Rosetta, revealing the potential interactions between E1 and E2. Moreover, the E1E2 heterodimer is applied to examine the interactions with the known HCV receptors, neutralizing antibodies as well as the inhibition of HCV infection, confirming the functionality of the E1E2 heterodimer and the binding profiles of E1E2 with the cellular receptors. Therefore, the expressed E1E2 heterodimer would be a valuable target for both viral studies and vaccination against HCV.