Yongzhe Zhu

Tupaia CD81, SR-BI, Claudin-1, and Occludin Support Hepatitis C Virus Infection

ABSTRACT Hepatitis C virus (HCV)-related research has been hampered by the lack of appropriate small-animal models. It has been reported that tree shrews, or tupaias (Tupaia belangeri), can be infected with serum-derived HCV. However, these reports do not firmly establish the tupaia as a reliable model of HCV infection. Human CD81, scavenger receptor class B type I (SR-BI), claudin 1 (CLDN1), and occludin (OCLN) are considered essential receptors or coreceptors for HCV cell entry. In the present study, the roles of these tupaia orthologs in HCV infection were assessed. Both CD81 and SR-BI of tupaia were found to be able to bind with HCV envelope protein 2 (E2). In comparison with human CD81, tupaia CD81 exhibited stronger binding activity with E2 and increased HCV pseudoparticle (HCVpp) cell entry 2-fold. The 293T cells transfected with tupaia CLDN1 became susceptible to HCVpp infection. Moreover, simultaneous transfection of the four tupaia factors into mouse NIH 3T3 cells made the cells susceptible to HCVpp infection. HCVpp of diverse genotypes were able to infect primary tupaia hepatocytes (PTHs), and this infection could be blocked by either anti-CD81 or anti-SR-BI. PTHs could be infected by cell culture-produced HCV (HCVcc) and did produce infectious progeny virus in culture supernatant. These findings indicate that PTHs possess all of the essential factors required for HCV entry and support the complete HCV infection cycle. This highlights both the mechanisms of susceptibility of tupaia to HCV infection and the possibility of using tupaia as a promising small-animal model in HCV study.

Association of heat-shock protein 70 with lipid rafts is required for Japanese encephalitis virus infection in Huh7 cells.

Japanese encephalitis virus (JEV) is an enveloped flavivirus and the most common agent of viral encephalitis. It enters cells through receptor-mediated endocytosis and low pH-triggered membrane fusion. Although lipid rafts, cholesterol-enriched lipid-ordered membrane domains, have been shown to participate in JEV entry, the mechanisms of the early events of JEV infection, including the cellular receptors of JEV, remain largely unknown. In the current study, it was demonstrated that heat-shock protein 70 (HSP70), rather than other members of the HSP70 family, was required for JEV entry into a human cell line. Cell-surface expression of HSP70 and a direct interaction between JEV envelope (E) protein and HSP70 were observed. Biochemical fractionation showed that HSP70 clearly migrated into the raft fraction after virus infection and co-fractioned with E protein. Depletion of cholesterol shifted the E protein and HSP70 to a non-raft membrane and decreased JEV entry without affecting virus binding to host cells. Notably, recruitment of HSP70 into lipid rafts was required for activation of the phosphoinositide 3-kinase/Akt signalling pathway in the early stage of JEV infection. These results indicate that lipid rafts facilitate JEV entry, possibly by providing a convenient platform to concentrate JEV and its receptors on the host-cell membrane.

Japanese Encephalitis Virus Enters Rat Neuroblastoma Cells via a pH-Dependent, Dynamin and Caveola-Mediated Endocytosis Pathway

ABSTRACT Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus and one of the most common agents of viral encephalitis. The infectious entry process of JEV into host cells remains largely unknown. Here, we present a systemic study concerning the cellular entry mechanism of JEV to B104 rat neuroblastoma cells. It was observed that JEV internalization was inhibited by chloroquine and ammonium chloride, both of which can elevate the pH of acidic organelles. However, JEV entry was not affected by chlorpromazine, overexpression of a dominant-negative form of EPS 15 protein, or silencing of the clathrin heavy chain by small interfering RNA (siRNA). These results suggested that JEV entry depended on the acidic intracellular pH but was independent of clathrin. We found that endocytosis of JEV was dependent on membrane cholesterol and was inhibited by inactivation of caveolin-1 with siRNA or dominant-negative mutants. It was also shown, by using the inhibitor dynasore, the K44A mutant, and specific siRNA, that dynamin was required for JEV entry. Phagocytosis or macropinocytosis did not play a role in JEV internalization. In addition, we showed that JEV entry into the neuroblastoma cells is not virus strain specific by assessing the effect of the pharmacological inhibitors on the internalization of JEV belonging to different genotypes. Taken together, our results demonstrate that JEV enters B104 cells through a dynamin-dependent caveola-mediated uptake with a pH-dependent step, which is distinct from the clathrin-mediated endocytosis used by most flaviviruses.

Three Different Functional Microdomains in the Hepatitis C Virus Hypervariable Region 1 (HVR1) Mediate Entry and Immune evasion

Background: HVR1 spans 27 residues at the N terminus of the HCV envelope glycoprotein E2 and is the most variable region within the HCV polyprotein. Results: Three independent functional microdomains were identified in HCV HVR1. Conclusion: Different microdomains in HVR1 cooperate to mediate HCV cell entry and immune evasion. Significance: The data provide novel insights into understanding the mechanisms of HCV infection and immune evasion. High genetic heterogeneity is an important characteristic of hepatitis C virus (HCV) that contributes to its ability to establish persistent infection. The hypervariable region 1 (HVR1) that includes the first 27 amino acid residues of the E2 envelope glycoprotein is the most variable region within the HCV polyprotein. HVR1 plays a major role in both HCV cell entry and immune evasion, but the respective contribution of specific amino acid residues is still unclear. Our mutagenesis analyses of HCV pseudoparticles and cell culture-derived HCV using the H77 isolate indicate that five residues at positions 14, 15, and 25–27 mediate binding of the E2 protein to the scavenger receptor class B, type I receptor, and any residue herein is indispensable for HCV cell entry. The region spanning positions 16–24 contains the sole neutralizing epitope and is dispensable for HCV entry, but it is involved in heparan binding. More importantly, this region is necessary for the enhancement of HCV entry by high density lipoprotein and interferes with virus neutralization by E2-neutralizing antibodies. Residues at positions 1–13 are also dispensable for HCV entry, but they can affect HCV infectivity by modulating binding of the envelope protein to scavenger receptor class B, type I. Mutations occurring at this site may confer resistance to HVR1 antibodies. These findings further our understanding about the mechanisms of HCV cell entry and the significance of HVR1 variation in HCV immune evasion. They have major implications for the development of HCV entry inhibitors and prophylactic vaccines.

Hepatitis C virus protects human B lymphocytes from Fas-mediated apoptosis via E2-CD81 engagement.

HCV infection is often associated with B-cell regulatory control disturbance and delayed appearance of neutralizing antibodies. CD81 is a cellular receptor for HCV and can bind to HCV envelope protein 2 (E2). CD81 also participates to form a B cell costimulatory complex. To investigate whether HCV influences B cell activation and immune function through E2 -CD81 engagement, here, human Burkitts lymphoma cell line Raji cells and primary human B lymphocytes (PHB) were treated with HCV E2 protein and cell culture produced HCV particles (HCVcc), and then the related cell phenotypes were assayed. The results showed that both E2 and HCVcc triggered phosphorylation of IκBα, enhanced the expression of anti-apoptosis Bcl-2 family proteins, and protected Raji cells and PHB cells from Fas-mediated death. In addition, both E2 protein and HCVcc increased the expression of costimulatory molecules CD80, CD86 and CD81 itself, and decreased the expression of complement receptor CD21. The effects were dependent on E2-CD81 interaction on the cell surface, since CD81-silenced Raji cells did not respond to both treatments; and an E2 mutant that lose the CD81 binding activity, could not trigger the responses of both Raji cells and PHB cells. The effects were not associated with HCV replication in cells, for HCV pseudoparticle (HCVpp) and HCVcc failed to infect Raji cells. Hence, E2-CD81 engagement may contribute to HCV-associated B cell lymphoproliferative disorders and insufficient neutralizing antibody production.

How hepatitis C virus invades hepatocytes: The mystery of viral entry

Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced.

Entry inhibitors: New advances in HCV treatment.

Hepatitis C virus (HCV) infection affects approximately 3% of the world’s population and causes chronic liver diseases, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although current antiviral therapy comprising direct-acting antivirals (DAAs) can achieve a quite satisfying sustained virological response (SVR) rate, it is still limited by viral resistance, long treatment duration, combined adverse reactions, and high costs. Moreover, the currently marketed antivirals fail to prevent graft reinfections in HCV patients who receive liver transplantations, probably due to the cell-to-cell transmission of the virus, which is also one of the main reasons behind treatment failure. HCV entry is a highly orchestrated process involving initial attachment and binding, post-binding interactions with host cell factors, internalization, and fusion between the virion and the host cell membrane. Together, these processes provide multiple novel and promising targets for antiviral therapy. Most entry inhibitors target host cell components with high genetic barriers and eliminate viral infection from the very beginning of the viral life cycle. In future, the addition of entry inhibitors to a combination of treatment regimens might optimize and widen the prevention and treatment of HCV infection. This review summarizes the molecular mechanisms and prospects of the current preclinical and clinical development of antiviral agents targeting HCV entry.

Significance of palmitoylation of CD81 on its association with tetraspanin-enriched microdomains and mediating hepatitis C virus cell entry

CD81, a co-receptor for hepatitis C virus (HCV), is a member of the tetraspanin superfamily and is heavily palmitoylated in the juxtamembrane cysteine residues. Palmitoylation plays an important role in protein-protein interactions and association with cholesterol-rich domains of membranes. In this study, Huh7 cells expressing wild-type or palmitoylation-defective CD81 were generated to analyze whether palmitoylation of CD81 is involved in HCV cell entry. Our data showed that de-palmitoylation of CD81 dramatically reduced its association with tetraspanin CD151, but did not influence CD81 partition in detergent-resistant membranes. Moreover, de-palmitoylated CD81 decreased the host cell susceptibility to HCV. Notably, CD151-specific antibodies and siRNA inhibited HCV cell entry, and detachment of CD81 with CD151 decreased the lateral movement of virus particle/CD81 complex to areas of cell-cell contact. These results suggest that palmitoylation of CD81 should facilitate HCV entry, at least in part, by regulating the association of CD81 with tetraspanin-enriched microdomains.

Serum adsorption, cellular internalization and consequent impact of cuprous oxide nanoparticles on uveal melanoma cells: implications for cancer therapy

AIM To investigate the biological fate of cuprous oxide nanoparticles (Cu2O-NPs) and to evaluate their potential in uveal melanoma therapy. MATERIALS & METHODS The protein corona, cellular uptake mechanism and localization of Cu2O-NPs were investigated. Furthermore, the effect of Cu2O-NPs on uveal melanoma cell proliferation, migration and invasion, and possible mechanisms were studied in detail. RESULTS Cu2O-NPs are able to adsorb serum proteins in cell culture medium, which are then internalized by uveal melanoma cells mainly through lipid raft-mediated endocytosis. Furthermore, Cu2O-NPs selectively inhibit cancer cell growth and impair the ability of uveal melanoma cell migration, invasion and the cytoskeleton assembly. The mechanism may be that Cu2O-NPs located in and damage mitochondria, autophagolysosomes and lysosomes, leading to elevated reactive oxygen species level and over-stimulated apoptosis and autophagy. CONCLUSION The data provide detailed information of Cu2O-NPs for further application and indicate that Cu2O-NPs could be a potential agent for uveal melanoma therapy.

Alanine scanning mutagenesis of hepatitis C virus E2 cysteine residues: Insights into E2 biogenesis and antigenicity

Envelope glycoprotein 2 (E2) of hepatitis C virus contains 18 conserved cysteine (Cys) residues in its ectodomain. By cysteine-alanine mutagenesis and function analysis, six Cys in H77 E2 (C494, C508, C552, C564, C607 and C644) were found to be indispensable for recognition by conformation-dependent mAb H53. Removal of any of these Cys residues did not affect E2 heterodimerization with E1, but notably reduced E1E2 transmembrane transportation. These Cys together with C429 and C503 were required for conformation-dependent mAb H48 recognition. All of the above Cys except C607 were required for H77 and Con1 E2 binding to CD81. None of individual mutation of above Cys affected the ability of E2 to induce neutralizing antibodies in mice. Mouse antibodies mainly recognize E2 linear epitopes and are unrelated to epitopes recognized by human E2 antibodies. The findings provide new insights for understanding the biogenesis of functional HCV envelope proteins and HCV neutralizing immunity.