Nonstructural protein 5A (NS5A) of the hepatitis C virus (HCV) possesses multiple and diverse functions in RNA replication, interferon resistance, and viral pathogenesis. Recent studies suggest that NS5A is involved in the assembly and maturation of infectious viral particles; however, precisely how NS5A participates in virus production has not been fully elucidated. In the present study, we demonstrate that NS5A is a prerequisite for HCV particle production as a result of its interaction with the viral capsid protein (core protein). The efficiency of virus production correlated well with the levels of interaction between NS5A and the core protein. Alanine substitutions for the C-terminal serine cluster in domain III of NS5A (amino acids 2428, 2430, and 2433) impaired NS5A basal phosphorylation, leading to a marked decrease in NS5A-core interaction, disturbance of the subcellular localization of NS5A, and disruption of virion production. Replacing the same serine cluster with glutamic acid, which mimics the presence of phosphoserines, partially preserved the NS5A-core interaction and virion production, suggesting that phosphorylation of these serine residues is important for virion production. In addition, we found that the alanine substitutions in the serine cluster suppressed the association of the core protein with viral genome RNA, possibly resulting in the inhibition of nucleocapsid assembly. These results suggest that NS5A plays a key role in regulating the early phase of HCV particle formation by interacting with core protein and that its C-terminal serine cluster is a determinant of the NS5A-core interaction.Hepatitis C virus (HCV) infection is a major public health problem and is prevalent in about 200 million people worldwide (27,40,42). Current protocols for treating HCV infection fail to produce a sustained virological response in as many as half of treated individuals, and many cases progress to chronic liver disease, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma (15,31,35,43).HCV is a positive-strand RNA virus classified in the Hepacivirus genus within the Flaviviridae family (55). Its approximately 9.6-kb genome is translated into a single polypeptide of about 3,000 amino acids (aa), in which the structural proteins core, E1, and E2 reside in the N-terminal region. A crucial function of core protein is assembly of the viral nucleocapsid. The amino acid sequence of this protein is well conserved among different HCV strains compared to other HCV proteins. The nonstructural (NS) proteins NS3-NS5B are considered to assemble into a membrane-associated HCV RNA replicase complex. NS3 possesses the enzymatic activities of serine protease and RNA helicase, and NS4A serves as a cofactor for NS3 protease. NS4B plays a role in the remodeling of host cell membranes, probably to generate the site for the replicase assembly. NS5B functions as the RNA-dependent RNA polymerase. NS5A is known to play an important but undefined role in viral RNA replication.NS5A is a phosphoprotein that can be...

A baculovirus (Autographa californica nucleopolyhedrovirus) vector containing a strong promoter, the CAG promoter, was developed to introduce foreign genes into mammalian cells. Recombinant baculoviruses carrying a reporter gene under the control of the CAG promoter were inoculated into various mammalian cell lines. High-level expression was observed not only in hepatocytes but also in other non-hepatic cell lines tested. Expression of the reporter gene was detected even 14 days after infection. The infectious titre of the recovered baculoviruses decreased significantly after infection, indicating that the baculoviruses did not repli-

Sodium taurocholate cotransporting polypeptide (NTCP) is a host cell receptor required for hepatitis B virus (HBV) entry. However, the susceptibility of NTCP-expressing cells to HBV is diverse depending on the culture condition. Stimulation with epidermal growth factor (EGF) was found to potentiate cell susceptibility to HBV infection. Here, we show that EGF receptor (EGFR) plays a critical role in HBV virion internalization. In EGFR-knockdown cells, HBV or its preS1-specific fluorescence peptide attached to the cell surface, but its internalization was attenuated. PreS1 internalization and HBV infection could be rescued by complementation with functional EGFR. Interestingly, the HBV/preS1–NTCP complex at the cell surface was internalized concomitant with the endocytotic relocalization of EGFR. Molecular interaction between NTCP and EGFR was documented by immunoprecipitation assay. Upon dissociation from functional EGFR, NTCP no longer functioned to support viral infection, as demonstrated by either (i) the introduction of NTCP point mutation that disrupted its interaction with EGFR, (ii) the detrimental effect of decoy peptide interrupting the NTCP–EGFR interaction, or (iii) the pharmacological inactivation of EGFR. Together, these data support the crucial role of EGFR in mediating HBV–NTCP internalization into susceptible cells. EGFR thus provides a yet unidentified missing link from the cell-surface HBV–NTCP attachment to the viral invasion beyond the host cell membrane.

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The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10--20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies. Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells. The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C.