Hepatitis E virus (HEV) is a causative agent of acute hepatitis. The crystal structure of HEV-like particles (HEV-LP) consisting of capsid protein was determined at 3.5-Å resolution. The capsid protein exhibited a quite different folding at the protruding and middle domains from the members of the families of Caliciviridae and Tombusviridae, while the shell domain shared the common folding. Tyr-288 at the 5-fold axis plays key roles in the assembly of HEV-LP, and aromatic amino acid residues are well conserved among the structurally related viruses. Mutational analyses indicated that the protruding domain is involved in the binding to the cells susceptive to HEV infection and has some neutralization epitopes. These structural and biological findings are important for understanding the molecular mechanisms of assembly and entry of HEV and also provide clues in the development of preventive and prophylactic measures for hepatitis E.capsid ͉ HEV ͉ VLP

Hepatitis C virus (HCV) is a major cause of chronic liver disease that frequently leads to steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC). HCV core protein is not only a component of viral particles but also a multifunctional protein because liver steatosis and HCC are developed in HCV core gene-transgenic (CoreTg) mice. Proteasome activator PA28␥/REG␥ regulates host and viral proteins such as nuclear hormone receptors and HCV core protein. Here we show that a knockout of the PA28␥ gene induces the accumulation of HCV core protein in the nucleus of hepatocytes of CoreTg mice and disrupts development of both hepatic steatosis and HCC. Furthermore, the genes related to fatty acid biosynthesis and srebp-1c promoter activity were up-regulated by HCV core protein in the cell line and the mouse liver in a PA28␥-dependent manner. Heterodimer composed of liver X receptor ␣ (LXR␣) and retinoid X receptor ␣ (RXR␣) is known to up-regulate srebp-1c promoter activity. Our data also show that HCV core protein enhances the binding of LXR␣/RXR␣ to LXR-response element in the presence but not the absence of PA28␥. These findings suggest that PA28␥ plays a crucial role in the development of liver pathology induced by HCV infection.fatty acid ͉ proteasome ͉ sterol regulatory element-binding protein (SREBP) ͉ RXR␣ ͉ LXR␣ H epatitis C virus (HCV) belongs to the Flaviviridae family, and it possesses a positive, single-stranded RNA genome that encodes a single polyprotein composed of Ϸ3,000 aa. The HCV polyprotein is processed by host and viral proteases, resulting in 10 viral proteins. Viral structural proteins, including the capsid (core) protein and two envelope proteins, are located in the N-terminal one-third of the polyprotein, followed by nonstructural proteins.HCV infects Ͼ170 million individuals worldwide, and then it causes liver disease, including hepatic steatosis, cirrhosis, and eventually hepatocellular carcinoma (HCC) (1). The prevalence of fatty infiltration in the livers of chronic hepatitis C patients has been reported to average Ϸ50% (2, 3), which is higher than the percentage in patients infected with hepatitis B virus and other liver diseases. However, the precise functions of HCV proteins in the development of fatty liver remain unknown because of the lack of a system sufficient to investigate the pathogenesis of HCV. HCV core protein expression has been shown to induce lipid droplets in cell lines and hepatic steatosis and HCC in transgenic mice (4-6). These reports suggest that HCV core protein plays an important role in the development of various types of liver failure, including steatosis and HCC.Recent reports suggest that lipid biosynthesis affects HCV replication (7-9). Involvement of a geranylgeranylated host protein, FBL2, in HCV replication through the interaction with NS5A suggests that the cholesterol biosynthesis pathway is also important for HCV replication (9). Increases in saturated and monounsaturated fatty acids enhance HCV RNA replication, whereas increases in polyunsaturat...

We have previously shown that mice inoculated intranasally with a wild-type baculovirus (Autographa californica nuclear polyhedrosis virus [AcNPV]) are protected from a lethal challenge by influenza virus. However, the precise mechanism of induction of this protective immune response by the AcNPV treatment remained unclear. Here we show that AcNPV activates immune cells via the Toll-like receptor 9 (TLR9)/ MyD88-dependent signaling pathway. The production of inflammatory cytokines was severely reduced in peritoneal macrophages (PECs) and splenic CD11c؉ dendritic cells (DCs) derived from mice deficient in MyD88 or TLR9 after cultivation with AcNPV. In contrast, a significant amount of alpha interferon (IFN-␣) was still detectable in the PECs and DCs of these mice after stimulation with AcNPV, suggesting that a TLR9/MyD88-independent signaling pathway might also participate in the production of IFN-␣ by AcNPV. Since previous work showed that TLR9 ligands include bacterial DNA and certain oligonucleotides containing unmethylated CpG dinucleotides, we also examined the effect of baculoviral DNA on the induction of innate immunity. Transfection of the murine macrophage cell line RAW264.7 with baculoviral DNA resulted in the production of the inflammatory cytokine, while the removal of envelope glycoproteins from viral particles, UV irradiation of the virus, and pretreatment with purified baculovirus envelope proteins or endosomal maturation inhibitors diminished the induction of the immune response by AcNPV. Together, these results indicate that the internalization of viral DNA via membrane fusion mediated by the viral envelope glycoprotein, as well as endosomal maturation, which releases the viral genome into TLR9-expressing cellular compartments, is necessary for the induction of the innate immune response by AcNPV.

The hepatitis C virus (HCV) nonstructural protein (NS)5AHepatitis C virus (HCV) infects 170 million people worldwide and frequently leads to cirrhosis or hepatocellular carcinoma (6, 29). HCV is classified in the family Flaviviridae and possesses a single-stranded positive-sense RNA with a length of 9.6 kb. The HCV genome encodes a single large precursor polyprotein composed of about 3,000 amino acids (aa) that is processed by cellular and viral proteases, resulting in at least 10 structural and nonstructural (NS) proteins (29). Details of HCV's replication cycle are unknown because of the low viral load in the sera of HCV-infected individuals and the lack of a reliable and robust cell culture system to support HCV infection and replication. The development of HCV RNA replicons in which a synthetic HCV genomic or subgenomic RNA replicates efficiently in the human hepatocarcinoma cell line Huh-7 has enabled the study of viral RNA replication in cell culture (4,20,24). The HCV RNA replication complex, composed of the viral NS proteins and host cellular proteins, replicates the viral RNA genome at the intracellular membrane. Thus far, the HCV replicon system has greatly contributed to the understanding of HCV replication and pathogenesis associated with the expression of viral NS proteins. Replication of positive-strand RNA viruses generally involves certain intracellular membrane structures, including the endoplasmic reticulum (ER), Golgi apparatus, endosome, and lysosome (39).Recently, several groups have succeeded in demonstrating cellfree replication activities of replication complexes in crude membrane fractions of HCV subgenomic replicon cells (2, 3, 14, 53). These cell-free systems provide semi-intact polymerase assays for biochemical dissection of HCV RNA replication and are a useful source for the isolation of HCV replication complexes. Replication complexes were detected in detergent-resistant membrane structures, most likely lipid raft structures (2, 14). Although HCV NS proteins presumably form a membrane-associated RNA replication complex with host proteins, the precise components and mechanisms for replication are poorly understood.HCV NS5A is a phosphoprotein that appears to possess multiple and diverse functions in viral replication, interferon resistance, and pathogenesis (26,35). Cell culture-adaptive mutations have been shown to cluster in the central portion of NS5A in subgenomic HCV replicons, indicating that NS5A is involved in the viral replication process either directly or by interacting with host cellular proteins (4, 55). This observation, together with the modulation of NS5A hyperphosphorylation by NS3, NS4A, and NS4B and physical interaction with other viral NS proteins, strongly supports the notion that NS5A is an essential component of the HCV replication complex (21,30,36). NS5A has been shown to be associated with a range of cellular proteins involved in cellular signaling pathways, such as interferon-induced kinase PKR (11), growth factor receptor-binding protein 2 (Grb2) (45), p53...

Japanese encephalitis virus (JEV) belongs to the genus Flavivirus within the family Flaviviridae. Members of the genus Flavivirus are predominantly arthropodborne viruses and frequently cause significant morbidity and mortality in mammals and birds (6). JEV is distributed in the south and southeast regions of Asia and kept in a zoonotic transmission cycle between pigs or birds and mosquitoes (6,50,57). JEV spreads to dead-end hosts, including humans, through the bite of JEVinfected mosquitoes and causes infection of the central nervous system, with a high mortality rate (6, 57). JEV has a single-stranded positive-strand RNA genome approximately 11 kb in length, which is capped at the 5Ј end but lacks modification of the 3Ј terminus by polyadenylation (34). The genomic RNA encodes a single large open reading frame, and a polyprotein translated from the genome is cleaved co-and posttranslationally by host and viral proteases to yield three structural proteins, the core, precursor membrane (prM), and envelope (E) proteins, and seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (53). Although the core protein has very little amino acid homology to other flaviviruses-for example, the core protein of JEV has only 25% homology to that of tick-borne encephalitis virus (TBEV)-the structural properties, such as the hydrophobicity profile, abundances of basic amino acid residues, and secondary structures, are very similar (11,20,36). The flavivirus core proteins commonly contain two hydrophobic sequences in the center and a carboxyl-terminal end, and the carboxyl-terminal hydrophobic region serves as a signal sequence of prM. The signal-anchor sequence is cleaved off by the viral protease NS2B-3, and this cleavage is required for the subsequent liberation of the amino terminus of prM by the host signal peptidase (35,52,63). The mature core protein, released from the endoplasmic reticulum (ER) membrane, is believed to bind to the genomic RNA via the basic amino acid clusters at the amino and carboxyl termini and forms nucleocapsids (23). The central hydrophobic region of the core protein may be associated with the ER membrane, and this interaction is believed to facilitate the assembly of nucleocapsid and two membrane proteins, prM and E, and to bud into the ER lumen as virions (39). The removal of the central hydrophobic region of the TBEV core protein increased the production of the subviral particles that consist of (pr)M and E proteins but that lack a core protein and genomic RNA (26,27).In addition to their role as structural proteins, core proteins of dengue virus (DEN) and Kunjin virus (KUN) are localized not only in the cytoplasm but also in the nucleus, especially in the nucleoli of several infected cell lines (4,38,55,59,61). Transport from the cytoplasm to the nucleus occurs through nuclear pore complexes that penetrate the double lipid layers of the nuclear envelope. Small molecules up to 9 nm in diameter (Ͻ50 kDa) can freely diffuse through the nuclear pore complexes, while most macromolecu...