Recent advances in reverse genetics of hepatitis C virus (HCV) made it possible to determine the properties and biochemical compositions of HCV virions. Sedimentation analysis and characterization of HCV RNAcontaining particles produced in the cultured cells revealed that HCV virions cover a large range of heterogeneous densities in sucrose gradient. The fractions of low densities are infectious, while the higher-density fractions containing the majority of HCV virion RNA are not. HCV core protein and apolipoprotein B and apolipoprotein E (apoE) were detected in the infectious HCV virions. The level of apoE correlates very well with HCV infectivity. Both apoE-and HCV E2-specific monoclonal antibodies precipitated HCV, demonstrating that HCV virions contain apoE and E2 proteins. apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity in a dose-dependent manner, resulting in a reduction of infectious HCV by nearly 4 orders of magnitude. The knockdown of apoE expression by specific small interfering RNAs (siRNAs) remarkably reduced the levels of intracellular as well as secreted HCV virions. The apoE siRNA suppressed HCV production by more than 100-fold at 50 nM. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles. Our findings also identified apoE as a novel target for discovery and development of antiviral drugs and monoclonal antibodies to suppress HCV virion formation and infection.Hepatitis C virus (HCV) is a major cause of liver diseases, affecting approximately 170 million people worldwide (59). Most (ϳ85%) acutely HCV-infected individuals become chronic carriers that can develop cirrhosis and hepatocellular carcinoma (50). HCV is an enveloped RNA virus with a singlestrand and positive-sense RNA genome and is classified as Hepacivirus in the Flaviviridae family (47). The genomic RNA consists of a long open reading frame and relatively short untranslated regions (UTR) at the 5Ј and 3Ј ends (11,32,36,46,53). The 5Ј and 3Ј UTR contain cis-acting RNA elements important for HCV polyprotein translation and RNA replication (16-18, 38, 39, 61, 62). The translation of HCV polyprotein is mediated by the internal ribosomal entry site within the 5Ј UTR (46, 58). Upon translation, the HCV polyprotein is cleaved by cellular peptidases and viral proteases into different viral proteins in the order of C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B (36, 38). A number of studies demonstrated that the NS3 to NS5B proteins are sufficient for HCV RNA replication (4, 6, 37), which occurs in the membranebound replication complex consisting of HCV RNA and proteins as well as cellular proteins (13,14,42,57). The core and NS5B coding regions also contain cis-acting RNA elements important for HCV RNA replication and regulation (40,63). Last, the newly synthesized HCV proteins and genomic RNA are packaged to form progeny virus particles. However, the molecular aspects underlying HCV virion assembly, matur...

Hepatitis C virus (HCV) NS5B protein possesses an RNA-dependent RNA polymerase (RdRp) activity, a major function responsible for replication of the viral RNA genome. To further characterize the RdRp activity, NS5B proteins were expressed from recombinant baculoviruses, purified to near homogeneity, and examined for their ability to synthesize RNA in vitro. As a result, a highly active NS5B RdRp (1b-42), which contains an 18-amino acid C-terminal truncation resulting from a newly created stop codon, was identified among a number of independent isolates. The RdRp activity of the truncated NS5B is comparable to the activity of the full-length protein and is 20 times higher in the presence of Mn 2؉ than in the presence of Mg 2؉ . When a 384-nucleotide RNA was used as the template, two major RNA products were synthesized by 1b-42. One is a complementary RNA identical in size to the input RNA template (monomer), while the other is a hairpin dimer RNA synthesized by a "copy-back" mechanism. Substantial evidence derived from several experiments demonstrated that the RNA monomer was synthesized through de novo initiation by NS5B rather than by a terminal transferase activity. Synthesis of the RNA monomer requires all four ribonucleotides. The RNA monomer product was verified to be the result of de novo RNA synthesis, as two expected RNA products were generated from monomer RNA by RNase H digestion. In addition, modification of the RNA template by the addition of the chain terminator cordycepin at the 3 end did not affect synthesis of the RNA monomer but eliminated synthesis of the self-priming hairpin dimer RNA. Moreover, synthesis of RNA on poly(C) and poly(U) homopolymer templates by 1b-42 NS5B did not require the oligonucleotide primer at high concentrations (>50 M) of GTP and ATP, further supporting a de novo initiation mechanism. These findings suggest that HCV NS5B is able to initiate RNA synthesis de novo.Hepatitis C virus (HCV) is the major causative agent of non-A, non-B viral hepatitis (23). Although acute HCV infection is often asymptomatic, nearly 80% of cases resolve to chronic hepatitis, which may lead to progressive liver disease, such as cirrhosis, and liver failure. Chronic HCV infection is also associated with the development of hepatocellular carcinoma (23). It is estimated that 170 million people worldwide and more than 4 million people in the United States are currently infected with HCV (2, 65). Obviously, HCV infection remains a major threat to the public health all over the world.HCV is an enveloped RNA virus containing a singlestranded positive-sense RNA genome approximately 9.5 kb in length (14,31,56). The RNA genome consists of a 5Ј-untranslated region (5Ј UTR) of 341 nucleotides (12, 13), a large open reading frame (ORF) encoding a single polypeptide of 3,010 to 3,040 amino acids (14, 31, 56), and a 3Ј-untranslated region (3Ј UTR) of variable length (33,57,66). HCV is similar in amino acid sequence and genome organization to flaviviruses and pestiviruses (41), and therefore HCV was classified ...

Chronic hepatitis C virus (HCV) infection is a major cause of liver disease worldwide.Restriction of HCV infection to human hepatocytes suggests that liver-specific host factors play a role in the viral life cycle. Using a yeast-two-hybrid system, we identified apolipoprotein E (apoE) as a liver-derived host factor specifically interacting with HCV nonstructural protein 5A (NS5A) but not with other viral proteins. The relevance of apoE-NS5A interaction for viral infection was confirmed by co-immunoprecipitation and co-localization studies of apoE and NS5A in an infectious HCV cell culture model system. Silencing apoE expression resulted in marked inhibition of infectious particle production without affecting viral entry and replication. Analysis of particle production in liver-derived cells with silenced apoE expression showed impairment of infectious particle assembly and release. The functional relevance of the apoE-NS5A interaction for production of viral particles was supported by loss or decrease of apoE-NS5A binding in assembly-defective viral mutants. H epatitis C virus (HCV) is a major cause of liver disease, including liver cirrhosis and hepatocellular carcinoma. 1 Current treatment by interferon-alpha and ribavirin is limited by resistance, toxicity, and high costs. 1,2 Novel treatment approaches are therefore urgently needed. HCV is an enveloped single-stranded RNA virus of positive polarity that is a member of the genus Hepacivirus within the family Flaviviridae. 3,4 The HCV RNA genome encodes a unique polyprotein of approximately 3000 amino acids and is flanked at its 5Ј and 3Ј ends by two highly conserved untranslated regions involved in the transla- Conclusion: These results suggest that recruitment of apoE by NS5A is important for viral assembly and release of infectious viral particles. These findings have important implications

cViruses are known to use virally encoded envelope proteins for cell attachment, which is the very first step of virus infection. In the present study, we have obtained substantial evidence demonstrating that hepatitis C virus (HCV) uses the cellular protein apolipoprotein E (apoE) for its attachment to cells. An apoE-specific monoclonal antibody was able to efficiently block HCV attachment to the hepatoma cell line Huh-7.5 as well as primary human hepatocytes. After HCV bound to cells, however, antiapoE antibody was unable to inhibit virus infection. Conversely, the HCV E2-specific monoclonal antibody CBH5 did not affect HCV attachment but potently inhibited HCV entry. Similarly, small interfering RNA-mediated knockdown of the key HCV receptor/coreceptor molecules CD81, claudin-1, low-density lipoprotein receptor (LDLr), occludin, and SR-BI did not affect HCV attachment but efficiently suppressed HCV infection, suggesting their important roles in HCV infection at postattachment steps. Strikingly, removal of heparan sulfate from the cell surface by treatment with heparinase blocked HCV attachment. Likewise, substitutions of the positively charged amino acids with neutral or negatively charged residues in the receptor-binding region of apoE resulted in a reduction of apoE-mediating HCV infection. More importantly, mutations of the arginine and lysine to alanine or glutamic acid in the receptor-binding region ablated the heparin-binding activity of apoE, as determined by an in vitro heparin pulldown assay. HCV attachment could also be inhibited by a synthetic peptide derived from the apoE receptor-binding region. Collectively, these findings demonstrate that apoE mediates HCV attachment through specific interactions with cell surface heparan sulfate. H epatitis C virus (HCV) is a leading cause of liver diseases, chronically infecting an estimated 130 million to 170 million people worldwide (71, 82). HCV infection results in acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma (59), which ranks as the fifth most common cancer and the third most frequent cause of cancer death worldwide. Hepatitis C is also the most common indication for liver transplantation (15). Coinfection of HCV and HIV is very common, particularly among drug abusers (3). Thus, HCV infection poses a major global health problem. Current standard therapy with pegylated alpha interferon (peg-IFN-␣) and ribavirin is less than 50% effective against HCV genotype 1, the dominant virus accounting for up to 70% of infections (27,41,54). Although two HCV NS3 protease-specific inhibitors, telaprevir and boceprevir, have recently been approved (33), their combination with peg-IFN-␣ and ribavirin has limitations such as severe side effects, long duration of treatment, and high cost. Discovery and development of more efficacious and safer anti-HCV drugs are urgently needed.HCV is the prototype virus of the Hepacivirus genus in the Flaviviridae family (68). It is an enveloped RNA virus containing a single positive-strand RNA genome that encodes...