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...

It has been reported that hepatitis C virus (HCV) may infect and replicate in human T cells, particularly in perihepatic lymph nodes, but the extent and consequence of T-cell infection in patients is unclear. This study is conducted to characterize the parameters and functional consequences of HCV infection in T lymphocytes. By using a lymphotropic HCV strain, we showed that HCV could infect T cell lines (Molt-4 and Jurkat cells) in vitro. Both positive- and negative-strand HCV RNA were detected for several weeks after infection. Viral proteins could also be detected by immunofluorescence studies. Moreover, infectious HCV particles were produced from Molt-4 cell cultures, and could be used to infect naïve T cell lines. HCV could also infect human primary CD4+ T cells, particularly naïve (CD45RA+CD45RO-) CD4+ cells, in culture. The amounts of STAT-1 and phosphorylated STAT-1 proteins in the infected Molt-4 cells were significantly less than those in uninfected cultures, suggesting the possibility of defect in interferon-gamma signaling. Indeed, T-bet and STAT-1 mRNA levels after interferon-gamma stimulation in infected Molt-4 were suppressed. In conclusion, HCV could infect and transiently replicate in T cells and that HCV replication suppressed the IFN-gamma/STAT-1/T-bet signaling due to the reduction of STAT-1 and inhibition of its activation (phosphorylation).

The machinery for hepatitis C virus (HCV) RNA replication is still poorly characterized. The relationship between HCV RNA replication and translation is also not clear. We have previously shown that a cellular protein polypyrimidine-tract-binding protein (PTB) binds to HCV RNA at several different sites and modulates HCV translation in several ways. Here we show that PTB also participates in RNA replication. By bromouridine triphosphate (BrUTP) labeling and confocal microscopy of cells harboring an HCV replicon, we showed that the newly synthesized HCV RNA was localized to distinct structures in the cytoplasm, which also contain PTB. Membrane flotation analysis demonstrated that a fraction of cytoplasmic PTB was associated with a detergent-resistant membrane (DRM) structure consisting of lipid rafts, which also contained HCV nonstructural proteins and the human vesicle-associated membrane proteinassociated protein (hVAP-33). PTB in the DRM was resistant to protease digestion, but became sensitive after treatment with the raft-disrupting agents. PTB in the DRM consisted of multiple isoforms and the brain-specific paralog. By using small interfering RNA (siRNA) of PTB, we showed that silencing of the endogenous PTB reduced the replication of HCV RNA replicon. In a cell-free, de novo HCV RNA synthesis system, HCV RNA synthesis was inhibited by anti-PTB antibody. These studies together indicated that PTB is a part of the HCV RNA replication complex and participates in viral RNA synthesis. Thus, PTB has dual functions in HCV life cycle, including translation and RNA replication.