Genetic interactions between hepatitis C virus replicons

To investigate interactions between hepatitis C virus (HCV) RNA replication complexes, a system was developed to simultaneously select different HCV subgenomic replicons within the same cell. Transcomplementation of defective replicons was not observed, suggesting an isolated and independent nature of the HCV RNA replication complex. In contrast, a high level of competition between replicons was observed, such that the presence and increased fitness of one replicon reduced the capacity of a second one to stably replicate. These results suggest that at least one factor in Huh7 cells required for HCV RNA replication is limiting and saturable.     

Protein-protein interactions between hepatitis C virus nonstructural proteins

Replication of the hepatitis C virus (HCV) genome has been proposed to take place close to the membrane of the endoplasmic reticulum in membrane-associated replicase complexes, as is the case with several other plus-strand RNA viruses, such as poliovirus and flaviviruses. The most obvious benefits of this property are the possibility of coupling functions residing in different polypeptidic chains and the sequestration of viral proteins and nucleic acids in a distinct cytoplasmic compartment with high local concentrations of viral components. Indeed, HCV nonstructural (NS) proteins were clearly colocalized in association with membranes derived from the endoplasmic reticulum. This observation, together with the demonstration of the existence of several physical interactions between HCV NS proteins, supports the idea of assembly of a highly ordered multisubunit protein complex(es) probably involved in the replication of the viral genome. The objective of this study, therefore, was to examine all potential interactions between HCV NS proteins which could result in the formation of a replication complex(es). We identified several interacting viral partners by using a glutathione S-transferase pull-down assay, by in vitro and ex vivo coimmunoprecipitation experiments in adenovirus-infected Huh-7 cells allowing the expression of HCV NS proteins, and, finally, by using the yeast two-hybrid system. In addition, by confocal laser scanning microscopy, NS proteins were clearly shown to colocalize when expressed together in Huh-7 cells. We have been able to demonstrate the existence of a complex network of interactions implicating all six NS proteins. Our observations confirm previously described associations and identify several novel homo- and heterodimerizations.     

Hepatocellular carcinoma in Japan and its linkage to infection with hepatitis C virus

Hepatitis B and C viruses are closely related to the development of liver cirrhosis and hepatocellular carcinoma. Recently these two viral agents were found to be the major causative agents of hepatocellular carcinoma in Japan. An increase in the number of HCV antibody-positive patients, but a decrease in the number of HBs antigen-positive patients with hepato cellular carcinoma has been noted over the last 15 years. In the late 1980s about 70% of the patients with hepatocellular carcinoma were found to be positive for HCV antibody and 24% for HBs antigen. Presence of several subtypes of HCV was reported. Hypervariable region (HVR) in a putative envelope, gp70, was detected within the same subtype. Variability of HVR seemed to be the result of spontaneous mutation caused after infection. Such changes with time in the sequence of the HCV genome in the blood of patients with type C hepatitis are likely to be due to immunological surveillance by the host.     

The molecular biology of hepatitis C virus

Hepatitis C virus (HCV) is one of the main causative agents for transfusion associated- and sporadic cases of non-A, non-B hepatitis throughout the world. HCV has a positive strand RNA of about 9400 nucleotides, as its genome, whose organization is similar to those of animal pestiviruses or human flaviviruses. In spite of the lack of the effective replication system in tissue culture cells, parts of the viral genome were expressed under the control of foreign promoters and the synthesized viral protein has been used for diagnostic assays.     

Analysis of molecular interactions of the p53-family p51(p63) gene products in a yeast two-hybrid system: homotypic and heterotypic interactions and association with p53-regulatory factors

p51 in the p53 tumor suppressor family, also referred to as p63, encodes multiple isoforms including p51A (TAp63gamma) and p51B (TAp63alpha). The p53 protein forms a tetramer, and its stability and activity are regulated by molecular association with viral and cellular proteins and by biochemical modifications. Using a yeast two-hybrid system, the p51A and p51B isoforms were examined for homotypic and heterotypic interactions in the p53 family proteins and for their affinity to the p53-regulatory factors. Results indicate a homotypic interaction dependent on the presumed oligomerization domain of the p51 proteins. The possibility of a weak heterotypic interaction between p51 and p73 proteins was suggested, while association between p51 and p53 appeared improbable. Furthermore, unlike p53, the p51 proteins failed to display an affinity to SV40 large T antigen or MDM2-family proteins. Having several features in common with p53, the p51 proteins may function in biological processes apart from p53.     

Salient molecular features of hepatitis C virus revealed

Hepatitis C virus (HCV) is a positive strand RNA virus with a narrow host and tissue tropism. It ranks among the most significant of human pathogens, causing inflammation, scarring and cancer of the liver. Recent investigations have shed light on some of the salient molecular features of this virus. These include a requirement for CD81 (a tetraspanin transmembrane protein for viral entry), a novel mechanism for the initiation of RNA synthesis, phosphorylation of a viral protein in the regulation of RNA amplification and virus assembly and, finally, a viral protease suppressing activation of the innate immune response in infected cells.     

Hepatocellular carcinoma in urban born blacks: frequency and relation to hepatitis B virus infection.

Chronic hepatitis B virus infection is far less common in urban born than in rural born southern African blacks, who also have a high incidence of hepatocellular carcinoma. A case-control study was carried out to determine the relative frequency of hepatocellular carcinoma and its relation to hepatitis B virus infection in urban born blacks. Three hundred and ninety two black patients with hepatocellular carcinoma and matched controls seen at two city hospitals were classified by questioning as urban born or rural born. The ratio of rural born to urban born blacks among the controls was 1.1:1.0 (207/185), whereas in the patients with cancer the ratio was 4.8:1.0 (324/68) (p less than 0.0001). Analysis of the prevalence of hepatitis B markers in 62 urban born and matched rural born blacks with hepatocellular carcinoma showed no differences in the frequency of current or past hepatitis B virus infection. It is concluded that urban born blacks are less likely than rural born blacks to develop hepatocellular carcinoma, but when they do the tumour is equally likely to be related to infection with hepatitis B virus. The findings lend further support to an important role for chronic hepatitis B virus infection in the aetiology of hepatocellular carcinoma.     

Analysis of hepatitis C virus RNA dimerization and core-RNA interactions

The core protein of hepatitis C virus (HCV) has been shown previously to act as a potent nucleic acid chaperone in vitro, promoting the dimerization of the 3′-untranslated region (3′-UTR) of the HCV genomic RNA, a process probably mediated by a small, highly conserved palindromic RNA motif, named DLS (dimer linkage sequence) [G. Cristofari, R. Ivanyi-Nagy, C. Gabus, S. Boulant, J. P. Lavergne, F. Penin and J. L. Darlix (2004) Nucleic Acids Res., 32, 2623–2631]. To investigate in depth HCV RNA dimerization, we generated a series of point mutations in the DLS region. We find that both the plus-strand 3′-UTR and the complementary minus-strand RNA can dimerize in the presence of core protein, while mutations in the DLS (among them a single point mutation that abolished RNA replication in a HCV subgenomic replicon system) completely abrogate dimerization. Structural probing of plus- and minus-strand RNAs, in their monomeric and dimeric forms, indicate that the DLS is the major if not the sole determinant of UTR RNA dimerization. Furthermore, the N-terminal basic amino acid clusters of core protein were found to be sufficient to induce dimerization, suggesting that they retain full RNA chaperone activity. These findings may have important consequences for understanding the HCV replicative cycle and the genetic variability of the virus.     

Microinjection technique used to study functional interaction between p53 and hepatitis B virus X gene in apoptosis

Microinjection of expression vectors into cultured cells has been utilized to study functional interaction of p53 and the hepatitis B virus HBx gene in apoptosis. This approach allows us to determine protein-protein interactions in primary cultured human cells at a single cell level, including fibroblasts, mammary epithelial cells, renal epithelial cells, and hepatocytes. In principle, this approach can be used to study functional interaction of p53 and any gene that is either pro- or anti-apoptotic. The use of primary cultured human cells minimizes ambiguous results associated with immortalized or tumorigenic cell lines. Moreover, it is an easy and effective way to introduce genes of interests into primary human cells with defined genetic defects, thereby facilitating the delineation of genetic pathways.     

Interplay between hepatitis C virus and ARF4

<正>Dear Editor,Hepatitis C virus(HCV)is a major cause of chronic liver diseases and hepatocellular carcinoma(HCC)with about71 million people globally infected.HCV encodes only10 viral proteins and its replication relies on host proteins.Many host factors including ADP-ribosylation factors     

Single-point mutations of hepatitis C virus NS3 that impair p53 interaction and anti-apoptotic activity of NS3

The N-terminal domain of NS3 of hepatitis C virus (HCV) possesses serine protease activity, which is essential for virus replication. This portion is also implicated in malignant transformation of hepatocytes. We previously demonstrated that an N-terminal portion of NS3 formed a complex with the tumor suppressor p53 and suppressed actinomycin D-induced apoptosis. We report here that single-point mutations of NS3 at position 106 from Leu to Ala (L106A), and position 43 from Phe to Ala (F43A) to a lesser extent, significantly impaired complex formation with p53. Moreover, the L106A mutation impaired an otherwise more distinct anti-apoptotic activity of NS3. F43A and L106A mutations also inhibited serine protease activity of NS3. These results collectively suggest the possibility that Leu106 and Phe43 are involved in p53 interaction and serine protease activity, and therefore, can be a good target for certain low-molecular-weight compound(s) to inhibit both oncogenic and replicative abilities of HCV.     

Biophysical characterization of hepatitis C virus core protein: implications for interactions within the virus and host

A primary function of the hepatitis C virus (HCV) core protein is to package the viral genome within a nucleocapsid. In addition, core protein has been shown to interact with more than a dozen cellular proteins, and these interactions have been suggested to play critical roles in HCV pathogenesis. A more complete knowledge of the biophysical properties of the core protein may help to clarify its role in HCV pathogenesis and nucleocapsid assembly and provide a basis for the development of novel anti-HCV therapies. Here we report that recombinant mature core protein exists as a large multimer in solution under physiological conditions. Far-UV circular dichroism (CD) experiments showed that the mature core protein contains stable secondary structure. Studies with truncated core protein demonstrated that the C-terminal region of the core protein is critical for its folding and oligomerization. Intrinsic fluorescence spectroscopy and near-UV CD analysis indicated that the tryptophan-rich region (residues 76-113) is largely solvent-exposed and not likely responsible for multimerization of the mature core protein in vitro.