UMLS:C0019204 - FACTA Search

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Query: UMLS:C0019204 (hepatocellular carcinoma)
71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We developed approaches using antisense RNA to inhibit hepatitis C virus (HCV) RNA translation and HCV core protein expression. An HCV genotype 1b cDNA comprising nt 1-1321 or a fusion construct consisting of HCV (nt 1-584) and luciferase cDNAs were inserted downstream of T7 and CMV promoter sequences and used to generate HCV RNA target molecules. Such constructs will produce HCV core or HCV coreluciferase fusion proteins in vitro or within transfected cells. Seven different antisense RNA constructs were designed to target the highly conserved 5' region of HCV RNA at nt positions 1-402. For in vitro experiments, synthesized HCV RNA target sequences and antisense RNAs were mixed at various molar ratios and subsequently translated in a rabbit reticulocyte lysate system. In cell culture studies, the HCV core-luciferase fusion cDNA was co-transfected with antisense RNA-producing constructs into human hepatocellular carcinoma (HCC) cells. Luciferase activity in cell lysates was measured to determine quantitatively antiviral effects within the cell. It was found that translation of HCV RNAs was efficiently inhibited by antisense RNA in vitro. The specificity of this inhibition was confirmed using control target RNA sequences or nonrelevant antisense RNA constructs. Co-transfection studies demonstrated that antisense RNA inhibited HCV core-luciferase fusion protein expression by 41-57% in HuH-7 HCC cells. These studies indicate that antisense RNA will find viral target RNA sequences in HuH-7 cells and inhibit HCV RNA translation. More important, these studies have defined critical viral RNA target sequences susceptible to antisense inhibitory effects within the cell.
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PMID:Antiviral effects of antisense RNA on hepatitis C virus RNA translation and expression. 1002 90

The nucleocapsid core protein of hepatitis C virus (HCV) has been shown to trans-act on several viral or cellular promoters. To get insight into the trans-action mechanism of HCV core protein, a yeast two-hybrid cloning system was used for identification of core protein-interacting cellular protein. One such cDNA clone encoding the DEAD box family of putative RNA helicase was obtained. This cellular putative RNA helicase, designated CAP-Rf, exhibits more than 95% amino acid sequence identity to other known RNA helicases including human DBX and DBY, mouse mDEAD3, and PL10, a family of proteins generally involved in translation, splicing, development, or cell growth. In vitro binding or in vivo coimmunoprecipitation studies demonstrated the direct interaction of the full-length/matured form and C-terminally truncated variants of HCV core protein with this targeted protein. Additionally, the protein's interaction domains were delineated at the N-terminal 40-amino-acid segment of the HCV core protein and the C-terminal tail of CAP-Rf, which encompassed its RNA-binding and ATP hydrolysis domains. Immunoblotting or indirect immunofluorescence analysis revealed that the endogenous CAP-Rf was mainly localized in the nucleus and to a lesser extent in the cytoplasm, and when fused with FLAG tag, it colocalized with the HCV core protein either in the cytoplasm or in the nucleus. Similar to other RNA helicases, this cellular RNA helicase has nucleoside triphosphatase-deoxynucleoside triphosphatase activity, but this activity is inhibited by various forms of homopolynucleotides and enhanced by the HCV core protein. Moreover, transient expression of HCV core protein in human hepatoma HuH-7 cells significantly potentiated the trans-activation effect of FLAG-tagged CAP-Rf or untagged CAP-Rf on the luciferase reporter plasmid activity. All together, our results indicate that CAP-Rf is involved in regulation of gene expression and that HCV core protein promotes the trans-activation ability of CAP-Rf, likely via the complex formation and the modulation of the ATPase-dATPase activity of CAP-Rf. These findings provide evidence that HCV may have evolved a distinct mechanism in alteration of host cellular gene expression regulation via the interaction of its nucleocapsid core protein and cellular putative RNA helicase known to participate in all aspects of cellular processes involving RNA metabolism. This feature of core protein may impart pleiotropic effects on host cells, which may partially account for its role in HCV pathogenesis.
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PMID:Hepatitis C virus core protein interacts with cellular putative RNA helicase. 1007 32

Hepatitis C virus (HCV) is one of the major causative agents of chronic liver disease with the potential for development of hepatocellular carcinoma. The putative core protein of the virus has many intriguing properties, including transcriptional regulation of cellular and unrelated viral promoters. To further characterize the transregulatory function, a number of chimeric constructs were made by fusion of the core gene to the DNA binding domain of the yeast transactivator factor GAL4. The fusion protein exhibited a repressor activity on the herpes simplex virus thymidine kinase promoter via the upstream GAL4 DNA binding sites. A structure /function analysis of HCV core mutants in the context of the GAL4 DNA binding domain revealed that the transcriptional repressor activity was located near the N-terminus (amino acids 26 85). Transcription was strongly inhibited upon transfer of this repressor domain to a heterologous activation domain, (3CGln) of Epstein Barr virus transcription factor EBNA3C. Results from this study suggest that the HCV core protein contains an overall repressor activity, and that the repressor domain is located near the N-terminus.
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PMID:Functional analysis of a transrepressor domain in the hepatitis C virus core protein. 1008 92

Hepatitis B virus (HBV) is one of the major causes of chronic liver diseases and hepatocellular carcinoma. In this study, we used a single chain antibody (sFv), which is a man-made antibody with a strong affinity of immunoglobulin, to inhibit HBV replication. Because HBV replication can only take place in the viral nucleocapsid made of HBV core protein (HBc), we generated anti-HBc sFv and examined whether intracellular anti-HBc sFv could inhibit viral replication in the human hepatoblastoma-derived cell line that produces HBV (HB611). With respect to HBV replication intermediates, both single-stranded and partially double-stranded DNA intermediates were markedly suppressed in the cells expressing anti-HBc sFv, although HBV RNA intermediates were not affected. This suggested that intracellular anti-HBc sFv inhibited HBV DNA replication by inhibiting reverse transcription from HBV pregenome RNA to single-stranded DNA. Because the sFv-HBc complex was detected in the cells expressing anti-HBc sFv by immunoprecipitation analysis but the quantity of intracellular HBc was not affected, the anti-HBc sFv was suggested to inhibit HBV DNA replication by interfering with the function of HBc. These results indicate that intracellular sFv against HBc might be effective as a novel active molecule for gene therapy of hepatitis B.
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PMID:Intracellular single-chain antibody against hepatitis B virus core protein inhibits the replication of hepatitis B virus in cultured cells. 1038 71

Accumulating evidence has demonstrated that aberration of the p53 tumour-suppressor gene is one of the pivotal genetic events in hepatocellular carcinogenesis. Recent reports suggest that the product of hepatitis B virus (HBV) interacts with p53 and that the hepatitis C virus (HCV) core protein reduces p53 expression. A novel p73 gene, which is related to p53, has recently been identified and mapped to chromosome 1p36.3, which is a locus of multiple tumour-suppressor genes for many cancers, including hepatocellular carcinoma (HCC) and neuroblastoma. Here, we investigated mRNA expression, allelotype and mutation of p73 in 48 HCCs obtained from untreated patients. Reverse transcriptase polymerase chain reaction (RT-PCR) revealed that p73 mRNA was expressed ubiquitously at low levels in all the tumour tissues, as well as in the adjacent normal liver tissues. The frequency of p73 loss of heterozygosity was observed in 20% of HCCs, but PCR-single strand conformation polymorphism (SSCP) analysis showed no mutations in the 48 tumours except for three types of polymorphisms. These results suggest that p73 may play a role in hepatocellular carcinogenesis in a different manner from a Knudson two-hit model. The regulatory mechanism of interaction between p73 and hepatitis viruses remains to be determined.
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PMID:Absence of mutation of the p73 gene localized at chromosome 1p36.3 in hepatocellular carcinoma. 1040 9

The functional role of the hepatitis B virus (HBV) pre-S region for assembly and appearance of the virus is not completely understood. In this study, 3 natural-occurring mutants were investigated. Three mutants of the pre-S region-a point mutation in the CCAAT box (MUT1), a 6-bp deletion (MUT2) 3' of the CCAAT box, and a 153-bp deletion (MUT3) in the preS2 domain-were cloned alone or in combinations in replication-competent HBV plasmids and transfected in hepatoma cells. The impact on HBV assembly and appearance was studied by Northern Blot, primer extension analysis, immunofluorescence studies, enzyme-linked immunosorbent assay, and electron microscopy. An inversed ratio of pre-S/S mRNA transcripts compared with wild-type (wt) HBV was found when either MUT1 or -2 were included into the plasmid. Intracellular localization with both mutants showed retention of large S-protein in the endoplasmic reticulum and nuclear accumulation of core protein. The extracellular amount of S-protein was reduced with MUT1 and -2 or combinations in which 1 of the mutants was included. However, the extracellular appearance of viral products was comparable with wtHBV. In contrast, MUT3 showed major changes. Virion-like particles had a fried-egg, and filaments a screw-like appearance. The S-promoter mutations MUT1 and MUT2 correlated with viral retention. MUT3 leads to malformed viral particles. Therefore, different regions in the pre-S domain are essential to determine the intracellular localization and extracellular appearance of HBV, and might contribute to the prognosis of chronic HBV infection.
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PMID:The pre-S region determines the intracellular localization and appearance of hepatitis B virus. 1042 62

Hepatitis C Virus (HCV) causes most cases of posttransfusion hepatitis. Chronic HCV infection is highly related to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Current therapies are only minimally effective and no vaccine has been developed. DNA-based immunization could be of prophylactic and therapeutic value for HCV infection. By intramuscular inoculation in BALB/c mice with an HCV recombinant plasmid pCI-HCV-C, we found significant levels of IgM antibody, but no significant IgG rise. After boost the immunized mice with recombinant HCV-core protein (cp1-10; 1-164aa), the anticore IgG, verified by Western-blotting, rose rapidly, which was two weeks earlier than that with control plasmid. Spleen cells from pCI-HCV-C immunized mice gave higher proliferation index (PI) than control (P < 0.05). The PI of cp1-10 boosted mice was even higher. Proliferation blocking assay with mAb proved the responding cell to be of CD4+ CD8- phenotype, supporting specific priming of T helper cells. A 51Cr-releasing CTL assay specific for HCV-core was developed, and a specific CTL response against HCV-core was demonstrated in both pCI-HCV-C immunized mice and mice boosted with cp1-10. Strong cytotoxic activity against peptide-pulsed p815 cells (H-2d), but not EL-4 cells (H-2b), suggested MHC class I restriction of the CTL activity. Blocking of CTL with mAb proved the effector cells to be of CD4- CD8+. Three CTL epitopes in HCV-core protein were demonstrated. We failed to detect CTL when immunized only with core protein. The results suggested that vaccination with HCV-core derived DNA sequences could be an effective method to induce humoral and cellular immune responses to HCV.
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PMID:Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization. 1046 52

The structure of hepatitis B virus (HBV) nucleocapsids has been revealed in great detail by cryoelectron microscopy. How nucleocapsids interact with surface antigens to form enveloped virions remains unknown. In this study, core mutants with N-terminal additions were created to address two questions: (1) can these mutant core proteins still form nucleocapsids and (2) if so, can the mutant nucleocapsids interact with surface antigens to form virion-like particles. One plasmid encoding an extra stretch of 23 aa, including six histidine residues, fused to the N terminus of the core protein (designated HisC183) was expressed in Escherichia coli and detected by Western blot. CsCl gradient and electron microscopy analyses indicated that HisC183 could self-assemble into nucleocapsids. When HisC183 or another similar N-terminal fusion core protein (designated FlagC183) was co-expressed with a core-negative plasmid in human hepatoma cells, both mutant core proteins self-assembled into nucleocapsids. These particles also retained kinase activity. Using an endogenous polymerase assay, a fill-in HBV DNA labelled with isotope was obtained from intracellular nucleocapsids formed by mutant cores. In contrast, no such signal was detected from the transfection medium, which was consistent with PCR and Southern blot analyses. Results indicate that core mutants with N-terminal extensions can form nucleocapsids, but are blocked during the envelopment process and cannot form secreted virions. The mutant nucleocapsids generated from this work should facilitate further study on how nucleocapsids interact with surface antigens.
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PMID:Hepatitis B viral core proteins with an N-terminal extension can assemble into core-like particles but cannot be enveloped. 1057 58

Assembly of replication-competent hepadnavirus nucleocapsids requires interaction of core protein, polymerase and encapsidation signal (epsilon) with viral pregenomic RNA. The N-terminal portion (aa 1-149) of the core protein is able to self-assemble into nucleocapsids, whereas the C-terminal portion (aa 150-183) is known to interact with pregenomic RNA. In this study, two hepatitis B virus (HBV) core mutants (C144Arg and C144Lys) in which the C-terminal SPRRR (Ser-Pro-Arg-Arg-Arg) motif was replaced by a stretch of arginine or lysine residues were generated to test their role in pregenome encapsidation and virus maturation. Mutant or wild-type core-expression plasmids were co-transfected with a core-negative plasmid into human hepatoma HuH-7 cells to compare trans-complementation efficiency for virus replication. Both low- and high-density capsids were present in -the cytoplasm and culture medium of HuH-7 cells in all transfections. Nucleocapsids formed by C144Arg and C144Lys, however, lost the endogenous polymerase activity to repair HBV DNA. Furthermore, in co-transfection of pHBVC144Arg or pHBVC144Lys with a plasmid which produces replication-competent nucleocapsids, the HBV DNA repairing signal was reduced 40- to 80-fold. This is probably due to formation of mosaic particles of wild-type and mutant cores. Results indicated that the SPRRR motif at the core protein C terminus is important for HBV DNA replication and maturation. Additionally, triple-plasmid transfection experiments showed that nucleocapsids containing various amounts of C144Arg and wild-type core proteins exhibited a bias in selecting a shorter pregenome for encapsidation and DNA replication. It is therefore suggested that unknown factors are also involved in HBV pregenome packaging.
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PMID:Hepatitis B virus maturation is affected by the incorporation of core proteins having a C-terminal substitution of arginine or lysine stretches. 1057 59

Recently we generated a panel of hepatitis B virus core gene mutants carrying single insertions or deletions which allowed efficient expression of the core protein in bacteria and self-assembly of capsids. Eleven of these mutations were introduced into a eukaryotic core gene expression vector and characterized by trans complementation of a core-negative HBV genome in cotransfected human hepatoma HuH7 cells. Surprisingly, four mutants (two insertions [EFGA downstream of A11 and LDTASALYR downstream of R39] and two deletions [Y38-R39-E40 and L42]) produced no detectable capsids. The other seven mutants supported capsid formation and pregenome packaging/viral minus- and plus-strand-DNA synthesis but to different levels. Four of these seven mutants (two insertions [GA downstream of A11 and EHCSP downstream of P50] and two deletions [S44 and A80]) allowed virion morphogenesis and secretion. The mutant carrying a deletion of A80 at the tip of the spike protruding from the capsid was hepatitis B virus core antigen negative but wild type with respect to virion formation, indicating that this site might not be crucial for capsid-surface protein interactions during morphogenesis. The other three nucleocapsid-forming mutants (one insertion [LS downstream of S141] and two deletions [T12 and P134]) were strongly blocked in virion formation. The corresponding sites are located in the part of the protein forming the body of the capsid and not in the spike. These mutations may alter sites on the particle which contact surface proteins during envelopment, or they may block the appearance of a signal for the transport or the maturation of the capsid which is linked to viral DNA synthesis and required for envelopment.
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PMID:Hepatitis B virus core gene mutations which block nucleocapsid envelopment. 1059 84

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