OBJECTIVETo explore the effect of hepatitis B virus (HBV) X protein (HBX) on expression of the host gene Wnt induced secreted protein-1 (WISP-1) that is related to the pathogenic process of hepatocellular carcinoma (HCC).

METHODSTumor and paratumor tissues were collected from HCC patients, and normal liver tissues were collected from healthy controls. Immunohistochemistry was used to evaluate the in vivo presence and expression levels of HBX and WISP-1 in the three tissue types. HepG2 cells stably transfected with pc-DNA3.1(+)-HBX or with pc-DNA3.1(+) only (G0, control) were generated and used to examine in vitro the HBX-induced changes in WISP-1 expression at the mRNA and protein levels by reverse transcription polymerase chain reaction and western blotting, respectively.

RESULTSThe HCC tissues showed significantly higher rates of positivity for WISP-1 expression than the non-tumor controls (76.6% vs. paratumor: 23.4% or normal tissues: 0%, x2= 35.967, P less than 0.01). HBX increased WISP-1 expression in HepG2 cells at both the mRNA (1170.33 +/- 41.26 vs. G0: 265.34 +/- 27.47, t = 31.63, P less than 0.01) and protein (240.33 +/- 11.37 vs. G0: 40.33 +/- 7.09, F = 600.57, P less than 0.01) levels.

CONCLUSIONHBV may up-regulate expression of the host gene WISP-1 through its X protein and thus promote the development of HCC.

Carcinoma, Hepatocellular ; Hepatitis B ; Hepatitis B virus ; metabolism ; Humans ; Liver Neoplasms ; Wnt1 Protein

OBJECTIVETo investigate the cleavage of HBV core protein in vivo by proprotein convertase furin or its family members and observe the intracellular localization of the putative cleaved product.

METHODSRecombinant HBV core protein was incubated with furin under different conditions in vitro, and the reaction was checked with Western blotting. The recombinant vectors expressed the putative cleaved fragment and intact core protein (serves as control) were constructed. The stable expression cell lines were established by transfecting constructs into HepG2 cell line, for which indirect immunofluorescence staining was used by monoclonal anti-HBc against the region shared by core protein and its cleaved product .The confocal microscopy was carried out to observe the intracellular distribution.

RESULTSHBV core protein was cleaved by furin in vitro under different tested conditions. The molecular weight of the major cleaved product just about 15,000 was in concordance with the expectation. The expressed cleaved fragment could react to the monoclonal antibody against core protein, and mainly located in cytosol in particle style just like the intact core protein.

CONCLUSIONHBV core protein can be cleaved by furin in vitro. The major cleaved product has similar antigenicity and subcellular distribution to core protein. These data suggest that proprotein convertase furin or its family members play important roles in HBV replication regulation, and the cleaved product may be involved in antiviral immunity of HBV infection. Further investigations are imperative.

Furin ; metabolism ; Genetic Vectors ; Hep G2 Cells ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Humans ; Microdissection ; Microscopy, Confocal ; Proprotein Convertases ; metabolism ; Transfection ; Virus Replication

Carcinoma, Hepatocellular ; virology ; Hepatitis B virus ; genetics ; pathogenicity ; Humans ; Liver Neoplasms ; virology ; Receptors, Virus ; metabolism ; Virus Integration

No abstract available.
DNA, Circular/*analysis ; Female ; Hepatitis B/*pathology ; Hepatitis B Surface Antigens/*genetics ; Hepatitis B virus/*metabolism ; Humans ; Male

Ebolavirus can cause hemorrhagic fever in humans with a mortality rate of 50%-90%. Currently, no approved vaccines and antiviral therapies are available. Human TIM1 is considered as an attachment factor for EBOV, enhancing viral infection through interaction with PS located on the viral envelope. However, reasons underlying the preferable usage of hTIM-1, but not other PS binding receptors by filovirus, remain unknown. We firstly demonstrated a direct interaction between hTIM-1 and EBOV GP in vitro and determined the crystal structures of the Ig V domains of hTIM-1 and hTIM-4. The binding region in hTIM-1 to EBOV GP was mapped by chimeras and mutation assays, which were designed based on structural analysis. Pseudovirion infection assays performed using hTIM-1 and its homologs as well as point mutants verified the location of the GP binding site and the importance of EBOV GP-hTIM-1 interaction in EBOV cellular entry.
Ebolavirus ; metabolism ; Flow Cytometry ; Glycoproteins ; metabolism ; Hepatitis A Virus Cellular Receptor 1 ; Hepatitis A Virus Cellular Receptor 2 ; Humans ; Membrane Glycoproteins ; metabolism ; Membrane Proteins ; metabolism ; Protein Binding ; Receptors, Virus ; metabolism ; Surface Plasmon Resonance ; Viral Envelope Proteins ; metabolism ; Viral Proteins ; metabolism

OBJECTIVETo investigate the effect of apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G) mediated antiviral activity against hepatitis B virus (HBV) and duck hepatitis B virus (DHBV).

METHODSTotal RNA was extracted from peripheral blood mononuclear cells (PBMCs), RT-PCR product was cloned into the EcoR I/Hind III restriction sites of the CMV-driven expression vector fused with a hemagglutinin fusion epitope tag at its carboxyl terminal. Replication competent 1.3 fold over-length HBV was constructed with full-length HBV of ayw subtype. The mammalian hepatoma cell HepG2 was cotransfected with the replication competent 1.3 fold over-length HBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. Levels of HBsAg and HBeAg in the media of the transfected cells were determined by ELISA, HBV DNA. RNA from intracellular core particles was examined using Northern and Southern blot analyses. Chicken hepatoma cell LMH was cotransfected with head-to-tail dimer of an EcoR I monomer of DHBV and various amounts of CMV-driven expression vector encoding APOBEC3G-HA. DHBV DNA from intracellular core particles was examined using Southern blot analysis.

RESULTSCMV-driven expression vector encoding APOBEC3G-HA and replication competent 1.3 fold over-length HBV were constructed. There was a dose dependent decrease in the levels of intracellular core-associated viral (HBV and DHBV) DNA and extracellular production of HBsAg and HBeAg. Levels of intracellular core-associated viral RNA were also decreased, but the expression of HBcAg remained almost unchanged.

CONCLUSIONAPOBEC3G suppresses HBV and DHBV replication and also suppresses HBsAg and HBeAg expression.

APOBEC-3G Deaminase ; Cytidine Deaminase ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B Virus, Duck ; physiology ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; physiology ; Humans ; RNA, Messenger ; genetics ; Virus Replication

Melamine in combination with cyanuric acid has been considered to be more toxic than either melamine or cyanuric acid alone. The objective of this study was designed to evaluate the combined genotoxicity and cytotoxicity of melamine (M) and cyanuric acid (C) at three mass ratios (1:1, 1:2, 2:1). MC (1:1), MC (1:2), and MC (2:1) were evaluated for their potential genotoxic risk, at gene level by Ames test, and at chromosomal level by micronucleus test. In order to evaluate cytotoxicity in HEK-293 cells, the MTT assay was included. Western blot was also employed to investigate the renal injury molecule-1 (Kim-1) expression in HEK-293 cells exposed to MC. Neither genotoxicity at gene level nor at chromosomal level was observed for MC (1:1), MC (1:2), and MC (2:1). Based on MTT assay, three ratios of MC at 82.5 and 165 µg/mL slightly inhibited viability of HEK-293 cells (P<0.05). MC (1:1) at 41.25 and 82.50 µg/mL could elevate the Kim-1 expression in HEK-293 cells.
Cell Survival ; drug effects ; HEK293 Cells ; Hepatitis A Virus Cellular Receptor 1 ; Humans ; Membrane Glycoproteins ; metabolism ; Receptors, Virus ; metabolism ; Triazines ; pharmacology

OBJECTIVETo study the effect of human spastic paraplegia 21 protein (SPG21) on the replication of hepatitis B virus(HBV) and its regulatory mechanism.

METHODSHBV infectious clone pHBV1.3 and its promoter pHBV-Luc were transfected respectively into HepG2 cells with SPG21 of different concentrations, HBsAg and HBeAg in the supernatants were measured by enzyme linked immunosorbent assay (ELISA), expression of HBV core mRNA and protein were detected by RT-PCR and western blot, covalently closed circular DNA(ccc DNA) levels were measured by real-time PCR, and HBV promoter activity was measured by luminometer fluorescence detector.

RESULTSExpression of HBsAg, HBeAg, HBV core protein and cccDNA were upregulated by SPG21 as well as HBV promoter activity in a dose-dependent approach. The activity of HBV promoter increased to 1.63, 3.09 and 4.66 times in HepG2 cells treated with 50mug/ml, 100mug/ml and 200mug/ml SPG21 respectively during 48 hour-treated ( P less than 0.05), as compared to the control group.

CONCLUSIONSSPG21 can enhance the replication of HBV in HepG2 cells.

Adaptor Proteins, Signal Transducing ; metabolism ; Hep G2 Cells ; Hepatitis B virus ; metabolism ; physiology ; Humans ; Transfection ; Virus Replication

OBJECTIVETo investigate the roles of the hepatitis B virus (HBV)-encoded X protein (HBx), including the full-length and truncated isoforms, and in conjunction with the host-encoded damaged DNA binding protein 1 (DDB1) in HBV replication.

METHODSRecominant expression plasmids carrying the wild-type HBV genome (pGEM-HBV1.2) or with deletion of the full-length HBx protein (pHBV-deltaX), or carrying the full-length HBx protein (pSI-X) or the HBx1to101 (pSI-X1to101) or HBx43to154 (pSI-X43to154) isoforms were constructed for transfection into HepG2 cells. The pcDNA6.2-GW/EmGFP-miR (DDB1-miRNA) vector was constructed for silencing of the DDB1 gene in co-transfected HepG2 cells. At 72 h after transfections, DDB1 silencing was confirmed by western blot analysis and real-time quantitive reverse transcription PCR, HBV DNA copies number was assessed by real time PCR, and levels of hepatitis B surface antigen (HbsAg) and hepatitis B e antigen (HbeAg) were determined by ELISA. Differences between groups was statistically analyzed by single-factor analysis of variance and the t-test.

RESULTSTransfection with pHBV-deltaX led to reductions in DDB1 mRNA (to 52.74% of that in the wild-type pGEM-HBV1.2 transfected cells), HBV replication (to 55.49%), HBsAg level (48.05%), and HBeAg level (46.22%). Co-transfection with pSI-X or pSI-X43to154, but not with pSI-X1to101, restored the pHBV-deltaX-induced reductions in DDB1 mRNA, HBV replication, HBsAg and HBeAg to wild-type levels. The quantity of DDB1 mRNA was approximately parallel with the quantity of HBV DNA copies in all the HepG2 transfection groups.

CONCLUSIONThe COOH-terminal amino acids of HBx are required for HBV replication in hepatocytes, possibly involving the host-encoded DDB1 protein.

DNA-Binding Proteins ; metabolism ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; metabolism ; physiology ; Host-Pathogen Interactions ; Humans ; Protein Isoforms ; metabolism ; Trans-Activators ; metabolism ; Transfection ; Virus Replication

OBJECTIVETo investigate the effect of miR-122 on the expression of hepatitis B virus (HBV) proteins.

METHODSAnti-sense oligodeoxynucleotide (ASODN) of two different sequences against miR-122, anti-miR-122 and LNA-antimiR-122 (Locked nucleic acid), human miR -122 (hsa-miR-122), or the negative control anti-GFP were designed and synthesized, then transfected into HepG2.2.15 cells. After 24 h and 48 h, the levels of HBsAg and HBeAg in the supernatant were determined with a time-resolved immunofluorometric assay (TRFIA). HBV DNA in supernatant and miR-122 in cells were measured by quantitative real-time PCR.

RESULTSAfter 48 h expressions of miR-122 in the LNA-antimiR-122 and anti-miR-122 groups were significantly suppressed and lower than those in the negative control (P<0.001), while the level of miR-122 in the hsa-miR-122 group was higher than that in the negative control (P<0.001). The expression of HBeAg and HBsAg in hsa-miR-122 group was lower than that in the negative control (P<0.01) 24 h and 48 h after transfection. The expression of HBeAg and HBsAg in the anti-miR-122 group and LNA-antimiR-122 group was significantly lower than that in negative control (P>0.001). The levels of viral DNA at both time-points in the various test groups were not significantly different from those of negative control group (P>0.05).

CONCLUSIONmiR-122 may regulate HBV antigens and potentially affect the progress of pathogenesis, which might be the new targets for treatment of HBV infection.

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; Humans ; MicroRNAs ; genetics ; metabolism ; Transfection