OBJECTIVETo investigate the biological role of auto-induced expression of hepatitis C virus (HCV) core protein (protein C) using a recombinant protein in an in vitro cell-based system.

METHODSThe PCR-amplified full-length HCV protein C gene (573 bp) was inserted into the pET28a prokaryotic expression vector. The recombinant plasmid was transformed into BL21(DE3)pLysS E. coli to achieve high-concentration expression of the recombinant C protein by auto-induction. The recombinant protein C was purified by Ni-NTA affinity chromatography, and tested in a protein binding assay for its ability to bind the HCV NS3 protein.

RESULTSThe transformed E. coli produced a large amount of recombinant protein C, as detected in the sonicated supernatant of the bacteria culture. The antigenic reactivity of the recombinant protein C was confirmed by western blotting. However, the recombinant protein C could not be purified by Ni-NTA affinity chromatography, but co-precipitated with the HCV NS3 protein.

CONCLUSIONSoluble recombinant protein C was successfully expressed by auto-induction, and shown to interact with the HCV NS3 protein, which provides a novel insight into the putative biological activity of this factor in HCV-related molecular processes. Future studies of this recombinant HCV protein C's crystal structure and antigenicity may provide further clues to its biological function(s) and potential for clinical applications.

Escherichia coli ; metabolism ; Genetic Vectors ; Hepacivirus ; Recombinant Proteins ; genetics ; metabolism ; Viral Core Proteins ; biosynthesis ; genetics ; metabolism ; Viral Nonstructural Proteins ; metabolism

OBJECTIVETo screen cellular proteins binding to the core region of hepatitis C virus (HCV) from human hepatoma cells.

METHODSUnlabeled and labeled RNA transcripts were prepared by in vitro transcription. Cytoplasmic extracts were prepared from human hepatoma cells HepG2. Ultraviolet (UV) cross-linking was used to screen the cellular proteins that would bind to the core region of HCV. Competition experiment was performed to confirm the specificity of the binding in which excess unlabeled RNA of HCV core region and plasmid RNA were used as competitors.

RESULTSTwo cellular proteins of 6.6 x 10(4) and 5.5 x 10(4) were found binding to the core region of HCV RNA by UV cross-linking assay. The unlabeled core region of HCV RNA could compete out this binding whereas the unlabeled plasmid RNA could not.

CONCLUSIONThe cellular proteins from HepG2 cells could bind to the core region of HCV RNA.

Binding Sites ; Cross-Linking Reagents ; chemistry ; Hepacivirus ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Ultraviolet Rays ; Viral Core Proteins ; genetics ; metabolism

OBJECTIVETo study the inhibition effect of HCV NS5A on p53 protein transactivity and its possible mechanism.

METHODSLuciferase reporter gene system was used for the study of p53 transactivity on p21 promoter and electrophorectic mobility-shift assay (EMSA) was applied to observe whether HCV NS5A could suppress the binding ability of p53 protein to its specific DNA sequence.

RESULTSEndogenous p53 protein could stimulate p21 promoter activity, and the relative luciferase activity increased significantly (3.49 x 10(5) vs 0.60 x 10(5), t = 5.92, P<0.01). Exogenous p53 protein also up-regulated p21 promoter driving luciferase expression, comparing to the control group (0.47 x 10(5)), the relative luciferase activity increased (5.63 x 10(5)) obviously (t = 10.12, P<0.01). HCV NS5A protein inhibited both endogenous and exogenous p53 transactivity on p21 promoter in a dose-dependent manner (F > or = 20.71, P<0.01). In the experiment of EMSA, p53 could bind to its specific DNA sequence, but when co-transfected with HCV NS5A expressing vector, the p53 binding affinity to its DNA decreased.

CONCLUSIONHCV NS5A can inhibit p53 protein transactivity on p21 promoter through its inhibiting of p53 binding ability to the specific DNA sequence.

Hepacivirus ; genetics ; Humans ; Promoter Regions, Genetic ; Transcriptional Activation ; drug effects ; Tumor Suppressor Protein p53 ; drug effects ; genetics ; metabolism ; physiology ; Viral Core Proteins ; genetics ; Viral Nonstructural Proteins ; genetics ; pharmacology

OBJECTIVETo study the roles of different truncated hepatitis C virus (HCV) core proteins (CORE) in the pathogenesis of HCV persistent infection and hepatocellular carcinoma (HCC) and to assess intracellular localization in transiently transfected cells.

METHODSSeven truncated GFP (green fluorescent protein)-CORE fusion protein expression plasmids were constructed, which contained HCV CORE sequences derived from tumor tissues (BT) and non-tumor tissues (BNT) from one patient infected with HCV. Amino acid (aa) lengths were BT: 1-172 aa, 1-126 aa, 1-58 aa, 59-126 aa, 127-172 aa; BNT: 1-172 aa and C191: 1-172 aa respectively. Subcellular localization of CORE-GFP was analyzed by con-focal laser scanning microscope. Apoptosis and necrosis were quantified by flow cytometry.

RESULTSDifferent truncated CORE-GFP localized mainly in the cytoplasm, but nuclear staining was also observed. HCV CORE could induce apoptosis and necrosis, and different truncated COREs could induce cell apoptosis and necrosis at different levels. Among the same length 1-172 aa of BT, BNT and C191, the cell apoptosis and necrosis percentage of BT is highest, and C191 is the lowest (BT>BNT>C191). To the different fragment COREs of BT, N-terminal of CORE induced apoptosis and necrosis higher, compared with that of C-terminal (1-172 aa>1-126 aa>1-58 aa>127-172 aa>59-126 aa).

CONCLUSIONThese results suggest HCV CORE could induce apoptosis and necrosis of cells, which might play an important role in the pathogenesis of HCV persistent infection and HCC and the different CORE domains of different HCV quasi-species might have some difference in their pathogenesis.

Apoptosis ; Cell Line, Tumor ; Hepacivirus ; genetics ; pathogenicity ; physiology ; Humans ; Necrosis ; virology ; Sequence Deletion ; genetics ; Viral Core Proteins ; chemistry ; genetics ; metabolism

OBJECTIVETo identify HCV core protein binding proteins.

METHODSThe library was amplified, purified, and then were transformed into yeast strain Y187. The reconstructed plasmid pGBKT7-core was transformed into yeast strain AH109 and screened on the nutrient deficiency medium SD/-Trp. The transformed AH109 mated with Y187 containing the library plasmid. The diploid yeast cells were plated on nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade and SD/-Trp/-Leu/-His/-Ade containing X-alpha-gal for selecting. The plasmids in diploid yeast cells were extracted and electrotransformed into E.coli DH5alpha. The plasmids in DH5alpha were extracted and sequenced.

RESULTSEleven proteins, including chymotrypsinogen B1 precursor, carboxypeptidase A1, trypsinogen 2, chymotryptic peptide C, trypsin 1, carboxypeptidase B1, kinesin superfamily proteins 3B, trypsin 2, mitochondria protein gene, elastase 3A and colipase were found to be able to bind to HCV core protein.

CONCLUSIONSProteins related with metabolism of glucose and lipid may bind to HCV core protein.

Gene Library ; Hepacivirus ; genetics ; Plasmids ; genetics ; Polymerase Chain Reaction ; Transformation, Genetic ; Two-Hybrid System Techniques ; Viral Core Proteins ; genetics ; metabolism

OBJECTIVETo clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.

METHODSGene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.

RESULTSpET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.

CONCLUSIONWe successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.

Blotting, Western ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; methods ; Humans ; Immunoglobulin M ; immunology ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Viral Core Proteins ; genetics ; immunology ; metabolism

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

To efficiently express nucleoprotein (NP) of influenza A virus A/Jingke/30/95 (H3N2) in E. coli for further immunogenicity study, three forms of NP gene, NP(His) (NP fused with 6 x His tag), NPwt (wild type NP, non-fused NP with native codon) and NP(O) (codon optimized, non-fused NP) were cloned by the technologies of restriction enzyme digestion, PCR, codon optimization and gene synthesis. Three recombinant plasmids were subsequently constructed based on the prokaryotic vector pET-30a, respectively. The comparative studies with these plasmids were carried out on the gene expression efficiency, induction temperature and time, purification process and immune reactivity. It was confirmed by restriction enzyme digestion and sequencing analysis that the three NP genes were inserted into the expression plasmid pET-30a correctly. SDS-PAGE showed that all three forms of NP gene could be efficiently ex pressed in E. coli, among which NP(O) was expressed with the highest expression level. The lower temperature fermentation (T=25 degrees C) and longer time induction (t=10 h) were necessary for high-level expression of protein in soluble form. The purity of tag-free NP was up to 90% through the two-step purification process with anion-exchange and gel filtration chromatography. It was indicated by Western blot that purified NP reacted well with the serum from mice immunized with PR8 virus. These results suggest that the codon-optimized influenza A virus NP gene can be efficiently expressed in E. coli and the expressed NP protein with specific immune reactivity could be purified from the supernatant of bacterial lysate.
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Humans ; RNA-Binding Proteins ; chemistry ; genetics ; isolation & purification ; metabolism ; Solubility ; Viral Core Proteins ; chemistry ; genetics ; isolation & purification ; metabolism

The core protein (CP) of the classical swine fever virus (CSFV) is one of its structural proteins. Apart from forming the nucleocapsid to protect internal viral genomic RNA, this protein is involved in transcriptional regulation. Also, during viral infection, the CP is involved in interactions with many host proteins. In this review, we combine study of this protein with its disorders, structural/functional characteristics, as well as its interactions with the non-structural proteins NS3, NS5B and host proteins such as SUMO-1, UBC9, OS9 and IQGAP1. We also summarize the important part played by the CP in CSFV pathogenicity, virulence and replication of genomic RNA. We also provide guidelines for further studies in the CP of the CSFV.
Animals ; Classical Swine Fever ; virology ; Classical swine fever virus ; genetics ; metabolism ; pathogenicity ; Genome, Viral ; Swine ; Viral Core Proteins ; chemistry ; genetics ; metabolism ; Virulence

OBJECTIVETo establish an experimental model of HCV C-HBV X co-expression protein and explore its effect on the expression of VEGF.

METHODSThe HBV X gene was recovered by enzyme excision and inserted into PBK-CMV and PBK-HCVC, and recombinant plasmids PBK-X and PBK-X-C were constructed. The plasmids PBK-CMV, PBK-X, PBK-HCVC and PBK-X-C were transfected into HepG2 cells with liposomes. After being selected by G418, resistant colonies were obtained. Reverse transcription PCR and Western blot were used to show HBV X and HCV core protein expression. VEGF was analyzed using immunohistochemical methods and Western blot.

RESULTSThe recombinant plasmid PBK-X-C expressed HBV X and HCV core protein efficiently under the control of the vectors promoter. VEGF and VEGF mRNA of the cells co-expressing HCV C-HBV X proteins were higher than those cells expressing HBV X, HCV C and vector alone.

CONCLUSIONHBV X-HCV C co-expression protein can increase the expression of VEGF of HepG2 cells. It suggests that HBV and HCV have a synergic action in the carcinogenesis.

Gene Expression ; Gene Expression Regulation, Viral ; Genetic Vectors ; Hep G2 Cells ; Hepacivirus ; genetics ; Humans ; Trans-Activators ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism ; Viral Core Proteins ; genetics