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

To develop a safe and broad-spectrum effective hepatitis C virus (HCV) T cell vaccine,we constructed the recombinant adenovirus-based vaccine that carried the hepatitis C virus truncated NS3 and core fusion proteins. The expression of the fusion antigen was confirmed by in vitro immunofluorescence and western blotting assays. Our results indicated that this vaccine not only stimulated antigen-specific antibody responses,but also activated strong NS3-specific T cell immune responses. NS3-specific IFN-γ+ and TNF-α+ CD4+ T cell subsets were also detected by a intracellular cytokine secretion assay. In a surrogate challenge assay based on a recombinant heterologous HCV (JFH1,2a) vaccinia virus,the recombinant adenovirus-based vaccine was capable of eliciting effective levels of cross-protection. These findings have im- portant implications for the study of HCV immune protection and the future development of a novel vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; CD4-Positive T-Lymphocytes ; immunology ; Cross Protection ; Female ; Genetic Vectors ; biosynthesis ; genetics ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Humans ; Interferon-gamma ; immunology ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; administration & dosage ; genetics ; immunology ; Viral Core Proteins ; administration & dosage ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; genetics ; immunology ; Viral Nonstructural Proteins ; administration & dosage ; genetics ; immunology

OBJECTIVETo explore the effects of hepatitis C virus (HCV) core protein on the activity of double-stranded RNA-dependent protein kinase (PKR).

METHODSThe human hepatoma cell line BEL-7402 was transfected with the HCV core gene-containing eukaryotic expression vector pCMH6K-Core (at various concentrations), or empty vector, or no vector; a group of cells was co-transfected with the luciferase reporter plasmid pGL3-promoter. The cells were treated with interferon (IFN) a-2b to induce the expression and activation of endogenous PKR, or left untreated to serve as controls. The effect of core protein on PKR phosphorylation was detected by western blotting. Luciferase activity was detected to reflect effects of the core protein on the synthesis of cellular proteins. The t-test and F test were used for statistical analyses.

RESULTSIn the case of IFNa stimulation, PKR phosphorylation levels were significantly lower in the HCV core protein expressing cells than in the cells transfected with empty plasmid or with no vector, but the total PKR expression level was not significantly different among these three groups of cells. Cells co-transfected with luciferase plasmid and the core protein expressing vector showed significantly higher levels of luciferase expression than the cells co-transfected with the empty vector. Moreover, the luciferase activity and core protein expression levels increased in a dose-dependent manner, with the luciferase activity of the cells treated with 0.5 mug, 1.0 mug and 1.5 mug pCMH6K-Core being 1.941 ± 0.199 times, 2.868 ± 0.275 times and 3.839 ± 0.338 times higher than that of the empty vector group (all P < 0.05).

CONCLUSIONIn the human hepatoma cell line BEL-7402, the HCV core protein can inhibit the activity of endogenous PKR, thereby promoting cell protein synthesis.

Cell Line, Tumor ; Genes, Reporter ; Genetic Vectors ; Humans ; Phosphorylation ; Protein Biosynthesis ; RNA-Binding Proteins ; metabolism ; Transfection ; Viral Core Proteins ; genetics ; metabolism

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

Nucleoprotein (NP) of influenza virus is highly conserved and type-specific. NP can trigger strong cell-mediated immune responses in host and is involved in the protection against the challenges with different subtype influenza viruses. Here, NP of an avian H5N1 (A/Hubei/1/2010, HB) was expressed by baculovirus surface-display technology and its immunogenicity as well as protective mechanism was investigated in mice infection model. Western blot and immunolabeled electron microscopy assay showed NP was displayed on baculovirus surface. ELISA results showed NP could induce high level of anti-NP IgG in the sera from NP-Bac-inoculated mice. Two cellular immune peptides (NP57-74 IQNSITIERMVLSAFDER and NP441-458 RTEIIKMMESARPEDLSF) were identified by IFN-gamma ELISPOT assay. NP57-66 and NP441-450 and NP protein could be able to trigger the activation of CD4+ and CD8+ T cells, and the response of CD8+ T was more predominant. The challenge study of mice-adapted virus A/PR/8/34 (H1N1) showed that NP-Bac could reduce viral load and attenuate the damage to lung tissue. 50% protection ratio against the virus could be detected.
Animals ; Antibodies, Viral ; immunology ; Baculoviridae ; genetics ; metabolism ; Cross Protection ; Enzyme-Linked Immunospot Assay ; Female ; Humans ; Immunity, Cellular ; Influenza A Virus, H1N1 Subtype ; genetics ; immunology ; Influenza A Virus, H5N1 Subtype ; genetics ; immunology ; Influenza, Human ; immunology ; virology ; Mice ; Mice, Inbred BALB C ; RNA-Binding Proteins ; genetics ; immunology ; T-Lymphocytes ; immunology ; Viral Core Proteins ; genetics ; immunology

Avian influenza virus subtype H9N2 has been circulating in multiple terrestrial birds and repeatedly infecting mammals, including swines and humans to pose a significant threat to public health. The cross-species infection of human, replication activity and tissue tropism of avian influenza virus H9N2 was evaluated in this study. The results showed that surgically removed human lung tissue samples were infected ex vivo by avian influenza virus subtype H9N2 (Ck/GX/1875/04, Ck/GX/187/05) and seasonal human influenza virus H3N2 (A/ST/602/05). Examination of nucleoprotein expression replication in the infected human lung tissue samples showed that the replication of avian influenza virus H9N2 and seasonal human influenza virus H3N2 were mainly prevalent in alveolar epithelial cells, respiratory bronchiole epithelial cells and bronchial epithelial cells. Double-immunostaining for viral antigens and cellular markers indicated that avian influenza virus subtype H9N2 replicated in type 2 alveolar epithelial cells. These findings suggest that the H9N2 virus may be better adapted to the human host and replicates efficiently in human lung epithelial cells. Moreover, H9N2 avian influenza virus repeatedly infecting human, may favor gene evolution and the potential emergence of pandemic influenza virus.
Animals ; Epithelial Cells ; virology ; Humans ; Influenza A Virus, H3N2 Subtype ; genetics ; physiology ; Influenza A Virus, H9N2 Subtype ; genetics ; isolation & purification ; physiology ; Influenza, Human ; virology ; Lung ; cytology ; virology ; RNA-Binding Proteins ; genetics ; metabolism ; Viral Core Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo investigate the biological function of the hepatitis C virus (HCV)-encoded F protein in hepatocytes.

METHODSThe full-length F gene was amplified by PCR from HCV genotype 1a and cloned into plasmid pSEB-3Flag by restriction enzyme digestion and ligation. Hepatoma cell lines, Huh7 and SMMC7721, were transfected with the resultant recombinant pSEB-3Flag-F or the original pSEB-3Flag (negative control) and screened with the selective antibiotic, blasticidin. Stable F gene and protein expression was verified by RT-PCR analysis. Analysis of cell growth and cell cycle was carried out by MTS assay, crystal violet staining and flow cytometry.

RESULTSHuh7 and SMMC7721 cells transfected with pSEB-3Flag-F plasmid (Huh7-F and SMMC7721-F, respectively) uniquely expressed the F gene and protein. The Huh7-F and SMMC7721-F cells showed significantly decreased proliferation rates, compared to the respective control groups. A similar HCV F-mediated growth-inhibiting activity was observed by the cell viability assay. Furthermore, cell cycle analysis revealed that the S-phase distribution was much lower in Huh7-F (47.12%) and SMMC7721-F (30.75%) cells than in the respective controls (55.35% and 33.23%, respectively) (P less than 0.05).

CONCLUSIONStable expression of the HCV F gene reduced the in vitro proliferation rate of hepatoma cell lines, indicating that the F protein may function as a growth inhibitor of infected cells.

Carcinoma, Hepatocellular ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Hepacivirus ; genetics ; Humans ; Liver Neoplasms ; metabolism ; pathology ; Reverse Transcriptase Polymerase Chain Reaction ; Viral Core Proteins ; genetics ; metabolism

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

OBJECTIVETo investigate the effect of core protein of hepatitis C virus (HCV) on the expression of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF).

METHODSHuh7.5.1 cells were transfected with plasmid flag2B-core carrying HCV core gene, expression of HIF-1alpha and VEGF were measured by reverse transcription-polymorphism chain reaction (RT-PCR) and western blot. Enzyme link immunoabsorbent assay (ELISA) were used to detect the level of VEGF in the supernatants.

RESULTSThe expression of HIF-1alpha and VEGF mRNA and protein were upregulated after flag2B-core was transfected into Huh7.5.1 cells, and VEGF level in the supernatant was significant elevated as compared to controls [(654.5+/-43.7) pg/ml vs (365.9+/-26.8) pg/ml, t = 653.1%, P less than 0.01]. The expression of HIF-1alpha and VEGF mRNA and protein were downregulated after flag2B-core and HIF-1alpha siRNA were co-transfected into Huh7.5.1 cells, and VEGF level in the supernatant was significantly reduced as compared to controls [(389.2+/-29.6) pg/ml vs (768.8+/-47.3)pg/ml, t = 1330.22, P less than 0.01].

CONCLUSIONSHCV core protein enhances the expression of HIF-1alpha and VEGF. HCV may regulate the expression of HIF-1alpha and VEGF via the core protein.

Cell Line, Tumor ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; RNA, Small Interfering ; Transfection ; Vascular Endothelial Growth Factor A ; metabolism ; Viral Core Proteins ; genetics

Animals ; Antiviral Agents ; pharmacology ; therapeutic use ; DNA-Directed RNA Polymerases ; antagonists & inhibitors ; Drug Discovery ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; metabolism ; Humans ; Influenza A virus ; drug effects ; genetics ; metabolism ; Influenza, Human ; drug therapy ; Molecular Targeted Therapy ; Neuraminidase ; antagonists & inhibitors ; RNA-Binding Proteins ; antagonists & inhibitors ; Signal Transduction ; drug effects ; Viral Core Proteins ; antagonists & inhibitors ; Viral Matrix Proteins ; antagonists & inhibitors