OBJECTIVETo screen and identify proteins that interact with hepatitis C virus NS3 by means of T7-phage display system.

METHODSHepatitis C virus NS3 was expressed by prokaryotic expression and used as a selected molecule to biopan the T7 select human liver cDNA library; the selected positive clones were identified using DNA sequencing and analyzed with BLAST program in GenBank.

RESULTSAfter BLAST analysis in all the positive clones, the proteins which interacted with the hepatitis C virus NS3 were found to be serpin peptidase inhibitor, clade A, member 1 (SERPINA1) and cyclophilin-LC.

CONCLUSIONT7-phage display system is a convenient, rapid and effective method for screening interacting proteins. The proteins thus selected will provide an important means for studying the pathogenesis and carcinogenesis of HCV.

Cell Line ; Gene Library ; Hepacivirus ; metabolism ; Humans ; Peptide Library ; Protein Interaction Mapping ; methods ; Viral Fusion Proteins ; genetics ; isolation & purification ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

Herpesviridae is a large family comprising linear, double-stranded DNA viruses. Herpesviridae contains three subfamilies: α-, β- and γ-herpesviruses. The glycoproteins gB, gH and gL of each subfamily form the "core fusion function" in cell-cell fusion. Other herpesviruses also need additional glycoproteins to promote fusion, such as gD of the Herpes simplex virus, gp42 of the Epstein-Barr virus, and gO or UL128-131 of the Human cytomegalovirus. In contrast, glycoproteins gM or gM/gN of herpesvirus inhibit fusion. We describe the molecular mechanisms of glycoprotein-induced fusion and entry of herpesviruses. It will be helpful to further study the pathogenic mechanism of herpesvirus.
Animals ; Cell Fusion ; Glycoproteins ; genetics ; metabolism ; Herpesviridae ; genetics ; metabolism ; Herpesviridae Infections ; physiopathology ; virology ; Humans ; Viral Proteins ; genetics ; metabolism

BACKGROUNDTo clone the type common antigen gD of human herpes simplex virus I, II (HSV-I, II), the authors constructed recombinant expression vector Pmal-c2/gD and induced to express the fusion protein MBP-gD.

METHODSThe authors extracted HSV DNA,amplified gD gene by PCR assay and directly cloned it into prokaryotic expression vector pMAL-c2, then transformed it into E.coli DH5alpha. After proved to be correct by PCR, double enzyme digestion and sequencing, the fusion protein is induced to express by IPTG and detected by both Western blot and ELISA.

RESULTSThe constructed expression vector pMAL-c2/gD can be expressed with high efficiency. The product expressed was about 35.5% of the total bacterium proteins by SDS?PAGE analysis and was found nearly 39% as soluble protein,61% as inclusion in cytoplasm.

CONCLUSIONSThe authors constructed recombinant expression vector pMAL-c2/gD, the Western blotting result showed that the recombinant protein could be identified with gD specific monoclonal antibody DL6. Therefore the protein was of natural antigenic structure of gD.

Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Humans ; Plasmids ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; Viral Envelope Proteins ; biosynthesis ; genetics

FMDV 2A peptide was introduced as a linker between GP5 and M protein of porcine reproduction and respiratory syndrome virus (PRRSV) to allow automatic self-cleavage the polyproteins. This strategy simultaneously displayed the neutralizing action of GP5 protein and cell-mediated immunity of M protein. We put them into the expression cassette of adenovirus vector. The results of RT-PCR, IFA and Western blotting showed that GP5 and M protein were not only expressed correctly, but also self-cleavaged and assemble heterodimers formation. To detect the advantages of rAd-GP5-2A-M, we also constructed some other recombinant adenoviruses (rAd-GP5, rAd-M and rAd-GP5-M) as control. After inoculated subcutaneously into BALB/c mice, the four recombinant adenoviruses can induce PRRSV-specific antibodies and cell-mediated immune response, but the level of humoral and cell-mediated immune response against PRRSV induced by rAd-GP5-2A-M is the strongest among the four recombinant adenoviruses. All of these suggested that it is possible to develop one multi-gene engineering vaccine utilizing FMDV 2A peptide, and also provided a novel strategy for developing other viral disease vaccine.
Adenoviridae ; genetics ; metabolism ; Animals ; Female ; Immunization ; Mice ; Mice, Inbred BALB C ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Swine ; Vaccines, Synthetic ; immunology ; Viral Envelope Proteins ; genetics ; immunology ; metabolism ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Viral Vaccines ; immunology

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Encephalitis Virus, Japanese ; enzymology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; Protease Inhibitors ; chemistry ; RNA Helicases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Serine Endopeptidases ; metabolism ; Viral Nonstructural Proteins ; metabolism

Influenza C virus, a member of the Orthomyxoviridae family, causes flu-like disease but typically only with mild symptoms. Humans are the main reservoir of the virus, but it also infects pigs and dogs. Very recently, influenza C-like viruses were isolated from pigs and cattle that differ from classical influenza C virus and might constitute a new influenza virus genus. Influenza C virus is unique since it contains only one spike protein, the hemagglutinin-esterase-fusion glycoprotein HEF that possesses receptor binding, receptor destroying and membrane fusion activities, thus combining the functions of Hemagglutinin (HA) and Neuraminidase (NA) of influenza A and B viruses. Here we briefly review the epidemiology and pathology of the virus and the morphology of virus particles and their genome. The main focus is on the structure of the HEF protein as well as on its co- and post-translational modification, such as N-glycosylation, disulfide bond formation, S-acylation and proteolytic cleavage into HEF1 and HEF2 subunits. Finally, we describe the functions of HEF: receptor binding, esterase activity and membrane fusion.
Animals ; Cattle ; Dogs ; Hemagglutinins, Viral ; chemistry ; metabolism ; Influenzavirus C ; physiology ; Orthomyxoviridae Infections ; metabolism ; virology ; Protein Conformation ; Protein Folding ; Protein Processing, Post-Translational ; Viral Fusion Proteins ; chemistry ; metabolism

LTB gene fragment was amplified by PCR from plasmid pMDTLT, and a recombinant plasmid pETLTBVP1 was constructed by inserting LTB gene fragment into VP1 gene expression plasmid pETVP1 constructed previously. The recombinant plasmids were transformed into E. coli BL21(DE3) and induced to express by IPTG. The recombinant protein existed in the inclusion body and its molecular weight was about 39 kD proved by SDS-PAGE analysis. Western blotting showed that the fusion protein could be reacted with both anti-FMDV and anti-cholera toxin serum demonstrating the immunoactivity of the fusion protein. Strong immune responses can be induced in mice inoculated with the fusion protein intraperitoneally, and the serum antibody level is higher than that of commercial foot-and-mouth disease vaccines.
Animals ; Antibodies, Viral ; blood ; Bacterial Toxins ; genetics ; immunology ; metabolism ; Capsid Proteins ; genetics ; immunology ; metabolism ; Enterotoxins ; genetics ; immunology ; metabolism ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; Female ; Gene Fusion ; genetics ; Mice ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo analyze the interaction of hepatitis C virus (HCV) core protein with HCBP1 and observe the expression and cellular localization of HCBP1.

METHODSThe cDNA fragments encoding HCV core protein and HCBP1 were amplified by PCR and subsequently cloned into pGEM T vector, respectively. After sequence verification, the two recombined vectors were respectively subcloned into two hybrid plasmids, pM and pVP16. pM-core, pVP16- HCBP1 and the reporter vector pG5CAT were co-transfected into COS-7 cells, and the interaction between HCV core protein and HCBP1 was assayed by detecting CAT gene expression after 48 h. The expression and subcellular localization of the fusion protein in the transfected COS-7 cells were analyzed by Western blotting and fluorescence microscopy, respectively.

RESULTSCAT-ELISA showed that the absorbance of the co-transfection group was significantly higher than that o f the negative control groups but lower than that of the positive control group. Western blotting confirmed the expression of fusion protein in the transfected COS-7 cells. Fluorescence microscopy showed that the fusion protein was distributed mainly in the cytoplasm, and in contrast, diffuse EGFP expression was detected in COS-7 cells transfected with the empty vector.

CONCLUSIONMammalian two-hybrid assay confirms the capacity of HCBP1 to bind HCV core protein, and the expression vector for HCBP1-EGFP fusion gene has been constructed successfully and expressed in COS-7 cells.

Animals ; Base Sequence ; COS Cells ; Cercopithecus aethiops ; Genetic Vectors ; Molecular Sequence Data ; Plasmids ; Protein Binding ; Receptors, Virus ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Transfection ; Viral Core Proteins ; metabolism

To investigate the transgenic expressing efficacy of helper-dependent adenoviral vector (HDAd) in vitro, we constructed a HDAd encoding enhanced green fluorescent protein (EGFP), denominated as HDAd/EGFP, performed large scale preparation and purification, and then identified the purified HDAd/EGFP under fluorescent microscope and electron microscope. After the concentration of HDAd/EGFP was determined by spectrophotometer, the transgenic expression efficiency of HDAd/EGFP was compared with first generation adenoviral vector encoding EGFP (FGAd/EGFP) in vitro. Therefore, we infected A549 cells with 2000 virus particles (vp) per cell by HDAd/EGFP and FGAd/EGFP respectively and analyzed EGFP expressing level by flow cytometry. Consequently, the fluorescent expression rate and fluorescent intensity of EGFP were higher in early infected A549 cells by HDAd/EGFP than by FGAd/EGFP. HDAd, capable of expressing transgene instantly and efficiently in vitro, is a potential vaccine vector.
Adenoviridae ; genetics ; metabolism ; Cell Line, Tumor ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; genetics ; Helper Viruses ; genetics ; metabolism ; Humans ; Transgenes ; Viral Fusion Proteins ; genetics ; metabolism

The respiratory syncytial virus (RSV) is one of the most common causes of acute infection of the lower respiratory tract among children. For viruses in the Paramyxoviridae subfamily, membrane fusion requires a specific interaction between two glycoproteins: the fusion protein and attachment protein. Membrane fusion of the RSV appears to be unique among paramyxoviruses in that fusion is accomplished by the fusion protein alone without help from the attachment protein. Here, we review recent achievements and advances in the study of membrane fusion triggered by the RSV published in high-impact-factor journals. We also review and make a comparative analysis of the popular hypotheses regarding membrane fusion of the RSV. Finally, we discuss the "hot topics" in current research and controversial data published in recent years in the hope of providing references for Chinese researchers.
Animals ; Humans ; Respiratory Syncytial Virus Infections ; virology ; Respiratory Syncytial Viruses ; genetics ; physiology ; Viral Fusion Proteins ; genetics ; metabolism ; Virus Internalization