Amino Acid Sequence ; Coronavirus/drug effects* ; Coronavirus Infections/drug therapy* ; Drug Design ; Humans ; Protein Structure, Secondary ; Spike Glycoprotein, Coronavirus/metabolism* ; Viral Fusion Protein Inhibitors/pharmacology* ; Viral Fusion Proteins/metabolism*

OBJECTIVETo eliminate the side effects of aluminum adjuvant and His-tag, we constructed chimeric VLPs displaying the epitope of EV71 (SP70) without His-tagged. Then evaluating whether the VLPs could efficiently evoke not only humoral but also cellular immune responses against EV71 without adjuvant.

METHODSThe fusion protein was constructed by inserting SP70 into the MIR of truncated HBcAg sequence, expressed in E. Coli, and purified through ion exchange chromatography and density gradient centrifugation. Mice were immunized with the VLPs and sera were collected afterwards. The specific antibody titers, IgG subtypes and neutralizing efficacy were detected by ELISA, neutralization assay, and EV71 lethal challenge. IFN-γ and IL-4 secreted by splenocytes were tested by ELISPOT assay.

RESULTSHBc-SP70 proteins can self-assemble into empty VLPs. After immunization with HBc-SP70 VLPs, the detectable anti-EV71 antibodies were effective in neutralizing EV71 and protected newborn mice from EV71 lethal challenge. There was no significant difference for the immune efficacy whether the aluminum adjuvant was added or not. The specific IgG subtypes were mainly IgG1 and IgG2b and splenocytes from the mice immunized produced high levels of IFN-γ and IL-4.

CONCLUSIONThe fusion proteins without His-tagged was expressed and purified as soluble chimeric HBc-SP70 VLPs without renaturation. In the absence of adjuvant, they were efficient to elicit high levels of Th1/Th2 mixed immune response as well as assisted by aluminum adjuvant. Furthermore, the chimeric VLPs have potential to prevent HBV and EV71 infection simultaneously.

Adjuvants, Immunologic ; Animals ; Antibodies, Neutralizing ; Antibodies, Viral ; blood ; Enterovirus A, Human ; genetics ; Enterovirus Infections ; immunology ; virology ; Epitopes ; immunology ; metabolism ; Escherichia coli ; metabolism ; Female ; Immunity, Cellular ; Immunity, Humoral ; Mice ; Recombinant Fusion Proteins ; immunology

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

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

To develop a recombinant lentivirus co-expressing structural protein of hepatitis C virus (HCV) and secreted Gaussia Luciferase (Gluc), we first constructed an expression vector that encoded HCV structural protein (C, E1, E2) and GLuc named pCSGluc2aCE1E2. The expression of HCV proteins and Gluc was confirmed by an immunofluorescence assay (IFA) and the detection of luciferase activity. Recombinant lentivirus (VSVpp-HCV) was developed by the co-transfection of pCSGluc2aCE1E2 into 293T cells with pHR'CMVA8.2 and pVSVG. The infectivity of VSVpp-HCV was confirmed by luciferase activity detection, IFA and western blotting. Virus-like particles were identified using electron microscopy after concentration. The results showed that the level of luciferase activity correlated with the expression of HCV protein after the infection of cells with lentivirus VSVpp-HCV. Therefore, the expression level of HCV proteins could be evaluated by detecting the luciferase activity of Gluc. In conclusion, this research pave a way for the development of transgenic mice that express HCV proteins and Gluc, which enable the evaluation of anti-HCV therapy and vaccine in vivo.
Animals ; Copepoda ; Genes, Reporter ; Genetic Vectors ; genetics ; metabolism ; Hepacivirus ; genetics ; metabolism ; Hepatitis C ; virology ; Humans ; Lentivirus ; genetics ; metabolism ; Luciferases ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

To prepare virus-like particles containing FluA/B mRNA as RNA standard and control in Influenza RNA detection, the genes coding the coat protein and maturase of E. coli bacteriophage MS2 were amplified and cloned into D-pET32a vector. Then we inserted 6 histidines to MS2 coat protein by QuikChange Site-Directed Mutagenesis Kit to construct the universal expressing vector D-pET32a-CP-His. In addition, the partial gene fragments of FluA and FluB were cloned to the down-stream of expressing vector. The recombinant plasmid D-pET32a-CP-His-FluA/B was transformed to BL21 with induction by IPTG. The virus-like particles were purified by Ni+ chromatography. The virus-like particles can be detected by RT-PCR, but not PCR. They can be conserved stably for at least 3 months at both 4 degrees C and -20 degrees C. His-tagged virus-like particles are more stable and easier to purification. It can be used as RNA standard and control in Influenza virus RNA detection.
Escherichia coli ; genetics ; metabolism ; Influenza A virus ; genetics ; metabolism ; Influenza B virus ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; RNA, Viral ; genetics ; metabolism ; Recombinant Fusion Proteins ; genetics ; metabolism ; Ribonucleases ; chemistry ; Virion ; 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

Heat-shock protein (Hsp) 70 potentiates specific immune responses to some antigenic peptides fused to it. Here, the prokaryotic plasmids harboring the envelope glycoprotein E0 gene of classical swine fever virus (CSFV) and/or the Hsp70 gene of Haemophilus parasuis were constructed and expressed in Escherichia coli Rosseta 2(R2). The fusion proteins were then purified. Groups of Balb/c mice were immunized with these fusion proteins, respectively, and sera collected 7 days after the third immunization. Immune effects were determined via an enzyme-linked immunosorbent assay and flow cytometric analyses. E0-Hsp70 fusion protein and E0+Hsp70 mixture significantly improved the titer of E-specific antibody, levels of CD4+ and CD8+ T cells, and release of interferon-γ. These findings suggested that Hsp70 can significantly enhance the immune effects of the envelope glycoprotein E0 of CSFV, thereby laying the foundation of further application in pigs.
Animals ; Antibodies, Viral ; blood ; CD4-Positive T-Lymphocytes ; cytology ; immunology ; CD8-Positive T-Lymphocytes ; cytology ; immunology ; Cell Proliferation ; Classical swine fever virus ; genetics ; Female ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Haemophilus parasuis ; genetics ; Immunization ; Interferon-gamma ; metabolism ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Recombinant Fusion Proteins ; genetics ; immunology ; Viral Envelope Proteins ; genetics

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

To study the impact of the enterovirus 71(EV71) on the nuclear transport mechanism,The pGFP-NLS vector with nuclear location signal(NLS) was constructed, RD cells transfected by the pGFP-NLS vector were inoculated with the EV71 or cotransfected by EV71-2A vector. The results showed that GFP protein with NLS was expressed in the cytoplasm due to the inhibition of nuclear transport. In order to further study the mechanism of the EV71 to prevent nuclear transport,Nup62 was detected by Western blotting after RD cells were infected with EV71 or transfected by EV71-2A vector. The results showed that decreased expression of Nup62 could be detected after infection with EV71 and transfection by EV71-2A vector. This study demonstrates that the cleavage of Nup62 by EV71 2A protease may be the mechanism of nuclear transport inhibition.
Active Transport, Cell Nucleus ; Cell Line, Tumor ; Cell Nucleus ; metabolism ; Enterovirus A, Human ; enzymology ; genetics ; metabolism ; Enterovirus Infections ; virology ; Gene Expression Regulation, Viral ; Genetic Vectors ; Green Fluorescent Proteins ; metabolism ; Humans ; Membrane Glycoproteins ; metabolism ; Nuclear Localization Signals ; metabolism ; Nuclear Pore Complex Proteins ; metabolism ; Peptide Hydrolases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Transfection