According to previous studies of SARS-CoV (Severe acute respiratory syndrome coronavirus), a variety of novel accessory genes have been identified in SARS-CoV genome, which were interspersed the structural genes of SARS-CoV and considered to be unique to the SARS-CoV genome. The predicted unknown proteins (PUPs) encoded by the accessory genes might play important roles in the SARS-CoV infection. Three of those genes, called X4, X5 and ORF10, were synthesized and introduced into E. coli to induce expression. SDS-PAGE and Western blotting revealed that the three genes have been expressed in E. coli. The induction of SARS PUPs genes expression in different temperatures, induction times, IPTG concentrations and A values of E. coli cells were performed. The optimal induction condition of SARS-CoV PUPs genes was characterized according to the orthorgonal analysis. The ratio of recombinant proteins of PUPs to total proteins is as follows: X4, 20%; X5, 27.8%; ORF10, 68.5% under the optimum conditions.
Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Viral ; Genes, Viral ; Genetic Vectors ; Genome, Viral ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; SARS Virus ; genetics ; Temperature ; Time ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

The newly emerged Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic respiratory virus with pathogenic mechanisms that may be driven by innate immune pathways. The goal of this study is to characterize the expression of the structural (S, E, M, N) and accessory (ORF 3, ORF 4a, ORF 4b, ORF 5) proteins of MERS-CoV and to determine whether any of these proteins acts as an interferon antagonist. Individual structural and accessory protein-coding plasmids with an N-terminal HA tag were constructed and transiently transfected into cells, and their native expression and subcellular localization were assessed using Wes tern blotting and indirect immunofluorescence. While ORF 4b demonstrated majorly nuclear localization, all of the other proteins demonstrated cytoplasmic localization. In addition, for the first time, our experiments revealed that the M, ORF 4a, ORF 4b, and ORF 5 proteins are potent interferon antagonists. Further examination revealed that the ORF 4a protein of MERS-CoV has the most potential to counteract the antiviral effects of IFN via the inhibition of both the interferon production (IFN-β promoter activity, IRF-3/7 and NF-κB activation) and ISRE promoter element signaling pathways. Together, our results provide new insights into the function and pathogenic role of the structural and accessory proteins of MERS-CoV.
Cell Line ; Coronavirus ; genetics ; pathogenicity ; Genes, Viral ; Humans ; Interferons ; antagonists & inhibitors ; Open Reading Frames ; Recombinant Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Regulatory and Accessory Proteins ; genetics ; metabolism ; Viral Structural Proteins ; genetics ; metabolism

OBJECTIVE: To investigate the molecular mechanism of Wnt5a and Epstein-Barr virus latent membrane protein 1 (LMP1) aberrant expression in the nasopharyngeal carcinogenesis and to estimate if it can act as a molecular marker for nasopharyngeal cancer (NPC). METHODS: Immunohistochemistry combined with previously made tissue microarrays were used to study the expression of Wnt5a and LMP1 in the nasopharyngeal carcinogenesis tissues. We investigated the role of over expression of Wnt5a and LMP1 in the development and progression of NPC and their relation with the clinicopathological features of NPC and whether they could act as molecular markers in benign and malignant NPC. RESULTS: The positive percentage of Wnt5a and LMP1 protein expression in the NPC was significantly increased as compared with that in atypically hyperplastic nasopharyngeal epithelium, hyperplastic nasopharyngeal epithelium and histologically normal nasopharyngeal epithelium (P<0.05, P<0.01, and P<0.01). Wnt5a and LMP1 proteins were significantly higher in atypically hyperplastic nasopharyngeal epithelium than those in the hyperplastic nasopharyngeal epithelium and normal nasopharyngeal epithelium (P<0.05 and P<0.01). The positive expression of Wnt5a and LMP1 proteins in clinical T3 and T4 staged NPC was higher than that in clinical T1 and T2 staged NPC (P<0.01 and P<0.05). The positive expression of Wnt5a protein in the NPC with lymph node metastasis was higher than that in the NPC without lymph node metastasis (P<0.01). The positive percentage of LMP1 protein was significantly increased in non-keratinizing carcinoma compared with undifferentiated carcinoma and keratinizing carcinoma (P<0.05 and P<0.05). The expression of Wnt5a protein in the NPC had significant positive correlation with LMP1 (r=0.354, P<0.001). Combined molecular phenotype of both Wnt5a and LMP1 expression was a good marker to distinguish NPC from non-cancerous nasopharyngeal epithelium. CONCLUSION The expression of Wnt5a and LMP1 protein in the NPC is positively correlated, and both wnt5a and LMP1 protein play important roles in the nasopharyngeal carcinogenesis either together or successively promoting the malignant transformation of nasopharyngeal epithelium and the development and progression of NPC. Both Wnt5a and LMP1 positive expression may act as good markers for NPC differential diagnosis.
Biomarkers, Tumor ; genetics ; metabolism ; Carcinogenesis ; Humans ; Nasopharyngeal Neoplasms ; genetics ; metabolism ; pathology ; Oncogene Proteins, Viral ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Tissue Array Analysis ; Viral Matrix Proteins ; genetics ; metabolism ; Wnt Proteins ; genetics ; metabolism ; Wnt-5a Protein

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

Six genes for nucleoprotein, phosphoprotein, matrix protein, hemagglutinin neuramindase protein, fusion protein and large protein were obtained by reverse transcription and PCR methods based on our previous work of sequencing full length genome of sendai virus BB1 strain (DQ219803 in GenBank). Sequencing showed the six genes were completely identical to that we reported. In order to supply the function necessary for rescuing and packaging of sendai virus vector in trans, the N, P, M, F, HN and L genes were separately cloned into an adenoviral shuttle expression vector pDC316 resulting in six recombinant adenoviral plasimds. Six replicating defective recombinant adenoviruses Ad5-N, Ad5-P, Ad5-M, Ad5-F, Ad5-HN and Ad5-L were obtained by separately cotransfection of pDC316 carrying N, P, M, F, HN and L genes with the adenoviral genomic plasmid pBHGloxdeltaE1, 3Cre into HEK293cells. Restrictive enzymatic results indicated that the six recombinant plasmids were correctly constructed. PCR results showed the recombinant adenoviruses contained the respective SeV genes . Western blotting as well as immunofluorescence assay indicated the expression of the corresponding proteins of sendai virus. These work laid the basis for the construction of the full length genome plasmid of sendai virus BB1 strain and the setup of SeV virus vector system based on SeV BB1 strain.
Adenoviridae ; genetics ; Animals ; Cell Line ; Cloning, Molecular ; Gene Expression Regulation, Viral ; Genetic Vectors ; genetics ; HN Protein ; genetics ; metabolism ; Humans ; Macaca mulatta ; Nucleoproteins ; genetics ; metabolism ; Phosphoproteins ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Ribosome Subunits, Large ; genetics ; metabolism ; Sendai virus ; genetics ; metabolism ; Viral Fusion Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism

The oncogenic Epstein-Barr virus (EBV) -encoded latent membrane protein 1 (LMP1) enables this virus's long-term survival within the cells of immune system. Mean while, LMP1 also plays a critical role for the transformation of resting B cells by EBV. It initiates the activation of signalling pathways, such as NF-kappaB, mitogen-activated protein kinase (MAPK), and JAK/STAT cascade by adaptor proteins including the tumor necrosis factor (TNF) receptor associated factors (TRAFs) and the TNF receptor associated death domain protein (TRADD). It increases the expression of adhesion molecules LFA-1, ICAM-1, and costimulatory molecule B7-1 of B cells, and regulates the antibody and cytokine secreted by B cells. LMP1 and CD40 have many common properties in signal transduction. Both of them co-localize in lipid rafts for signal transduction. Considering its close relationship with CD40, the research on LMP1 has become a hot spot in the immunology field.
Animals ; B-Lymphocytes ; immunology ; CD40 Antigens ; genetics ; physiology ; Gene Expression Regulation, Viral ; Herpesvirus 4, Human ; genetics ; metabolism ; physiology ; Humans ; Signal Transduction ; Viral Matrix Proteins ; genetics ; physiology

Animals ; Humans ; Influenza A virus ; immunology ; Influenza Vaccines ; immunology ; Protein Engineering ; Viral Matrix Proteins ; chemistry ; genetics ; immunology ; metabolism

Influenza virus assembly requires the completion of viral protein and vRNP transport to the assembly site at the plasma membrane. Therefore, efficient regulation of intracellular transport of the viral proteins and vRNPs to the surface of the host cell is especially important for virus morphogenesis. Influenza A virus uses the machineries of host cells to transport its own components including ribonucleoproteins (vRNPs) and three transmembrane proteins hemagglutinin (HA), neuraminidase (NA) and matrix 2 protein (M2). It has been shown that newly synthesized vRNPs are associated with active form of Rab11 and accumulate at recycling endosomes adjacent to the microtubule organizing center (MTOC) following nuclear export. Subsequently, they are transported along the microtubule network toward the plasma membranes in cargo vesicles. The viral transmembrane proteins are translated on the rough endoplasmic reticulum and transported to the virus assembly site at the plasma membrane. It has been found that several host factors such as ARHGAP21 and GTPase Cdc42 are involved in regulation of intracellular trafficking of influenza A virus transmembrane proteins including NA. In this review, we will highlight the current knowledge about anterograde transport and its regulation of the influenza A virus transmembrane proteins and genome in the host cytoplasm.
Cytoplasm ; metabolism ; GTP Phosphohydrolases ; metabolism ; GTPase-Activating Proteins ; metabolism ; Genome, Viral ; Hemagglutinin Glycoproteins, Influenza Virus ; metabolism ; Humans ; Influenza A virus ; genetics ; pathogenicity ; physiology ; Neuraminidase ; metabolism ; Protein Transport ; Ribonucleoproteins ; metabolism ; Viral Matrix Proteins ; metabolism ; cdc42 GTP-Binding Protein ; metabolism

Latent Epstein-Barr virus (EBV) infection has a substantial role in causing many human disorders. The persistence of these viral genomes in all malignant cells, yet with the expression of limited latent genes, is consistent with the notion that EBV latent genes are important for malignant cell growth. While the EBV-encoded nuclear antigen-1 (EBNA-1) and latent membrane protein-2A (LMP-2A) are critical, the EBNA-leader proteins, EBNA-2, EBNA-3A, EBNA-3C and LMP-1, are individually essential for in vitro transformation of primary B cells to lymphoblastoid cell lines. EBV-encoded RNAs and EBNA-3Bs are dispensable. In this review, the roles of EBV latent genes are summarized.
Epstein-Barr Virus Infections/complications/virology ; Epstein-Barr Virus Nuclear Antigens/genetics/metabolism ; *Genes, Viral ; Herpesvirus 4, Human/*physiology ; Humans ; MicroRNAs/genetics ; Neoplasms/etiology ; Protein Binding ; RNA, Viral/genetics ; Viral Matrix Proteins/genetics/metabolism ; *Virus Latency

HA and M2 genes derived from human highly pathogenic avian influenza H5N1 virus (A/Anhui/ 1/2005) isolated from China, were amplified and cloned into the DNA vaccine expression vector pVRC. In order to improve the expression of hemagglutinin, the human codon usage preference was made and the whole length of HA gene of H5NI (A/Anhui/1/2005) influenza virus was synthesized,named HA/YH/K, and inserted to pVRC vector, the expression of HA/YH/K protein in eukaryotic cells was significantly improved according to internal control of actin protein. Furthermore, the M2 and HA/YH/K genes were cloned into bicistronic eukaryotic expressing vector pIRES to yield the recombinant plasmid pIRES-HA/ YH/K-M2/YS/K, which could expressed HA and M2 protein simultaneously by transfection of one plasmid. Western blot and IFA showed that the recombinant pIRES-HA/YH/K-M2/YS/K plasmid was successfully expressed in several mammalian cells (Hela, MDCK and 293FT). The above results may help to identify the function and pathogenic mechanism of HA, M2 genes derived from HPAI H5N1 (Anhui strain) and pave a way for the development of novel bivalent vaccines against human highly pathogenic avian influenza virus and for preparedness for influenza pandemic.
Animals ; Cell Line ; Gene Expression ; Genetic Engineering ; Genetic Vectors ; genetics ; metabolism ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; metabolism ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; isolation & purification ; metabolism ; Influenza, Human ; virology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Viral Matrix Proteins ; genetics ; metabolism