Influenza B virus ; genetics ; pathogenicity ; Phylogeny ; RNA, Viral ; genetics ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo investigate biological functions of hepatitis C virus (HCV) non-structural protein 4A (NS4A).

METHODSYeast-two hybrid technique was performed to seek proteins in hepatocytes interacting with HCV NS4A. HCV NS4A bait plasmid was constructed by ligating the NS4A gene with carrier plasmid pGBKT7, then it was transformed into yeast AH109 (alpha type). The transformed yeast cells were amplified and mated with yeast cells Y187 (alpha type) containing liver cDNA library plasmid pACT2 in 2 x YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-alpha-gal for selection two times. After extracting plasmid from blue colonies, plasmid DNA was transformed into competent E.coli and analyzed by DNA sequencing and bioinformatics methods.

RESULTSAmong twenty-two positive colonies there were eleven positive for metallothionein 2A, three for eukaryotic translation elongation factor 1 alpha 1, two for albumin, two for RNA binding motif protein 21, two for myomesin, one for cytochrome C oxidase II, and one for ATPase.

CONCLUSIONSGenes of HCV NS4A interacting proteins in hepatocytes were successfully cloned and the results pave the way for studying the biological functions of NS4A and associated proteins.

Carrier Proteins ; genetics ; Cloning, Molecular ; Hepacivirus ; genetics ; Hepatocytes ; metabolism ; Humans ; Two-Hybrid System Techniques ; Viral Nonstructural Proteins ; Viral Proteins ; genetics

Escherichia coli ; genetics ; Humans ; Recombinant Fusion Proteins ; immunology ; Viral Nonstructural Proteins ; genetics ; immunology

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

Apoptosis ; Cell Line ; Hepacivirus ; genetics ; Humans ; Serum ; metabolism ; Viral Nonstructural Proteins ; genetics

OBJECTIVETo confirm the activity of non structural protein 1 (NSP1) of Rotavirus (RV) as E3 ubiquitin ligase by experiments and to provide some clues for NSP1 on the pathogenic mechanisms and replication of RV.

METHODSThe whole gene and RING deleted mutation gene of NSP1 were coloned into pEGFPC1 expression plasmid, and transfected into human embryonic kidney (HEK) 293 FT cells with pBlue-Script-HA-Ubiquitin. The expression of proteins were proved by using con-focal microscope and western blotting. The ubiquination of proteins were detected by co-immunoprecite.

RESULTSThe cellular proteins of HEK293FT are ubiquinated by NSP1 protein and NSP1 protein was self-ubiquinated also.

CONCLUSIONSIt revealed that RV NSP1 had the activity of E3 ubiquitin ligase and it may play a role on the modulate mechanisms of ubiquination.

HEK293 Cells ; Humans ; Rotavirus ; enzymology ; genetics ; Ubiquitin-Protein Ligases ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

Until now, more than seventeen parvovirus have been reported which can infect mammals and poultries. The infected cells appeared different properties of apoptosis and death, present a typical cytopathic effect. NS1 is a major nonstructural protein of parvovirus, with a conservative structure and function, which plays an important role in the viral life cycle. In addition to the influence on viral replication, the NS1 also participates in apoptosis induced by viruses. Parvovirus induced apoptosis which is mainly mediated by mitochondrial pathway, this review summarized the latest research progresses of parvovirus induced apoptosis.
Animals ; Apoptosis ; Humans ; Parvoviridae Infections ; physiopathology ; veterinary ; virology ; Parvovirus ; genetics ; metabolism ; Viral Nonstructural Proteins ; genetics ; metabolism

OBJECTIVETo clone 1b type of HCV NS3-4b Gene and express in HEK 293 cells, lay the foundation for further study of the HCV NS3-4b recombinant adeno-associated virus vaccine and its dendritic cell vaccine.

METHODSHCV 1b patients' serum was collected, and full length NS3-4b segment was amplified by RT-PCR and cloned into adeno-associated virus' expression vector pAAV. CMV. EGFP in order to express in HEK 293 cells. At last, it was validated whether express or not by Western Blot.

RESULTSThe 1b type gene NS3-4b were amplified and consistent to the expected size (2838 bp), the recombinant plasmid has been confirmed its successful restructured by double enzyme and sequencing, at last, Western Blot map can see objective protein expression after it transfect HEK 293 cells.

CONCLUSIONThe adeno-associsted virus recombination HCV NS3-4b plasmid have successfully constructed and it can express in eukaryotic cells.

Dependovirus ; genetics ; Genetic Vectors ; HEK293 Cells ; Hepacivirus ; genetics ; Humans ; Plasmids ; Vaccines, Synthetic ; immunology ; Viral Nonstructural Proteins ; genetics ; Viral Vaccines ; immunology

OBJECTIVETo investigate whether the nonstructural protein 5A (NS5A) encoded by the hepatitis C virus RNA genome affects the expression of hepcidin gene.

METHODSHCV NS5A expression plasmid (pCN5A) and pRc/CMV were transfected into QSG7701 cells individually, RT-PCR was employed to detect the HCV NS5A and hepcidin mRNA transcription. Western blot was used for detection of HCV NS5A and hepcidin proteins. Iron was stained to evaluate the intracellular iron level.

RESULTSHCV NS5A plasmid was successfully transfected into QSG7701 cells, which was evidenced by HCV NS5A mRNA and protein from the transfected cells. The hepcidin mRNA relative quantification in untransfected cells, pRc/CMV transfected cells and pCNS5A transfected cells were 0.711+/-0.049, 0.718+/-0.052 and 0.264+/-0.030 respectively. The transcription of hepcidin mRNA decreased remarkably in the cells transfected with pCNS5A plasmid as compared to the untransfected cells and pRc/CMV transfected cells (P less than 0.01). The level of hepcidin protein expression was found also significantly lower in the pCN5A plasmid transfected cells as compared to the untransfected cells and pRc/CMV transfected cells. The intracellular iron staining was remarkably higher in the pcNS5A transfected cells than untransfected or pRc/CMV transfected cells.

CONCLUSIONSHCV NS5A inhibits the transcription of hepcidin mRNA and expression of hepcidin protein, inducing hepatic intracellular iron storage.

Antimicrobial Cationic Peptides ; genetics ; Cell Line ; Gene Expression Regulation, Viral ; Hepacivirus ; genetics ; Hepcidins ; Humans ; Plasmids ; Transfection ; Viral Nonstructural Proteins ; genetics

Recently, the interactions between hepatitis C virus (HCV) genes and the host cell factors were the focus of this field. Cell factors in the different biochemical pathway were approved to be interfered when HCV infection. To make sure which HCV gene(s) was the major factor during the interaction process, ten eukaryotic expression plasmids containing different functional genes of HCV: Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B were transfected into the CHO-K1 cells respectively. Then ten stable cell lines expressing different HCV functional proteins were constructed under the selective pressure of G418. DNA and mRNA of the HCV genes were both detected by PCR and RT-PCR respectively in the corresponding stable cell lines, freezation and anabiosis would not lose the HCV genes. Besides, the El, E2 and NS5B proteins were detected by Western-blot which demonstrated that the HCV genes have formed stable expression in the host cells. The activity of UDP-glucose ceramide glucosyltransferase (UGCG) in the stable cell lines increased in different degree by TLC assay. For example, the activity of UGCG in CHO-K1-E2 and CHO-K1-p7 was doubled according to the control cells,and in CHO-K1-NS2 and CHO-K1-NS5A was about 1.6 times compared with the control cells. The establishment of the stable cell lines containing different single HCV gene will provide foundation for investigating the interactions between the virus and the host factors, and for the filtration of antiviral medicine.
Animals ; CHO Cells ; Cricetinae ; Cricetulus ; Glucosyltransferases ; biosynthesis ; metabolism ; Hepacivirus ; genetics ; metabolism ; Transfection ; Viral Envelope Proteins ; biosynthesis ; genetics ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; Viral Proteins ; biosynthesis ; genetics