Quantification of cytotoxic T lymphocytes (CTL) is extremely important due to the pivotal role they play in controlling pathogen infection and anti-tumor actions. Previously used methods for detecting specific CTL are usually indirect. In recent years, tetramer technology has been developed to directly visualize antigen-specific CTL efficiently, and become the critical approach in studying T cell immune responses. A simplified procedure for preparing tetramers is reported here in this paper and a tetramer loaded with human cytomegalovirus (HCMV) peptide was successfully obtained using this procedure, which possessed binding activity with specific CTL. The heavy chain of HLA-A * 0201 gene was cloned by RT-PCR from HLA-A2+ donor. An expression vector, encoding the extracellular domain of HLA-A * 0201 heavy chain (A2) fused with a BirA substrate peptide (BSP) at its carboxyl terminus, was constructed by PCR with cloned A2 gene as the template. The A2 heavy chain was expressed in Escherichia coli mostly in the form of inclusion body and purified by washing inclusion body. The monomer of soluble A2 loaded with peptide was reconstructed by dilution from the heavy chain in the presence of light chain beta2-microglobulin and HLA-A2 restricted HCMV pp65(495-503) peptide (NLVPMVATV, NLV). Refolded A2-NLV monomer was biotinylated with a commercial BirA and purified by low pressure anion exchange chromatography on a Q-Sepharose (fast flow) column. The tetramer was then formed by mixing A2-NLV monomer with streptavidin-PE in a ratio of 4:0.8 leading to more than 85% multiplication as revealed by SDS-PADE under non-reducing conditions without boiling the sample. Flow cytometry analysis indicated that this tetramer could bind to specific CTL from HLA-A2+ donor. In conclusion, a simplified procedure is established to prepare HLA-A2 tetramer, which may not only facilitate the application of tetramer technology for studying specific T lymphocyte immune response but A2-NLV itself be applied clinically to monitor CMV-specific CTL in stem cell and organ transplantation.
Cloning, Molecular ; Cytomegalovirus ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; HLA-A Antigens ; biosynthesis ; genetics ; immunology ; HLA-A2 Antigen ; Humans ; Phosphoproteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; T-Lymphocytes, Cytotoxic ; immunology ; metabolism ; Viral Matrix Proteins ; biosynthesis ; genetics

M2 protein of influenza A virus is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in influenza virus replication. It is also a target molecule of anti-virus drugs. We extracted the viral genome RNAs from MDCK cells infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M2 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into a prokaryotic expression vector pET-28a(+) (designated pET-28a(+)-M2) and a eukaryotic expression vector p3xFLAG-CMV-7.1 (designated p3xFLAG-CMV-7.1-M2), respectively. The resulted constructs were confirmed by restriction enzyme digestion and DNA sequencing analysis. We then transformed the plasmid pET-28a(+)-M2 into Escherichia coli BL21 (DE3) strain and expressed it by adding 1 mmol/L of IPTG (isopropyl-beta-D-thiogalactopyranoside). The recombinant M2 protein was purified from the induced bacterial cells using Ni(2+) affinity chromatography. Wistar rats were immunized with the purified M2 protein for producing polyclonal antibodies specific for it. Western blotting analysis and immunofluorescence analysis showed that the produced antibodies were capable of reacting with M2 protein expressed in p3xFLAG-CMV-7.1-M2-transfected cells as well as that synthesized in SIV-infected cells. We also transfected plasmid p3xFLAG-CMV-7.1-M2 into Vero cells and analyzed its subcellular localization by immunofluorescence. The M2 protein expressed in the Vero cells was 20 kDa in size and dominantly localized in the cytoplasm, showing a similar distribution to that in SIV-infected cells. Western blotting analysis of SIV-infected cells suggested that M2 was a late phase protein, which was detectable 12 h post-infection, later than NS1, NP and M1 proteins. It would be a potential molecular indicator of late phases replication of virus. Our results would be useful for studying the biological function of M2 protein in SIV replication.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Cercopithecus aethiops ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; RNA ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Swine ; Transfection ; Vero Cells ; Viral Matrix Proteins ; biosynthesis ; genetics ; Virus Replication ; genetics

Carcinoma, Squamous Cell ; metabolism ; pathology ; Cell Line, Tumor ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Oncogene Proteins, Viral ; genetics ; Papillomavirus E7 Proteins ; Plasmids ; RNA, Messenger ; metabolism ; Repressor Proteins ; genetics ; Transfection ; Vascular Endothelial Growth Factor A ; biosynthesis ; genetics

OBJECTIVETo construct a recombinant lentivirus vector of latent membrane protein 1 (LMP1) and detect the expression of LMP1 in vitro.

METHODSThe LMP1 fragment including all the exons was amplified by PCR and inserted to the downstream of CMV promoter in the lentivirus vector pCDF. The three plasmids (packaging plasmid pFIV-34N, envelope plasmid pVSV-G and target plasmid pCDF-LMP1) were packaged into 293FT cells via liposome. The virus supernatant was harvested, concentrated and titrated. Mouse B lymphoma cell line A20 was transfected with the recombinant lentivirus vector of LMP1, and the expression of LMP1 in A20 cells was detected by RT-PCR and Western blotting.

RESULTSDNA sequencing confirmed that the sequence of PCR-amplified LMP1 was consistent with the GenBank data. The LMP1 gene fragment was cloned into pCDF in the right direction, and the open reading frame of LMP1 was maintained. The 3 plasmids were effectively transferred into 293FT cells, which emitted green fluorescence in the cytoplasm and on the cell membrane under fluorescence microscope. The titer of the lentivirus vector reached 10(7) Tu/ml with a transfection efficiency 90% in A20 cells. LMP1 expression was detected by RT-PCR and Western blotting in transfected A20 cells.

CONCLUSIONThe recombinant lentivirus vector of LMP1 constructed can be effectively transfected into A20 cells, which provides a basis for exploring the role of LMP1 in the pathogenesis of lymphoma.

Blotting, Western ; Cell Line, Tumor ; Cloning, Molecular ; Genetic Vectors ; genetics ; Green Fluorescent Proteins ; biosynthesis ; genetics ; Humans ; Lentivirus ; genetics ; metabolism ; Lymphoma, B-Cell ; pathology ; Recombinant Proteins ; biosynthesis ; genetics ; Recombination, Genetic ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Viral Matrix Proteins ; biosynthesis ; genetics

Interferon regulatory factor 7 (IRF7) is one of the transcriptional factors for the activation of type I Interferon (IFN) genes. It is known that IRF7 and the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) are highly expressed in EBV type III latency cells, and LMP1 induces mRNA expression of IRF7. In this study, the expression pattern of endogenous IRF7 was observed in several B cell lines with or without EBV infection by immunofluorescence staining. IRF7 was localized in the cytoplasm of EBV-negative B cells and EBV type I latency B cell lines. However, IRF7 was located both in the cytoplasm and nucleus of EBV type III latency cell lines. In the Jijoye cell (type III latency cell), IRF7 was colocalized with LMP1 in the cytoplasm in a capping configuration, and their interaction was confirmed by co-immunoprecipitation of LMP1 and IRF7. This colocalization was confirmed by co-transfection of IRF7 and LMP1 plasmids in EBV-negative B cells. These results suggest that the IRF7 and LMP1 interact with each other, and this may relate to the mechanism whereby LMP1 exerts functional effects in B-lymphocytes.
Viral Matrix Proteins/*biosynthesis/metabolism ; Trans-Activation (Genetics) ; Signal Transduction ; RNA, Messenger/metabolism ; Plasmids/metabolism ; Microscopy, Fluorescence ; Interferon Regulatory Factor-7/*biosynthesis ; Immunoprecipitation ; Humans ; Herpesvirus 4, Human/metabolism ; *Gene Expression Regulation ; Cytoplasm/metabolism ; Cell Line, Tumor ; B-Lymphocytes/metabolism/virology

The M2 ion channel protein is an important target against influenza A virus. In this study, H5N1 influenza A virus M2 ion channel (H5M2) gene was cloned into pcDNA4 vector. The HEK293 stable cell line expressing H5M2 was successfully established. The expression of H5M2 ion channel protein was induced only by tetracycline and confirmed by imuunofluorescence and Western blot. The ion channel activity of H5M2 was confirmed by whole cell patch-clamp recording. Fifty micromol per liter amantadine blocked the H5M2 channel conductance completely in HEK293 cells. This stable cell line may provide a model for screening inhibitors of M2 ion channel.
Base Sequence ; Cell Line ; Humans ; Influenza A Virus, H5N1 Subtype ; metabolism ; Ion Channels ; antagonists & inhibitors ; Kidney ; cytology ; Molecular Sequence Data ; Patch-Clamp Techniques ; Viral Matrix Proteins ; biosynthesis ; genetics

OBJECTIVETo investigate effect of AP-1 and Ets binding site adjacent to matrix metalloproteinase-9 (MMP-9) promoter on activation of MMP-9 transcription of nasopharyngeal carcinoma cells transfected with EBV-encoded latent membrane protein 1 (LMP1), and to ascertain if cross-talk between c-Jun and Ets1 is involved in LMP1-regulating expression of MMP-9.

METHODSSite-directed mutagenesis technique was used to establish a series of mutants, including MMP-9-CAT-Ets(-540)mt, MMP-9-CAT-AP-1(-533)mt and MMP-9-CAT-AP-1(-533)/Ets(-540)mt. After the mutants were transfected into LMP1-expressing NPC HNE2 cells regulated by Tet-on system (pTet-on-LMP1 HNE2), CAT activity of these mutants were assayed with induction of LMP1. With blockade of c-Jun or Ets1 antisense oligonucleotides, the activity of MMP-9 induced by LMP1 was assayed with gelatin zymography.

RESULTSThe CAT activity of MMP-9-Ets(-540)mt-CAT, MMP-9-AP-1(-533)mt-CAT, MMP-9-AP-1(-533)/Ets(-540) mt-CAT decreased significantly compared to MMP-9-CAT wt. After blockade with c-Jun or Ets1 antisense oligonucleotides, activity of MMP-9 induced by LMP1 decreased significantly, especially with combined blockade of c-Jun and Ets1.

CONCLUSIONThe results suggest that transcription factor AP-1 and Ets play an crucial role in activation of MMP-9 transcription induced by LMP1, and cross-talk between c-Jun/Ets1 is involved in expression of MMP-9 mediated by LMP1.

Herpesvirus 4, Human ; genetics ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; genetics ; Nasopharyngeal Neoplasms ; metabolism ; virology ; Proto-Oncogene Protein c-ets-1 ; genetics ; Proto-Oncogene Proteins c-jun ; genetics ; Transfection ; Tumor Cells, Cultured ; Viral Matrix Proteins ; genetics

OBJECTIVETo clarify if Epstein-Barr virus encoded LMP1 induces matrix metalloproteinase 9 expression via NF-kappa B or AP-1 signaling pathway, which gives evidence to the elucidation of the mechanism of LMP1- mediated carcinogenesis.

METHODSTo determine whether LMP1 or its mutants contribute to MMP9 production via NF-kappa B or AP-1 transcription factor, MMP9-chloramphenicol acetyl transferase (CAT), NF-kappa B mut 9-CAT, AP-1 mut MMP9-CAT were transfected into human nasopharyngeal carcinoma cells stably expressing LMP1 (HNE2-LMP1) or its mutants, [HNE2-LMP1 (1-185), HNE2-LMP1 (1-231), HNE2-LMP1 delta 187-351] by electroporation technic. The difference of MMP9 reporter activity among those cell lines was detected by CAT assay and expression of MMP9 was determined in nasopharyngeal carcinoma cells stably expressing LMP1 or its mutants by zymographic analysis. In the meantime, efforts were made to demonstrate if LMP1 regulates NF-kappa B or AP-1 activation using reporter gene analysis.

RESULTSIn contrast with vector-transfected cells, MMP9 CAT activity in HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231), HNE2-LMP1 delta 187-351 increased 7.2, 1.3, 3.3, 4.0 times respectively. Zymographic analysis demonstrated that the 92 kDa MMP9 expression was induced in HNE2-LMP1, HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells, whereas it was negative in HNE2-pSG5 and HNE2-LMP1 (1-185) cells. As compared to the HNE2 cells, NF-kappa B or AP-1 reporter activity in HNE2-LMP1 cells were increased 13.8, 8.4 fold respectively. Moreover, In contrast with MMP9 CAT-transfected cells, MMP9 CAT activity in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-LMP1, HNE2-LMP1 (1-185), HNE2-LMP1(1-231) and HNE2-LMP1 delta 187-351 cells were significantly decreased by 18.1% or 16.3%, 35.0% or 33.3%, 29.1% or 26.1% from the original level. However, there was no difference in NF-kappa B mut MMP9-CAT or AP-1 mut MMP9-CAT transfected HNE2-pSG5, HNE2-LMP1 (1-185) cells.

CONCLUSIONIn nasophargyngeal carcinoma, Epstein-Barr virus-encoded LMP1 induces MMP9 transcription and enzymatic activity via an NF-kappa B or AP-1 signaling pathway, which may contribute to invasiveness and metastasis.

Gene Expression ; drug effects ; Herpesvirus 4, Human ; chemistry ; Humans ; Matrix Metalloproteinase 9 ; biosynthesis ; NF-kappa B ; metabolism ; Nasopharyngeal Neoplasms ; pathology ; Signal Transduction ; Transcription Factor AP-1 ; metabolism ; Tumor Cells, Cultured ; Viral Matrix Proteins ; pharmacology

Matrix protein 2(M2) is an integral tetrameric membrane protein of influenza A virus, which functions as ion channel. M2 sequence has shown remarkable conservation, so there has been growing interest in it as "universal" vaccine. In order to establish a stable 293 cell line that express M2 protein under the control of the tetracycline operator, M2 gene was obtained by PCR amplification from the plasmid containing the segment 7 of influenza A virus strain A/PR/8/34 firstly. The PCR product was cloned into BamH I/Not I restriction site of pcDNA5/FRT/TO vector, and cotransfected with pOG44 which express Flp recombinase into Flp-In T-REx-293 cell. Integration of pcDNA5/FRT/TO-M2 into the cell genome at the Flp Recombination Target (FRT) site brought the SV40 promoter and the initiation codon in frame with the hygromycin resistance gene. Thus, stable cell lines were selected for hygromycin resistance. The expression of M2 protein from hygromycin-resistant cell was induced by addition of tetracycline into the cell culture media, and then tested by indirect immunofluorescence assay (IFA). 16 strains with high expression of M2 were selected. After subculturing for more than ten passages, the cell lines still stably expressed M2 protein. No M2 protein could be detected without tetracycline induction, suggesting that the expression was strictly controlled by tetracycline operator. The cell lines expressing M2 will be useful for further functional studies of M2 protein, detection of immune response against natural structure M2 protein and development of live attenuated influenza virus vaccine with reverse genetics technique.
Animals ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; genetics ; HEK293 Cells ; Humans ; Influenza A virus ; genetics ; metabolism ; Influenza Vaccines ; genetics ; Operator Regions, Genetic ; Recombinant Proteins ; biosynthesis ; genetics ; Tetracycline ; pharmacology ; Transfection ; Viral Matrix Proteins ; biosynthesis ; genetics

Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.
Animals ; Antibodies, Monoclonal ; biosynthesis ; Chick Embryo ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Influenza A Virus, H3N2 Subtype ; genetics ; physiology ; Rats ; Rats, Wistar ; Recombinant Proteins ; genetics ; immunology ; metabolism ; Swine ; Viral Matrix Proteins ; genetics ; immunology ; metabolism ; Virus Replication ; genetics