To develop polyclonal antibodies against predicted B cell epitopes in HIV-1 accessory protein Vpr, the prepared consensus Vpr amino acid sequence was used to predict potential B cell epitopes by online softwares (ABCpred and Bcepred), the synthesized polypeptides of B-cell epitopes were subsequently conjugated with keyhole limpet hemocyanin (KLH) and then used to immunize rabbits. The antibody titers were determined by ELISA, and antibody specifity was analyzed by Western-Blotting and immunoprecipitation, respectively. Amino acid residues 3-19 (N) and 82-95 (C) of Vpr were predicted as the potential B cell epitopes. After inoculation of the conjugation of synthesized peptide to KLH, the antibody titers in rabbit sera against N and C peptides reached more than 1:100000 by ELISA. Western-Blotting analysis showed that the polyclonal antibodies reacted with both wild Vpr and fusion protein of GFP with Vpr, no matter Vpr was derived from HIV-1 B subtype or CRF07_BC recombinant form; Immunoprecipitation analysis showed similar reactions to Western-Blotting results. Two B cell epitopes of Vpr were successfully predicted by Bio-informatics methods and polyclonal antibodies against those peptides could be successfully prepared.
Animals ; Antibodies, Viral ; blood ; immunology ; Cell Line ; Enzyme-Linked Immunosorbent Assay ; HIV Infections ; blood ; immunology ; virology ; HIV-1 ; genetics ; immunology ; Humans ; Peptides ; genetics ; immunology ; Rabbits ; vpr Gene Products, Human Immunodeficiency Virus ; genetics ; immunology

Vpr, an auxiliary protein of HIV-1(Human immunodeficiency virus type 1), exerts important functions to promote viral replication and AIDS progression. In this study, we performed a yeast two-hybrid screening assay using human cDNA library to further investigate the molecular mechanism of various functions of Vpr RelB, a key protein in NF-kappaB signaling pathway, was identified as a Vpr interaction protein by co-immunoprecipitation. Further investigations indicated that RelB not only promoted the Vpr-mediated activation of NF-kappaB reporter gene, but also enhanced the transactivation of HIV LTR. Moreover, the results showed that RelB promoted Vpr-induced cell cycle G2/M arrest. Collectively, these results indicated that RelB might interact with Vpr and regulate its transcriptional activation and cell cycle arrest.
Cell Cycle Checkpoints ; Cell Division ; G2 Phase ; HIV Long Terminal Repeat ; HeLa Cells ; Humans ; NF-kappa B ; genetics ; Transcription Factor RelB ; physiology ; Transcriptional Activation ; vpr Gene Products, Human Immunodeficiency Virus ; physiology

Gene therapy offers the promise of curing the HIV-infected patients. Specific, potent, and sustained short hairpin RNA (shRNA)-mediated gene silencing is crucial for the successful application of RNA interference technology to therapeutic interventions. To reduce the probability of viral escape mutants, in this study, we constructed lentiviral vector containing vpr and tat shRNA, respectively, furthermore the bispecific lentiviral vector harboring vpr and tat shRNA expression cassettes from U6 promotor and H1 promotor was cotransfected with recombinant plasmid expressing the vpr and tat gene. The result showed that the bispecific lentiviral vector plvx-vpr-tatshRNA could inhibit the vpr and tat effectively,with ratios of 89.20% and 62.00% respectively. When cotransfected with pNL4-3 in 293T cell, plvx-vpr-tatshRNA showed higher efficacy in down regulating the HIV NL4-3 packaging production than the plvx-vprshRNA or plvx-tatshRNA individually. MT4 cell clones transduced with recombinant lentiviral vectors were screened and challenged with HIV NL4-3. P24 ELISA test showed that MT4 transduced with the combinational lentiviral vector could inhibit virus replication efficiently.
Cell Line ; Down-Regulation ; Genetic Therapy ; Genetic Vectors ; genetics ; metabolism ; HIV Infections ; therapy ; virology ; HIV-1 ; genetics ; metabolism ; Humans ; Lentivirus ; genetics ; metabolism ; RNA Interference ; RNA, Small Interfering ; genetics ; metabolism ; therapeutic use ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism ; vpr Gene Products, Human Immunodeficiency Virus ; genetics ; metabolism

OBJECTIVEGoal of this study was to test the potential regulatory effects of Vpr on Vif and Vif-mediated degradation of APOBEC3G.

METHODSThe Vpr effect was first tested in a fission yeast RE007 strain that carries a single integrated copy of vpr gene in the chromosome and transformed with a vif-expressing plasmid. Similar tests were also carried out in a muristerone A vpr-inducing HEK293 mammalian cell line that were transfected with the plasmids expressing vif and/or APOBEC3G. Western Blot analyses were used to measure the corresponding protein levels under different experimental conditions.

RESULTSExpression of HIV-1 vpr appears to enhance the protein levels of Vif both in fission yeast and mammalian cells. A similar enhancement effect of APOBEC3G by Vpr was also detected in mammalian cells. Interestingly, however, the increased Vif protein level by Vpr did not result in more APOBEC3G degradation than without Vpr, indicating a potential regulatory effect of Vpr on Vif-mediated proteolysis of APOBEC3G.

CONCLUSIONTo our knowledge, this is the first report describing a potentially conserved and regulatory effect of HIV-1 Vpr on Vif and Vif-mediated protein degradation of APOBEC3G.

APOBEC-3G Deaminase ; Animals ; Cell Line ; Cytidine Deaminase ; biosynthesis ; metabolism ; Gene Expression ; Gene Products, vif ; biosynthesis ; metabolism ; Gene Products, vpr ; metabolism ; HIV-1 ; Humans ; Schizosaccharomyces ; genetics

OBJECTIVETo study the selective effect to tumor cells mediated by a recombinant adenoviral vector carrying E2F-1 promoter.

METHODSThe AdEasy-1 adenoviral vector system was used in this experiment. Several recombinant adenovirus with tumor-targeting E2F-1 promoter were constructed and then the E2F-1 promoter gene was checked by PCR and sequencing. The two adenovirus expressing GFP gene which is regulated by E2F-1 promoter or CMV promoter were used to respectively transfect tumor cells and non-proliferating normal cells, then observed and analyzed the different results caused by different promoters. Vpr gene was cloned into the targeting recombinant adenovirus. The new adenovirus named rvAdE2F-1/vpr was used to transfect tumor cells SMMC-7721, LS174T and non-proliferating normal cells H292, L-02. The surviving rate of each group was registered; the level of E2F-1 protein expressed in normal and tumor cell lines were checked by Western Blot.

RESULTE2F-1 promoter can regulate the downstream gene GFP selectively expressed in LS174T and its activity in LS174T was similar with CMV promoter's; Vpr gene regulated by E2F-1 promoter can suppress the proliferation of tumor cells and no toxicity to normal cells; In all of the tumor cells, a much higher level of E2F-1 was expressed compared with normal cell lines. E2F-1 promoter's activity correlated well with E2F-1 protein levels.

CONCLUSIONSE2F-1 promoter can control a selective cell killing to cancer cells, with no effect to normal cells. The system of E2F-1 promoter is a useful method for tumor-targeting gene therapy.

Adenoviridae ; genetics ; Blotting, Western ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; E2F1 Transcription Factor ; genetics ; metabolism ; Gene Products, vpr ; genetics ; physiology ; Genetic Therapy ; methods ; Genetic Vectors ; Green Fluorescent Proteins ; genetics ; metabolism ; Humans ; Neoplasms ; genetics ; pathology ; therapy ; Promoter Regions, Genetic ; genetics ; Recombinant Fusion Proteins ; genetics ; metabolism ; Transfection

OBJECTIVETo study the anti-glioma effect of recombinant adenovirus mediated combined gene therapy of bFGF-siRNA and HIV1-Vpr in vivo.

METHODSMouse glioma model was established by injecting 5 × 10(6) LN229 cells into BALB/c-nu nude mice. 30 nude mice were randomly divided into 5 groups: the negative control group, mock group, bFGF-siRNA group, Vpr group and combined therapy group, which at regular intervals were injected with PBS, rAd5-null, rAd5-bFGF-siRNA, rAd5-Vpr, rAd5-bFGF-siRNA plus rAd5-Vpr, respectively. The tumor volume was recorded every third day to draw a growth curve. After four weeks treatment, the mice were killed and specimens were taken. HE, immunohistochemical and TUNEL staining were performed to observe the cell morphology, detect the changes of relevant target proteins and cell apoptosis, respectively. Also the ultrastructural changes were observed by electron microscopy.

RESULTSThe tumor growth inhibition rates were 36.9%, 37.2% and 58.6% in the bFGF-siRNA group, Vpr group and combined therapy group, respectively, and the combined therapy group showed the most significant effect (P < 0.05). Also the results of HE, immunohistochemical and TUNEL staining revealed that the combined therapy group had the best effects on proliferation inhibition and apoptosis induced in glioma cells (P < 0.05). The most significant ultrastructural changes were observed in the combined therapy group.

CONCLUSIONThe combined gene therapy of bFGF-siRNA with Vpr shows a prominent and synergistic anti-glioma effect compared with that of mono-gene therapy in nude mice.

Adenoviridae ; genetics ; Animals ; Apoptosis ; Brain Neoplasms ; metabolism ; pathology ; therapy ; Cell Line, Tumor ; Cell Proliferation ; Fibroblast Growth Factor 2 ; genetics ; metabolism ; Gene Products, vpr ; genetics ; metabolism ; Genetic Therapy ; Glioma ; metabolism ; pathology ; therapy ; HIV-1 ; genetics ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; RNA, Small Interfering ; genetics ; Random Allocation ; Recombinant Proteins ; genetics ; metabolism