OBJECTIVETo study the effects of HIV Tat protein on CCR5 expression of monocytes and HIV infection in monocytes.

METHODSMembrane expression of CCR5 on monocytes was analyzed by flow cytometry. Stimulated with HIV Tat protein, monocytes were infected with monocyte-tropic HIV(Ba-L) and HIV gag p24 level in the supernatant was measured by ELISA methods.

RESULTSHIV Tat protein increased CCR5 expression in human monocytes,which was inhibited by rabbit anti-Tat polyclonal antibody. Tat protein also increased p24 level after monocyte-tropic HIV-1(Ba-L) infected monocytes.

CONCLUSIONTat increases CCR5 expression and HIV-1 infection in monocytes, which indicates that HIV Tat might be a key protein in HIV-1 infection.

Flow Cytometry ; Gene Products, tat ; pharmacology ; HIV ; HIV Infections ; metabolism ; Humans ; Monocytes ; metabolism ; Receptors, CCR5 ; biosynthesis ; genetics ; tat Gene Products, Human Immunodeficiency Virus

We studied the construction of fusion protein TAT-RIG-I-GFP prokaryotic expression vector and verified the function of TAT in transmembrane delivery. First, four pairs of specific primers were designed, and the RIG-I gene of Mallard Duck (Anas platyrhynchos) was cloned. Then, the pET-TAT-RIG-I-GFP and pET-RIG-I-GFP prokaryotic expression vectors were constructed. Meanwhile, they were converted to E. coli BL21 (DE3), which were induced to be expressed after culture. After the purification of His-60 nickel affinity chromatography column and the identification of SDS-PAGE, the purified TAT-RIG-I-GFP and RIG-I-GFP proteins were incubated to DF-1 cells. Finally, fluorescence microscopy was used to observe whether the corresponding fluorescence was produced in DF-1 cells. The results showed that pET-TAT-RIG-I-GFP fusion with TAT showed obvious green fluorescence in DF-1 cells. However, the pET-RIG-I-GFP without TAT cannot display green fluorescence. This shows that TAT-fused protein have successfully delivered DF-1 cells and play a key role in transmembrane delivery. In conclusion, these results provide a solid material basis for further study of antiviral drugs in poultry.
Cell Membrane ; DNA Primers ; Escherichia coli ; Gene Expression ; Gene Products, tat ; Genetic Vectors ; Recombinant Fusion Proteins

Anti-HIV Agents ; pharmacology ; Ciprofloxacin ; analogs & derivatives ; pharmacology ; Gene Products, tat ; chemistry ; genetics ; Genes, tat ; HIV-1 ; drug effects ; genetics ; Humans ; Oligopeptides ; pharmacology ; Organophosphates ; pharmacology ; Transcription, Genetic ; Transcriptional Activation ; Uridine ; analogs & derivatives ; pharmacology ; tat Gene Products, Human Immunodeficiency Virus

OBJECTIVETo characterize HIV-1 specific CTL responses to regulatory proteins Tat and Rev in HIV-B'/C virus-infected ART-naive individuals.

METHODSHIV-1-specific CTL responses were analyzed by IFN-gamma ELISPOT assay using overlapping peptides spanning the consensus sequences of HIV-1 clade C Tat and Rev proteins. Statistical analysis and graphical presentation were performed using SIGMAPLOT 10.0 and SIGMASTAT 3.5. For samples with a positive response, the magnitude of CTL responses was compared between HIV-1 C proteins by Wilcoxon rank sum test, and the significance threshold was P<0.05.

RESULTSTat and Rev were frequently recognized, with 23% and 52% of the tested individuals having detectable responses to these proteins, respectively. Several immunodominant regions were detected in Rev. No significant correlation was observed between the magnitude and breadth of CTL responses to regulatory proteins and the control of virus replication in this study.

CONCLUSIONTat and Rev can serve as targets for HIV-1-specific CTL, and several immunodominant regions are detectable in Rev. Further characterization of epitopes and their role in virus control may shed light on pathogenesis of HIV-1 natural infection and also be useful for the design and testing of candidate vaccines.

Amino Acid Sequence ; Gene Products, rev ; immunology ; Gene Products, tat ; immunology ; HIV ; physiology ; HIV Infections ; immunology ; Humans ; Molecular Sequence Data ; T-Lymphocytes, Cytotoxic ; immunology ; Virus Replication

Few papers have reported that the HIV-1 replication was inhibited by p53 in the infected cells. However, the detail mechanism for the p53-medicated HIV-1 suppression has not yet been clearly demonstrated. In our previous report, we addressed that p53-mediated Tat suppression is very likely associated with PKR. In the present study, we found that the amounts of p53 in the HIV-1 infected cells increased over 10 times in the early stages of infection as much as those in normal cells. Particularly noteworthy is that the both exogenous p53 and endogenous p53 enhanced PKR expression in the transformed or treated cells, and the amounts of PKR induced by p53 were almost equivalent to those induced by interferon. In the PKR promoter studies using Ppkr-CAT (CAT reporter system under the control of PKR promoter), CAT activity induced by p53 was stronger than that by interferon, suggesting that the p53-mediated PKR expression might be more efficient than interferon under the control of PKR promoter. Co-immunoprecipitation experiments showed that PKR directly binds to Tat protein. We established eIF-2alpha dominant negative (S51A) Jurkat cells (JK/eIF2alpha-51A) to block the PKR-mediated cell cycle arrest or apoptosis. In the JK/eIF2alpha-51A cells, not only p53 but also PKR inhibited the Tat activity. Taken together, our results demonstrate that the HIV-1 infection induces p53, which enhances PKR expression by promoter activation, followed by the inhibition of the Tat activity, finally resulting in the inhibition of HIV-1 replication. Detail mechanisms for the PKR-mediated Tat inactivation are under investigation.
Animals ; Apoptosis ; Cats ; Cell Cycle Checkpoints ; Eukaryotic Initiation Factor-2 ; Gene Products, tat ; HIV-1* ; Humans ; Immunoprecipitation ; Interferons ; Jurkat Cells

HIV-1 Tat protein has been implicated as a causative agent in the pathogenesis of HIV-1-associated neurocognitive disorder (HAND) and Alzheimer's disease (AD)-like pathology in HIV-1 infected patients. Here, we provide insights into the potential roles of extracellular HIV-1 Tat protein in amyloid beta (Abeta) generation and Tau phosphorylation, two major neuropathological features of AD. Exposure of the rat hippocampal slices to the full-length HIV-1 Tat protein (Tat1-86) for 3 days led to the increased levels of Abeta precursor protein (APP) accumulation, which accompanied by Abeta generation in the hippocampus, the brain region most commonly damaged in HIV-1-associated dementia (HAD). Moreover, extracellular HIV-1 Tat significantly stimulated the level of phosphrylated Tau (pTau) identified using immunoblotting with AT8 antibody, which recognizes abnormally hyperphosphorylated Tau. Collectively, our data suggest that HIV-1 Tat plays important roles in increasing the levels of APP accumulation, Abeta generation and Tau phosphorylation in the hippocampus, and thereby might contribute to the development of AD-like pathology in HIV-1-infected patients.
Alzheimer Disease ; Amyloid* ; Animals ; Brain ; Dementia ; Gene Products, tat* ; Hippocampus ; HIV-1* ; Humans ; Immunoblotting ; Pathology ; Phosphorylation* ; Rats*

Gene Products, tat ; genetics ; Gene Targeting ; Hepatitis B virus ; genetics ; growth & development ; Humans ; Recombinant Fusion Proteins ; genetics ; Ribonucleases ; genetics ; Transduction, Genetic ; Virus Replication ; drug effects

OBJECTIVETo develop a novel gene delivery vector TAT-PEI-beta-CyD.

METHODSbeta-cyclodextrin (beta-CyD) was linked by low molecular weight (PEI 600) via 1, 1-carbonyldiimidazole (CDI), and TAT peptide (RRRQRRKKRC) was coupled to PEI 600 by [N-succinimidy-3-(2-pyridyldithio) propionate, SPDP]. The copolymer was characterized by (1)H-NMR and FT-IR. Physiochemical characteristics of TAT-PEI-beta-CyD/DNA complexes were tested by agarose gel electrophoresis and particle size measurements. Cell viability and transfection efficiency were evaluated in A293 and B16 cells using PEI 25 kDa as a control.

RESULTTAT peptide was successfully coupled to PEI-beta-CyD. The result of gel electrophoresis showed that the TAT-PEI-beta-CyD was able to condense DNA efficiently at N/P ratio of 4. The particle size of TAT-PEI-beta-CyD/DNA complexes was around 100 nm. The cytotoxicity of TAT-PEI-beta-CyD was lower than that of PEI 25 kDa. The transfection efficiency of TAT-PEI-beta-CyD was higher than that of PEI 25 kDa in A293 and B16 cells at N/P ratio of 30.

CONCLUSIONThe novel vector TAT-PEI-beta-CyD has been developed successfully with low cytotoxicity and high transfection efficiency.

Cell Line ; Gene Transfer Techniques ; Genetic Therapy ; methods ; Humans ; Peptide Fragments ; chemistry ; Polyethyleneimine ; chemistry ; beta-Cyclodextrins ; chemistry ; tat Gene Products, Human Immunodeficiency Virus ; chemistry

Bone morphogenetic proteins(BMPs) are regarded as members of the transforming growth factor-beta superfamily with characteristic features in their amino acid sequences. A number of studies have demonstrated the biologic activities of BMPs, which include the induction of cartilage and bone formation. Recently there was a attempt to overcome a limitation of mass production, and economical efficieny of rh-BMPs. The method producing PTD by using bacteria have advantages of acquiry a mass of proteins. Hences, a new treatment which deliver protein employed by protein transduction domain(PTD) has been tried. The purpose of this study was to evaluate the bone regenerative effect of TATBMP-2 and TAT-HA2-BMP-2 employed by PTD from HIV-1 TAT protein for protein translocation in the rat calvarial model. An 8mm calvarial, critical size osteotomy defect was created in each of 32 male Spraque-Dawley rats(weight 250~300g). The animals were divided into 4 groups of 32 animals each (4 animals/group/healing interval). The defect was treated with TATBMP-2/ACS(Absorbable collagen sponge) (TATBMP-2 0.1mg/ml), TAT-HA2-BMP-2/ACS(TAT-HA2-BMP-2 0.1mg/ml), ACS alone or left untreated for surgical control(negative control). The rats were sacrificed at 2 or 8 weeks postsurgery, and the results were evaluated histologically. The results were as follows: New bone formation were not significantly greater in the TATBMP-2/ACS group relative to negative, and positive control groups. New bone was evident at the defect sites in TAT-HA2-BMP-2/ACS group relative to negative, positive control and TATBMP-2 groups. There were a little bone regeneration in TATBMP-2 groups. While, enhanced local bone formation were observed in TAT-HA2-BMP-2 group. But, The results was not the same in all rat defects. Therefore, further investigations are required to develop a method, which disperse homogenously, and adhere to target cells.
Amino Acid Sequence ; Animals ; Bacteria ; Bone Morphogenetic Proteins ; Bone Regeneration ; Cartilage ; Collagen ; Gene Products, tat ; HIV-1 ; Humans* ; Male ; Osteogenesis ; Osteotomy ; Protein Transport ; Rats

BACKGROUND: Cytotoxic T lymphocytes (CTLs) appear to play an important role in the control and prevention of human cytomegalovirus (HCMV) infection. The pp65 antigen is a structural protein, which has been defined as a potential target for effective immunity against HCMV infection. Incorporation of an 11 amino acid region of the HIV TAT protein transduction domain (Tat) into protein facilitates rapid, efficient entry into cells. METHODS: To establish a strategy for the generation of HCMV-specific CTLs in vitro, recombinant truncated N- and C-terminal pp65 protein (pp65 N&C) and N- and C-terminal pp65 protein fused with Tat (Tat/pp65 N&C) was produced in E.coli system. Peripheral blood mononuclear cells were stimulated with dendritic cells (DCs) pulsed with pp65 N&C or Tat/pp65 N&C protein and immune responses induced was examined using IFN-gamma ELISPOT assay, cytotoxicity assay and tetramer staining. RESULTS: DCs pulsed with Tat/pp65N&C protein could induce higher T-cell responses in vitro compared with pp65N&C. Moreover, the DCs pulsed with Tat/pp65 N&C could stimulate both of CD8+ and CD4+ T-cell responses. The T cells induced by DCs pulsed with Tat/pp65 N&C showed higher cytotoxicity than that of pp65-pulsed DCs against autologous lymphoblastoid B-cell line (LCL) expressing the HCMV-pp65 antigen. CONCLUSION: Our results suggest that DCs pulsed with Tat/pp65 N&C protein effectively induced pp65-specific CTL in vitro. Tat fusion recombinant protein may be useful for the development of adoptive T-cell immunotherapy and DC-based vaccines.
B-Lymphocytes ; Cytomegalovirus ; Dendritic Cells ; Enzyme-Linked Immunospot Assay ; HIV ; Humans ; Immunotherapy ; T-Lymphocytes ; T-Lymphocytes, Cytotoxic ; tat Gene Products, Human Immunodeficiency Virus ; Vaccines