1.Effect of HIV Tat protein on CCR5 expression in monocytes and infection with monocyte-tropic HIV strains.
Yi-da YANG ; Lin ZHENG ; Guo-cai LU ; Ya-gang CHEN ; Maria SALVATO
Journal of Zhejiang University. Medical sciences 2004;33(6):532-545
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
2.Transactivation of HIV-1 transcription and inhibitors.
Acta Pharmaceutica Sinica 2006;41(4):289-295
Anti-HIV Agents
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pharmacology
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Ciprofloxacin
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analogs & derivatives
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pharmacology
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Gene Products, tat
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chemistry
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genetics
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Genes, tat
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HIV-1
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drug effects
;
genetics
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Humans
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Oligopeptides
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pharmacology
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Organophosphates
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pharmacology
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Transcription, Genetic
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Transcriptional Activation
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Uridine
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analogs & derivatives
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pharmacology
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tat Gene Products, Human Immunodeficiency Virus
3.Peptide TAT modified polyethylenimine-beta-cyclodextrin for gene delivery.
Li-Hua LAI ; Qi-Ying JIANG ; Dan CHEN ; Yi-Ping HU ; Hai YU ; Qing-Qing WANG ; Gu-Ping TANG
Journal of Zhejiang University. Medical sciences 2009;38(1):15-23
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
4.Enhanced Induction of T Cell Immunity Using Dendritic Cells Pulsed with HIV Tat and HCMV-pp65 Fusion Protein In Vitro.
Jung Sun PARK ; Soo Young PARK ; Hyun Il CHO ; Hyun Jung SOHN ; Tai Gyu KIM
Immune Network 2011;11(3):182-189
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
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Cytomegalovirus
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Dendritic Cells
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Enzyme-Linked Immunospot Assay
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HIV
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Humans
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Immunotherapy
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T-Lymphocytes
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T-Lymphocytes, Cytotoxic
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tat Gene Products, Human Immunodeficiency Virus
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Vaccines
5.Cloning of PTD-NPY fusion gene and its secretory expression in Pichia pastoris.
Yucheng SUN ; Fengqiu ZHOU ; Jiayu WAN ; Hongwei GAO
Chinese Journal of Biotechnology 2008;24(9):1620-1624
The PTD-NPY fusion gene derived from HIV-1 TAT protein transduction domain and rat neuropeptide Y was amplified by overlap extension PCR, digested and subcloned into yeast expression vector pPICZ alpha A to construct recombinant expression plasmid pPICZ alpha-PTD-NPY. The cloned PTD-NPY fusion gene was identified by PCR and restriction enzyme digestion and sequenced. The exact recombinant plasmid was linearized by Sac I and integrated by electrotransformation into the genome of Pichia pastoris GS115 cells. Then, these positive recombinant yeast cells were induced by 10 mL/L methanol to express soluble PTD-NPY fusion protein. After 120 h of methanol induction, the SDS-PAGE electrophoresis result indicated PTD-NPY fusion protein was efficiently secreted into the medium. Western blotting analysis proved that the expressed fusion protein had specific NPY binding activity. The successful expression of PTD-NPY fusion protein in Pichia pastoris provided basis for its further application study.
Animals
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Cloning, Molecular
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Gene Fusion
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Genetic Vectors
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genetics
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Neuropeptide Y
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genetics
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Pichia
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genetics
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metabolism
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Polymerase Chain Reaction
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methods
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Rats
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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Transduction, Genetic
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tat Gene Products, Human Immunodeficiency Virus
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genetics
6.Effects of HIV-1 tat on secretion of TNF-α and IL-1β by U87 cells in AIDS patients with or without AIDS dementia complex.
Li ZHAO ; Shuang Shuang PU ; Wen Hua GAO ; Yuan Yuan CHI ; Hong Ling WEN ; Zhi Yu WANG ; Yan Yan SONG ; Xue Jie YU ;
Biomedical and Environmental Sciences 2014;27(2):111-117
OBJECTIVETo explore the role of HIV-1 tat gene variations in AIDS dementia complex (ADC) pathogenesis.
METHODSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of an AIDS patient with ADC and an AIDS patient without ADC were cloned for sequence analysis. HIV-1 tat gene sequence alignment was performed by using CLUSTAL W and the phylogentic analysis was conducted by using Neighbor-joining with MEGA4 software. All tat genes were used to construct recombinant retroviral expressing vector MSCV-IRES-GFP/tat. The MSCV-IRES-GFP/tat was cotransfected into 293T cells with pCMV-VSV-G and pUMVC vectors to assemble the recombinant retrovirus. After infection of gliomas U87 cells with equal amount of the recombinant retrovirus, TNF-α, and IL-1β concentrations in the supernatant of U87 cells were determined with ELISA.
RESULTSHIV-1 tat genes derived from peripheral spleen and central basal ganglia of the AIDS patient with ADC and the other one without ADC exhibited genetic variations. Tat variations and amino acid mutation sites existed mainly at Tat protein core functional area (38-47aa). All Tat proteins could induce U87 cells to produce TNF-α and IL-1β, but the level of IL-1β production was different among Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen. The level of Tat proteins derived from the ADC patient's spleen, basal ganglia, and the non-ADC patient's spleen were obviously higher than that from the non-ADC patient's basal ganglia.
CONCLUSIONTat protein core functional area (38-47aa) may serve as the key area of enhancing the secretion of IL-1β. This may be related with the neurotoxicity of HIV-1 Tat.
AIDS Dementia Complex ; metabolism ; pathology ; virology ; Adult ; Amino Acid Sequence ; Basal Ganglia ; virology ; Cell Line, Tumor ; Gene Expression Regulation, Viral ; Genes, tat ; HIV-1 ; genetics ; pathogenicity ; Humans ; Interleukin-1beta ; biosynthesis ; genetics ; secretion ; Middle Aged ; Molecular Sequence Data ; Neuroglia ; pathology ; secretion ; Spleen ; virology ; Tumor Necrosis Factor-alpha ; biosynthesis ; genetics ; secretion ; tat Gene Products, Human Immunodeficiency Virus ; genetics ; physiology
7.Action of protein phosphatase-1 on Tat-dependent HIV-1 transcription and its related inhibitors.
Acta Pharmaceutica Sinica 2009;44(12):1343-1347
Host cell protein phosphatase-1 (PP1) is an important regulator of human immunodeficiency virus-1 (HIV-1) transcription. PP1 is involved in the regulation of HIV-1 transcription, and dephosphorylates RNA polymerase II C-terminal domain (RNAPII CTD) or CycT1-dependent kinase 9 (CDK9) to increase Tat-dependent HIV-1 transcription. In this review, we discuss the action of PP1 in Tat-induced HIV-1 transcription and related to PP1 inhibitors.
Anti-HIV Agents
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pharmacology
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Enzyme Inhibitors
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pharmacology
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HIV-1
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genetics
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Humans
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Okadaic Acid
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pharmacology
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Protein Phosphatase 1
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antagonists & inhibitors
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chemistry
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physiology
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Pyrans
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pharmacology
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Spiro Compounds
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pharmacology
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Transcription, Genetic
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tat Gene Products, Human Immunodeficiency Virus
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physiology
8.Design, synthesis and evaluation of novel 2H-1, 4-benzodiazepine-2-ones as inhibitors of HIV-1 transcription.
Yan-Boi TANG ; Chuan-Ming ZHANG ; Cheng FANG ; Chun HU ; Li HUANG ; Chin-Ho CHEN ; Zhi-Yan XIAO
Acta Pharmaceutica Sinica 2011;46(6):688-694
HIV-1 trans-activator of transcription (Tat) plays a critical role in HIV-1 transcription. Based on the beta-turn motif present in HIV-1 Tat, a series of novel benzodiazepine analogs were designed as beta-turn mimetics and prepared from p-chloro-nitrobenzene/2-phenylacetonitrile, p-toluidine/benzoyl chloride, or (Z)-7-nitro-5-phenyl-1H-benzo[e][1, 4]diazepin-2(3H)-one (nitrazepam) through different synthetic routes. Preliminary biological evaluation indicated that compound 30 exhibited inhibitory activity on HIV-1 tat-mediated LTR transcription with EC50 of 25.0 micromol x L(-1) and showed no obvious cytotoxic effects on TZM-BI cells under the concentration of 100 micromol x L(-1).
Benzodiazepinones
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chemical synthesis
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chemistry
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pharmacology
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Cell Line, Tumor
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HIV Long Terminal Repeat
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genetics
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HIV-1
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genetics
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Humans
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Transcription, Genetic
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drug effects
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tat Gene Products, Human Immunodeficiency Virus
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antagonists & inhibitors
9.Analysis of genetic diversity and amino acid sequence of HIV-1tat in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC.
Shuang-Shuang PU ; Yi-ping LI ; Yu-fen YAN ; Hong-ling WEN ; Zhi-yu WANG ; Yan-yan SONG ; Hong-zhi XU ; Li ZHAO
Chinese Journal of Experimental and Clinical Virology 2011;25(4):251-253
OBJECTIVETo study the diversity of HIV-1 tat gene in CNS and peripheral tissue of a patient with ADC and a patient with non-ADC, so as to research HIV evolution, the mechanism of CNS invasion and the pathogenesis of ADC.
METHODSThe tat gene was amplified with nested PCR from genomic DNA which was extracted from spleen and basal ganglia of one non-ADC patient with a wide range of cerebral artery atherosclerosis and one ADC patient. PCR products were cloned into the PGEM-T vector, after transformation and selection by ampicillin and blue/white spotting. Five of positive clones were sequenced. HIV-1 tat sequences were processed with BioEdit and MEGA4. With the softwares, neighbor-joining tree, p-distances, values of ds/dn, and analysis of amino acid motifs were all done, so as to research the diversity of HIV-1 tat gene in CNS and peripheral tissue.
RESULTSGene mutation of HIV-1 tat exist in the two patients, the mutation process of tat isolated from ADC patient suffered more compartmentalization than tat isolated from non-ADC patient, the differences of tat genes between CNS and peripheral tissue in ADC patient were greater than the non-ADC patient. Ds/dn showed that the virus gene mutation played a major role, the body intend to remove harmful non-synonymous mutations.
CONCLUSIONSThe compartmentation of tat gene in CNS and peripheral tissue of the two patients was different, the reason may be related to the pathway of HIV into the CNS, the relationship between HIV gene mutation in CNS and ADC still need more investigation.
AIDS Dementia Complex ; virology ; Adult ; Amino Acid Sequence ; Central Nervous System ; virology ; Female ; Genetic Variation ; HIV-1 ; genetics ; isolation & purification ; Humans ; Male ; Middle Aged ; Molecular Sequence Data ; Peripheral Nervous System ; virology ; tat Gene Products, Human Immunodeficiency Virus ; genetics
10.Prokaryotic expression of fusion protein TAT-EDAG and study on its transduction activity.
Sheng-Hua ZHOU ; Dong-Chang HE ; Li-Jun ZHANG ; Li-Dong CHEN
Chinese Journal of Biotechnology 2006;22(4):598-603
HIV-TAT protein transduction domain (PTD) is a new kind of peptide that is responsible for transduction of proteins through the plasma membrane with high efficiency. Linked covalently to proteins, peptides, nucleic acid, it could transduce into nearly all kinds of cells and tissues with high efficiency and without any damages. In this study, we constructed the TAT-EDAG and TAT-GFP prokaryotic expression vectors and expressed soluble TAT-EDAG and TAT-GFP fusions in E. coli BL21 (DE3) successfully. By using the Ni-NTA-agrose purification system under the native condition we got the purified fusion proteins whose purification were higher than 90%. After desalting we found the TAT-GFP could transduced successfully into the mouse fibroblast cells and the TAT-EDAG could transduced into HL-60 cells in vitro. It will be useful to amplify the HSCs in vitro in the next step.
Animals
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Escherichia coli
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genetics
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Fibroblasts
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metabolism
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HL-60 Cells
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Humans
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Mice
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Nuclear Proteins
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genetics
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Recombinant Fusion Proteins
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biosynthesis
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isolation & purification
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Transduction, Genetic
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tat Gene Products, Human Immunodeficiency Virus
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genetics