• Journal of Microbiology and Biotechnology
• /
• v.19 no.10
• /
• pp.1103-1108
• /
• 2009

The human immunodeficiency virus (HIV)-l protein Tat acts as a transcription transactivator that stimulates expression of the infected viral genome. It is released from infected cells and can similarly affect neighboring cells. The nucleocapsid is an important protein that has a related significant role in early mRNA expression, and which contributes to the rapid viral replication that occurs during HIV-1 infection. To investigate the interaction between the Tat and nucleocapsid proteins, we utilized cDNA micro arrays using pTat and flag NC cotransfection in HEK 293T cells and reverse transcription-polymerase chain reaction to validate the micro array data. Four upregulated genes and nine downregulated genes were selected as candidate genes. Gene ontology analysis was conducted to define the biological process of the input genes. A proteomic approach using PathwayStudio determined the relationship between Tat and nucleocapsid; two automatically built pathways represented the interactions between the upregulated and downregulated genes. The results indicate that the up- and downregulated genes regulate HIV-1 replication and proliferation, and viral entry.

• Journal of Microbiology and Biotechnology
• /
• v.17 no.1
• /
• pp.154-161
• /
• 2007

Human immunodeficiency virus type 1 (HIV-1) infections are responsible for a substantial number of deaths annually and represent a significant threat to public health. According to the latest study, the Tat (Transactivator of transcription) protein is essential in transcription and replication of viral genes, and is among the early expression genes involved in the life cycle of HIV. The virion NC (nucleocapsid) plays an important role in early mRNA expression and contributes to the rapid viral replication that occurs during HIV-1 infection. Therefore, we attempted to elucidate the relationship between the Tat protein and nucleocapsid protein. In a comparison of two independently prepared and hybridized samples, flag NC overexpressed HEK 293T cells and pTat overexpressed HEK 293T cells, and hybridization showed the differences in expression in each case. Among the microarray results confirmed with real-time reverse transcriptase assay, twelve genes were identified to be involved according to their gene expression profiles. Of approximately 8,208 human genes that were analyzed, we monitored candidate genes that might have been related to NC and Tat genes from gene expression profiles. Additionally, the pathways could be viewed and analyzed through the use of Pathway Studio software. The pathways from the gene list were built and paths were found among the molecules/cell objects/processes by the curation method.

• Journal of Pharmaceutical Investigation
• /
• v.34 no.2
• /
• pp.101-106
• /
• 2004

This work aims at examining the cellular uptake behavior of poly(D,L-lactide-co-glycolide) (PLGA) nanoparticles derivatized with a protein transduction domain (PTD) using HeLa cells. For this purpose, $Tat_{49-57}$ peptide derived from transcriptional activation (Tat) protein of HIV type-1 was covalently conjugated to the terminal end of PLGA. Nanoparticles were ten prepared with the $Tat_{49-57}-PLGA$ conjugates by a spontaneous phase inversion method. The prepared particles had a mean diameter of ca. 84 nm, as measured by dynamic light scattering. The interaction of the Tat-PLGA nanoparticles with cells was examined by using confocal laser scanning microscopy. It was found tat Tat-PLGA nanoparticles incubated with HeLa cells could efficiently translocate into cytoplasm, while plain PLGA nanoparticles showed negligible cellular uptake. In addition, even at $4^{\circ}C$ or in the presence of sodium azide significant cellular internalization of Tat-PLGA nanoparticles was still observed. These results indicate that a non-endocytotic translocation mechanism might be involved in the cellular uptake of Tat-PLGA nanoparticles.

• Journal of Microbiology
• /
• v.42 no.4
• /
• pp.328-335
• /
• 2004

The human immunodeficiency virus type 1 (HIV-I) Tat protein transduction domain (PTD), which con­tains rich arginine and lysine residues, is responsible for the highly efficient transduction of protein through the plasma membrane. In addition, it can be secreted from infected cells and has the ability to enter neighboring cells. When the PTD of Tat is fused to proteins and exogenously added to cells, the fusion protein can cross plasma membranes. Recent reports indicate that the endogenously expressed Tat fusion protein can demonstrate biodistribution of several proteins. However, intercellular transport and protein transduction have not been observed in some studies. Therefore, this study exam­ined the intercellular transport and protein transduction of the Tat protein. The results showed no evi­dence of intercellular transport (biodistribution) in a cell culture. Instead, the Tat fusion peptides were found to have a significant effect on the transduction and intercellular localization properties. This sug­gests that the HIV-1 PTD passes through the plasma membrane in one direction.

• IMMUNE NETWORK
• /
• v.11 no.3
• /
• pp.182-189
• /
• 2011

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.

• Molecules and Cells
• /
• v.19 no.2
• /
• pp.191-197
• /
• 2005

The human immunodeficiency virus type 1 (HIV-1) Tat protein transduction domain (PTD) is responsible for highly efficient protein transduction across plasma membranes. In a previous study, we showed that Tat-Cu,Zn-superoxide dismutase (Tat-SOD) can be directly transduced into mammalian cells across the lipid membrane barrier. In this study, we fused the human SOD gene with a Tat PTD transduction vector at its N- and/or C-terminus. The fusion proteins (Tat-SOD, SOD-Tat, Tat-SOD-Tat) were purified from Escherichia coli and their ability to enter cells in vitro and in vivo compared by Western blotting and immunohistochemistry. The transduction efficiencies and biological activities of the SOD fusion protein with the Tat PTD at either terminus were equivalent and lower than the fusion protein with the Tat PTD at both termini. The availability of a more efficient SOD fusion protein provides a powerful vehicle for therapy in human diseases related to this anti-oxidant enzyme and to reactive oxygen species.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2006.05a
• /
• pp.17-19
• /
• 2006

• Molecules and Cells
• /
• v.21 no.1
• /
• pp.104-111
• /
• 2006

Gene therapy with nonviral vectors using the suicide gene/prodrug activating system of herpes simplex virus type-1 thymidine kinase (HSV1-TK)/ganciclovir (GCV) is inefficient in killing malignant tumor cells due to two major factors: (a) an unsatisfactory bystander effect; (b) short-lived expression of the protein. To study the capacity of the protein transduction domain (PTD) of HIV-1 TAT protein to enhance HSV1-TK/GCV cancer gene therapy, we constructed three fusion proteins TAT-TK, TK-TAT and TK. TAT-TK retained as much enzyme activity as TK, whereas that of TK-TAT was much lower. TAT-TK can enter HepG2 cells and much of it is translocated to the nucleus. The transduced HepG2 cells are killed by exogenously added GCV and have bystander effects on untransduced HepG2 cells. Most importantly, the introduced recombinant protein is stable and remains functional for several days at least, probably because nuclear localization protects it from the cytoplasmic degradation machinery and provides access to the nuclear transcription machinery. Our results indicate that TAT fusion proteins traffic intercellularly and have enhanced stability and prodrug cell killing activity. We conclude that TAT has potential for enhancing enzyme prodrug treatment of liver cancers.

• Proceedings of the SCSK Conference
• /
• 2003.09a
• /
• pp.630-644
• /
• 2003

A human Cu, Zn-superoxide dismutase (Cu, Zn-SOD) was fused with a Tat PTD of HIV-1 to produce a novel anti-aging ingredient, Tat-SOD for cosmeceuticals. Test of stability and evaluation of transduction efficacy and enzymatic activity suggest Tat-SOD is an effective active ingredient for anti-aging treatment.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2001.11a
• /
• pp.90-96
• /
• 2001