The signal which triggers replication of the HIV genome in T4 cells is an attractive target for the design of new promising anti-AIDS chemotherapy. A number of recent investigations have shown that polyclonal mitogens, tumor promoters, certain cytokines, and other stimuli promote the infection of T4 cells by HIV, and stimulate marked increases in the expression of previously latent HIV genome in these lymphocytes. Nevertheless, it is still uncertain whether these structurally diverse classes of agonists stimulate replication of the virus via a common or distinct molecular mechanisms. This study will examine the role of the signal transducing enzyme. PKC, in the infection of T4 cells by HIV-1, and in trans-activation of latent viral genome in the lymphocytes. Mitogen-responsive T4 cells lines will be infected with a standard preparation of HIV-1 in the presence of PKC-activating agents, or PKC inhibitors. Time-dependent internalization of the HIV-1 nucleocapsid, and RNA template-directed reverse transcription of the first copies of proviral DNA intermediates, as well as virus replication in T4 cells will be analyzed by means of the following techniques: a) immunofluorescence; b) Taq DNA polymerase-dependent chain reaction (PCR) using oligonucleotide primer pairs complementary to conserved sequences in the LTR, GAG, and ENV genes; c) MTT chromogenic test for virus-induced lysis of target cells; and d) p55 antigen capture immunoassay. To assess the role of PKC in trans-activation and replication of the latent HIV-1 genome, T4 cells will be stably transfected with HIV-pBR322 plasmid constructs which express the TAT, ENV as well as heterologous CAT reporter gene ligated at positions down stream from the LTR. DNA- transfected cells will be incubated with the modulators of PKC. The trans-activating effects of the ligands on the expression of the HIV-1 genome will be quantified by means of several techniques: by a) liquid scintillation assay for the LTR-directed expression of the CAT reporter gene; and b) northern blot hybridization using the ENV and TAT cDNA probes. In addition, a potential role of PKC-dependent secretion of specific cytokines in the regulation of the HIV-1 replication will be examined. MRNA isolated from normal peripheral blood mononuclear cells following in vitro infection with HIV-1 will be analyzed by means of northern blot hybridization to quantify the levels of expression of the genes encoding IL 2, IL 6, INF-alpha, INF-gamma, and TNF-alpha. The goal of this investigation is to elucidate the cellular and molecular mechanisms which determine the latent and lytic course of HIV-1 infection.

Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
1996
Total Cost
Indirect Cost
Fried, Eric S; Li, Yue-Ming; Gilchrist, M Lane (2017) Phase Composition Control in Microsphere-Supported Biomembrane Systems. Langmuir 33:3028-3039
Gilchrist, M Lane; Ahn, Kwangwook; Li, Yue-Ming (2016) Imaging and Functional Analysis of ?-Secretase and Substrate in a Proteolipobead System with an Activity-Based Probe. Anal Chem 88:1303-11
Fried, Eric S; Luchan, Joshua; Gilchrist, M Lane (2016) Biodegradable, Tethered Lipid Bilayer-Microsphere Systems with Membrane-Integrated ?-Helical Peptide Anchors. Langmuir 32:3470-5
Banerjee, Shaibal; Sinha, Saikat; Pradhan, Padmanava et al. (2016) Regiospecifically Fluorinated Polycyclic Aromatic Hydrocarbons via Julia-Kocienski Olefination and Oxidative Photocyclization. Effect of Fluorine Atom Substitution on Molecular Shape. J Org Chem 81:3983-93
Beck, Cade; Singh, Tanya; Farooqi, Angela et al. (2016) Controlled microfluidics to examine growth-factor induced migration of neural progenitors in the Drosophila visual system. J Neurosci Methods 262:32-40
Salas-Ramirez, Kaliris Y; Bagnall, Ciara; Frias, Leslie et al. (2015) Doxorubicin and cyclophosphamide induce cognitive dysfunction and activate the ERK and AKT signaling pathways. Behav Brain Res 292:133-41
Thomson, Paul F; Parrish, Damon; Pradhan, Padmanava et al. (2015) Modular, Metal-Catalyzed Cycloisomerization Approach to Angularly Fused Polycyclic Aromatic Hydrocarbons and Their Oxidized Derivatives. J Org Chem 80:7435-46
Small, Chiyedza; Ramroop, Johnny; Otazo, Maria et al. (2014) An unexpected link between notch signaling and ROS in restricting the differentiation of hematopoietic progenitors in Drosophila. Genetics 197:471-83
Zhong, Lina; Tu, Raymond; Gilchrist, M Lane (2013) Tether-supported biomembranes with ?-helical peptide-based anchoring constructs. Langmuir 29:299-307
Guleyupoglu, Berkan; Schestatsky, Pedro; Edwards, Dylan et al. (2013) Classification of methods in transcranial electrical stimulation (tES) and evolving strategy from historical approaches to contemporary innovations. J Neurosci Methods 219:297-311

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