Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology
Anthony Obi, Jr. - Texas Southern University
Intercellular communication between HIV infected and uninfected cells is critical for HIV-1 transmission and AIDS progression. LFA1 is expressed on the cell surface and its expression is inhibited by HIV-1 infection, which disrupts the intercellular communication between LFA1 and Icam1. HIV-1 Nef is also important for intercellular communication by modulating the expression of cytokines and chemokines or by formation of a protrusion. However the molecular mechanism of how HIV infected cells reduce LFA1 expression on the cell surface and thereby block intercelluar communication is unknown. We believe HIV-1 Nef and LFA1 are key regulators in the molecular process of intercellular interaction and key to the spread of HIV-1. Purpose: The aim of this study is to analyze the effects of HIV-1 Nef on the expression of the intracellular adhesion protein, LFA1. Methods: HIV-1 Nef expressing plasmid DNA was constructed by the standard cloning method and transfected into 293T cells by the calcium phosphate method. At 60 hours post transfection, the cells were washed with PBS and lysed with lysis buffer (0.15M NaCl, 0.5% NP40, 2mM EDTA, 10mM Tris-Cl pH 7.5) to collect cellular proteins. The protein concentration in the lysate was determined by colormetric analysis and 50 μg of protein was applied to10% SDS PAGE for separation of the proteins. Western Blot was performed to visualize the protein band. Briefly membranes were incubated with the indicated antibodies and visualized with Bio-Rad ECL Clarity solution. Relative amount of the proteins was quantified using Bio-Rad image analysis system. Results: Our flow cytometric analysis indicated that the amount of LFA1 on the surface of Jurkat cell was reduced by the expression of HIV-1 Nef. Whether the diminution of LFA1 was due to the reduced intracellular gene expression or down modulation of surface presentation LFA1 the molecular mechanism is under investigation.
Funder Acknowledgement(s): Dr. Inwoo Park
Faculty Advisor: Ayodotun Sodipe, firstname.lastname@example.org
Role: I did all parts of the research listed above.