IDENTIFICATION OF CD8+ CELL ANTI HIV FACTOR

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Introduction Individuals who have been infected with HIV and remain healthy for over 10 years are considered long-term survivors. These asymptomatic individuals have a strong cell-mediated immune response that suppresses HIV replication. This activity is lost as the infected person advances to disease. The antiviral response is mediated by a secreted CD8+ cell antiviral factor (CAF) which blocks transcription via the HIV promoter. CAF is produced at low levels by CD8+ cells from these healthy infected individuals. The protein appears to be unlike any of the known cytokines, chemokines and human growth factors. CAF identification will most likely be achieved through protein purification and mass spectrometry. Methods CD8+ cells from healthy HIV-infected individuals are grown in culture medium lacking serum and where possible, albumin. Fluids are assayed for antiviral activity by standard procedures in the laboratory. Active fluids are then concentrated and fractionated by various biochemical techniques including ion exchange, chromatography, and by sizing columns Fractions with enriched CAF activity are analyzed by 2-D gel and evaluated by mass spectrometry. Results Thus far, we have been able to show that CAF has a molecular size of about 50 kD. By differential 2D gel analysis, there appears to be at least 20 different spots that are associated with CAF activity. Studies are in progress to analyzed by mass spectrometry. Those protein spots that are unique to CAF-containing fluids will be further evaluated. Discussion By mass spectrometry, we hope to be able to identify the protein (CAF) that mediates this anti-HIV activity.