302 Tb4 is an HIV-1 Inhibitory Factor in Transformed CD8+ Cell Supernatants M.E. Klotman*1, A. Mosoian1, A. Teixeira1, T. Chang1, A.A. High2, D.F. Hunt2, J. Shabanowitz2 1Mt Sinai Sch of Med, New York, NY and 2Univ of Virginia Sch of Med, Charlottesville
Background: CD8+ lymphocytes from normal and human immunodeficiency virus type 1 (HIV-1) infected patients (pts) produce a number of soluble factors that suppress the replication of HIV-1. Several of these factors have been identified as members of the b-chemokine family and shown to inhibit viral entry. There are other, unidentified factors that are responsible for the anti-HIV-1 activity produced by CD8+ cells.
Methods: To identify these additional factors, we fractionated the supernatant of the Herpes virus saimiri (HVS) transformed CD8+ cell line K#1 50K, previously shown to have anti HIV-1 activity, not attributable to b-chemokines or interferon a, b, and g. Chromatographic fractions were screened for HIV-1 inhibitory activity in macrophages with each fractionation step, and the final fractions were analyzed by mass spectroscopy to identify the component(s). Mechanism of inhibition was analyzed in a single pass infection measuring reverse transcribed HIV DNA and extrachromasomal circular (ec) HIV DNA and in a transcription assay measuring luciferase.
Results: Thymosin b4 (Tb4) was the predominant protein in one of the final active fractions. Synthetic Tb4 inhibited HIV-1 Bal in primary macrophages and primary lymphocytes in a dose dependent manner with > 90 % inhibition at a dose of 200-400 ng/ml. In macrophages Tb4 inhibited virus following entry but prior to complete formation of the ec DNA.
Conclusion: Our data indicate that Tb4 is one of the active HIV-1 inhibitory components found in the transformed CD8+ cell supernatant and inhibits the virus following entry but prior to integration and transcription.
