Role of Sub-Cellular Localization and Cellular Factors In Regulation of HIV-1 Tat Protein Stability

Human Immunodeficiency Virus (HIV) -1 Tat protein plays an indispensable role in viral gene transcription. Tat is reported to produce a favorable environment for viral replication by modulating a variety of cellular processes. A vast amount of literature is available on the function and effects of Tat, however; intracellular regulation of Tat protein is not precisely understood. In this study, degradation of HIV-1 Tat protein was studied in detail. Tat was predominantly localized to the cell nucleus and nucleolus. Tat showed a relatively short half life of (3.5 ± 0.07) h. 26S proteasomal inhibitor, MG132 rescued Tat from degradation indicating that degradation occurs via the proteasomal pathway. Proteasomal pathway is active both in the cytoplasm and nucleus. Nuclear and cytoplasmic protein extracts prepared following CHX treatment showed marked degradation of Tat in cytoplasm whereas nuclear Tat showed minimal degradation. Degradation pattern of cytoplasmic Tat mutants, one lacking nuclear localization signal and other containing a nuclear export signal, showed reduced half life. Interestingly, cytoplasmic localization of Tat mutants resulted in an enhanced rate of degradation. These data demonstrate that cytoplasmic localization compromises Tat stability. siRNA mediated knock down of exportin-1 (Xpo1), which is the most common nuclear export pathway in cells, resulted in suppression of Tat degradation. Similarly, use of Leptomycin B (LMB), an inhibitor of Xpo1 mediated nuclear export rescued Tat from degradation. The data suggest that Tat directly or indirectly utilizes Xpo1 pathway of nuclear export. In co-immunoprecipitation experiments, HIV-1 derived Tat was found to interact with Xpo1. It was also observed that, in addition to proteasomal degradation some fraction of Tat protein was released from the cells in extracellular milieu. Western blot analysis demonstrated that Tat was present in cell supernatants collected from Tat expressing cells. Interestingly, Tat coming from producer cells could enter neighboring cells and induce HIV-1 LTR-transactivation. Hence, the fate of Tat protein in infected cells is to go through the proteasomal degradation pathway in cytoplasm or get released out of the cells where it can show effects on bystander cells