HIV virions were sedimented at 100,000 for 1.5 h and resuspended in PBS at 10 g of p24/ml. marrow-liver-thymus mice up to 11 weeks after treatment cessation. Despite the absence of rebound viremia, we were able to recover infectious HIV from PBMC with heat shock. Replication-competent virus was detected in spleen cells from these nonviremic Hsp90 inhibitor-treated mice, indicating the presence of a tissue reservoir of persistent contamination. Our novel findings provide evidence that inhibition of Hsp90 activity prevents HIV gene expression in replication-competent cellular reservoirs that would typically cause rebound in plasma viremia after antiretroviral therapy cessation. Alternating or supplementing Hsp90 inhibitors with current antiretroviral therapy regimens could conceivably suppress rebound viremia from persistent HIV reservoirs. HIV host factor by pharmacologic inhibition and by siRNA-mediated silencing of cellular Hsp90 in primary human cells (20). Hsp90 is usually a unique member of the heat shock protein family of cellular chaperones in that it uses the energy generated by ATP hydrolysis to activate its client proteins (18, 22, 23). The Hsp90 inhibitors we used (17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and AUY922) have a high affinity for the unique ATP-binding pocket created by Hsp90 dimerization, and these competitive inhibitors specifically block the ATPase activity of the mature Hsp90 protein complex (24). Highly specific second-generation Hsp90 TH 237A inhibitors currently being evaluated in clinical trials do not interact with other heat shock proteins or cellular factors and have improved bioavailability and significantly reduced toxicity (24, 25). Heat shock has previously been shown to control HIV reactivation from latency (26), and a recent study suggested that Hsp90 inhibitors prevent HIV gene expression by suppressing NF-B activation (27). The chaperone function of cellular Hsp90 is not restricted to activating HIV transcription, because we previously exhibited that replication-incompetent HIV with mutant capsids could be rescued by increased Hsp90 activity (21, 28). We and others also found that Hsp90 is usually incorporated within the mature virion (21, 29), and there is growing evidence that several virus families exploit cellular Hsp90 for folding and assembly of virus structural proteins and for maturation of viral enzymes (30,C32). Heat shock induces cellular transcription through a rapid increase in Hsp90 activity (33, 34). Previous studies have exhibited that heat shock increases HIV production and that Hsp90 colocalizes with the site of HIV transcription. In this study, we provide novel evidence that 39.5 C accelerates transcription from the HIV promoter through specific inducible host transcription factors and that inhibition of Hsp90 greatly reduces gene expression. Inhibition of Hsp90 with specific inhibitors in clinical development, tanespimycin (17-AAG) and AUY922, durably prevented viral rebound in HIV-infected humanized mice even after Hsp90 inhibitor treatment was discontinued. Replication-competent HIV was isolated from the mouse spleens despite undetectable HIV RNA or infected cells in the peripheral blood, indicating the establishment of a persistent tissue reservoir. HIV transcription in the spleen reservoir was reduced by Hsp90 inhibition, but replication-competent virus was readily isolated when the spleen cells were activated by heat shock and by treatment with suberoylanilide hydroxamic acid (SAHA). Here, we present evidence for a persistent HIV-infected tissue reservoir and show that administration of Hsp90 inhibitors for brief periods (2 weeks) prevents rebound in plasma viremia for many weeks after treatment cessation. The TH 237A ability of Hsp90 inhibitors to suppress HIV transcription was confirmed in chronically infected cell lines, and we demonstrate that Hsp90 inhibition directly affects HIV transcription. Heat shock conditions increased Hsp90 activity in chronically infected cells, and increased virus production at 39.5 C is the direct result of accelerated HIV transcription. Experimental Procedures Cell Lines, Virus Stocks, and Reagents HIV-infected 8E5/LAV cells and ACH-2 cells and uninfected Jurkat E6-1 cells were obtained from the National Institutes of TP53 Health AIDS Reagent Program (Division of AIDS, NIAID, National Institutes of Health) and were cultured in RPMI 1640 supplemented with 10% fetal bovine serum. Human peripheral blood mononuclear cells (PBMC) were isolated from healthy donors and stimulated with phytohemagglutinin (PHA) for 3 days, and PBMC from six donors were pooled and cryopreserved. The pNL4-3 (35) and pYK-JRCSF (36,C38) plasmids were obtained from the AIDS Reagent Program TH 237A and used to generate infectious virus stocks by transfecting HEK 293T cells. The virus titer in the infectious culture supernatant was decided in PHA-stimulated PBMC by end point dilution.