Latent HIV proviruses are silenced as the result of deacetylation and methylation of histones located at the viral long terminal repeat (LTR). Inhibition of histone deacetylases (HDACs) leads to the reemergence of HIV-1 from latency, but the contribution of histone lysine methyltransferases (HKMTs) to maintaining HIV latency remains uncertain. Chromatin immunoprecipitation experiments using latently infected Jurkat T-cell lines demonstrated that the HKMT enhancer of Zeste 2 (EZH2) was present at high levels at the LTR of silenced HIV proviruses and was rapidly displaced following proviral reactivation. Knockdown of EZH2, a key component of the Polycomb repressive complex 2 (PRC2) silencing machinery, and the enzyme which is required for trimethyl histone lysine 27 (H3K27me3) synthesis induced up to 40% of the latent HIV proviruses. In contrast, there was less than 5% induction of latent proviruses following knockdown of SUV39H1, which is required for H3K9me3 synthesis. Knockdown of EZH2 also sensitized latent proviruses to external stimuli, such as T-cell receptor stimulation, and slowed the reversion of reactivated proviruses to latency. Similarly, cell populations that responded poorly to external stimuli carried HIV proviruses that were enriched in H3K27me3 and relatively depleted in H3K9me3. Treating latently infected cells with the HKMT inhibitor 3-deazaneplano-cin A, which targets EZH2, led to the reactivation of silenced proviruses, whereas chaetocin and BIX01294 showed only minimal reactivation activities. These findings suggest that PRC2-mediated silencing is an important feature of HIV latency and that inhibitors of histone methylation may play a useful role in induction strategies designed to eradicate latent HIV pools. Current highly active antiretroviral therapies (HAARTs) for HIV infection rely on cocktails of potent antiviral drugs to reduce virus in the peripheral circulation to below detectable levels (74). Unfortunately, this regimen fails to eradicate the virus. Even after decades of effective HAART, high levels of virus replication invariably resume when antiretroviral treat-ment is interrupted (13, 17). The viral rebound appears to be due to reactivation of virus from a long-lived pool of latently infected cells (21). Genetic evidence strongly suggests that the latent virus resides primarily in the pool of resting memory CD4 T cells, since both the residual virus recovered from treated patients (7) and the rebounding virus recovered during the short treatment interruptions (31) have much greater se-quence homogeneity than would be expected for a viral pop-ulation replicating at low levels. Eliminating the latent reser-voir is particularly challenging since the reservoir is established early during infection (12), is extremely stable, with an esti-mated half-life of 44 months (64), and can be replenished during episodes of viremia (14) or by homeostatic replacement of latently infected cells (11). Since intensification of antiviral regimens has essentially no impact on eradicating the latent pool from the infected host (18), there is a pressing need to develop entirely novel forms of therapy that purge the pool of latent proviruses (62, 69). Latent HIV infections arise when the expression of the viral trans-activator protein, Tat, which stimulates transcriptional elongation, is restricted leading to ineffective HIV transcrip-tion (reviewed in reference 35). The formation of chromatin blocks to HIV transcription are believed to be one of the primary events leading to Tat restriction and the silencing of HIV proviruses (reviewed in references 27 and 54). Although most latent HIV-1 proviruses are found integrated into actively transcribed genes (28, 48), latent HIV-1 proviruses character-istically acquire heterochromatic structures. Typically, the long terminal repeats (LTRs) of latent proviruses accumulate high levels of histone deacetylases (HDACs) and deacetylated his-tones (71, 75, 79), as well as methylated histones (19, 53, 60, 72). In addition to histone methylation, hypermethylation of CpG islands near the HIV-1 promoter correlates with silencing of HIV-1 transcription in both Jurkat cells and primary isolates from HIV-1-positive aviremic patients (4, 36). Treatment of these cells with the DNA methylation inhibitor 5-aza-deoxycy-tidine (5-aza-CdR) leads to enhanced reactivation and out-growth of silenced proviruses (4, 36). Additional mechanisms that can contribute to HIV-1 silencing include transcriptional interference when the viruses have integrated into actively transcribed genes (29, 47). However, even in these circum-stances the latent proviruses also acquire restrictive chromatin structures that are essential for the maintenance of HIV-1 latency (36, 60). Thus, epigenetic silencing of HIV-1 proviruses appears to be a general feature of all HIV-1 infections, which