The latent reservoir for HIV-1 in resting memory CD4+ T cells remains a major barrier to virus eradication. The lack of a convenient in vitro latency model in primary resting CD4+ T cells has hampered progress in exploring ways to overcome HIV-1 latency. In this study, we took advantage of the genetically modified primary CD4+ T cells to establish an in vitro HIV-1 latency system. We demonstrated that Bcl-2–transduced cells can reach a profoundly quiescent state in which HIV-1 latency can be established. More importantly, using this model, we performed small-molecule screening and identified 5HN as an activator of latent HIV-1 that, unlike previously described activators, does not induce global T cell activation. These results illustrate the application of this approach to the discovery of novel agents for the eradication of latent HIV-1.
Overexpression of Bcl-2 allows the primary CD4+
T cells to survive in a quiescent state, a key step to establishing HIV-1 latency in vitro. Although overexpression of Bcl-2 may alter the physiology of CD4+
T cells, our extensive analysis revealed that the Bcl-2–transduced cells are similar to freshly isolated CD4+
T cells in both the activated and resting states. Bcl-2 is a downstream antiapoptotic effector of IL-7, an essential cytokine in maintaining the survival of resting naive and memory T cells in vivo. Prior studies have shown that the Bcl-2 transgene can partly compensate for deprivation of IL-7, indicating the important role of Bcl-2 in the homeostasis of resting T cells (35
). Because IL-7 also activates latent HIV-1 (12
), decoupling Bcl-2 from other IL-7 signaling pathways provided us an opportunity to measure the HIV-1 LTR activity in cells that are in a profoundly quiescent state.
Another concern is that a modified HIV-1 vector was used instead of wild-type virus. Although the modifications increased the yield of latently infected cells, there is the possibility that modified HIV-1 may behave differently than wild-type virus. We have preserved portions of the HIV-1 sequence relevant to the regulation of viral gene expression (LTR, tat
, and rev
). Therefore, this model is most useful in studying the upregulation of HIV-1 gene expression in latently infected cells, as we have done here. We have shown that the signaling pathways that lead to reactivation of latent HIV-1 are well preserved in this in vitro model. We tested an array of small molecules and cytokines and observed responses very similar to those seen in other primary cell models and latently infected CD4+
T cells from patients on HAART (10
). This result justifies the use of this model in investigating the reactivation of latent HIV-1. In order to increase the yield of latently infected cells, we inactivated genes whose products have been associated with cytopathicity. For this reason, the model may not be ideal for studying the fate of cells reactivated from latency or the efficiency with which latency is established. Importantly, we have recently shown that latently infected cells can be obtained using the same approach with a vector in which all of the HIV-1 ORFs except env
are functional (L. Shan et al., unpublished observations). However, as expected, the yield of latently infected cells is substantially lower.
Although several in vitro HIV-1 latency models in primary CD4+
T cells have been described recently, certain factors limit their utility in high-throughput screening for activators of latent HIV-1. Several studies have demonstrated that HIV-1 can establish latency by direct infection of resting CD4+
T cells in vitro (40
). This strategy does have some advantages because it avoids the activation of T cells and utilizes wild-type HIV-1. However, the efficiency with which latency is established may be lower than that of the activation-dependent pathway described here, and this system is still subject to the short life span of resting CD4+
T cells in an in vitro culture system. Bosque et al. infected activated CD4+
T cells and maintained them in the presence of IL-2 (25
). Because IL-2 also activates latent HIV-1, it is uncertain whether IL-2 disturbs the quiescent state of these cells. Some models were created by infection of thymocytes with HIV-1 (24
). However, the majority of the resulting cells were resting naive CD4+
T cells rather than memory CD4+
T cells, which harbor latent HIV-1 in vivo. Marini et al. used low doses of IL-7 to generate and maintain memory CD4+
T cells in vitro up to 2 months and established viral latency among these cells (26
). Although interesting, this model only yields approximately 20% viable cells and therefore may not produce enough latently infected cells for high-throughput screening. In addition, IL-7 can activate latent HIV-1 (12
). This may also confound the results of studies examining the reactivation of latent HIV-1 by other agents. Therefore, our primary cell HIV-1 latency model is the only one in which cells are maintained in vitro in the absence of activating cytokines.
The system described here can serve as an efficient platform to screen for activators of latent HIV-1 that do not induce global T cell activation. Bcl-2 transduction allows us to generate sufficient numbers of latently infected cells for high-throughput screening. In addition, the resting phenotype of the resulting cells recapitulates the quiescent state of primary resting CD4+ T cells. By flow cytometry analysis, we can easily detect changes in parameters that measure T cell activation, for instance, cell size. We illustrated the potential application of this in vitro system by identifying 5HN as an activator of latent HIV-1 that does not induce global T cell activation. 5HN can strongly reactivate latent HIV-1 with an efficiency similar to that of TCR stimulants. High reactivation efficiency is essential for eradicating HIV-1 because even a small number of viruses released from any residual latently infected cells can rebound exponentially after discontinuation of HAART.
Our data suggest that 5HN activates latent HIV-1 through the ROS and NF- κB signaling pathways. The role of ROS in the activation of NF-κB was first described by Schreck et al. (59
). Some investigators have proposed a central role for ROS in mediating the ability of extrinsic stimuli to activate NF-κB, although even after more than a decade of studies, there is still uncertainty about the target protein that senses ROS and transduces the signals to activate NF-κB (58
). The potential toxicity of ROS also creates concern about the therapeutic application of 5HN. Previous studies revealed differential cellular responses to the different levels of ROS. In response to low levels of ROS, cells upregulate the antioxidant genes, while high levels of ROS induce apoptosis. Interestingly, intermediate levels can stimulate some inflammatory genes, such as NF-κB and AP-1 (58
). This may explain why 5HN has a narrow therapeutic window. In addition, 5HN is chemically reactive and affects several cellular proteins (70
). Although we showed that 5HN activates latent HIV-1 independent of Pin1, it is possible that other mechanisms are involved. Further studies are required to determine whether we can decouple the toxicity of 5HN from its ability to activate latent HIV-1. One intriguing finding is that 5HN activates latent HIV-1 without causing nonspecific T cell activation. Given that NF-κB is a key transcription factor in controlling innate and adaptive immunity, it is surprising that 5HN stimulates latent HIV-1 by activating NF-κB without inducing global T cell activation. This can be explained by our observation that 5HN activates latent HIV-1 independent of NFAT and PKCθ. PKCθ is a master regulator of T cell activation and activates multiple signaling pathways, including the NF-κB, AP-1, and ERK1/2 pathways (62
). The importance of NFAT proteins in T cell activation is also well documented (71
). Bypassing PKCθ and NFAT may allow 5HN to avoid full-blown T cell activation. This finding also suggests that reactivation of latent HIV-1 does not necessarily require T cell activation and that the pathways leading to the activation of latent HIV-1 can be separated from those involving in T cell activation.
Because of the high cost and potential toxicities of long-term HAART and the disappointing results from the clinical trials of HIV-1 vaccines and microbicides (72
), there is still a pressing need for pursuing the goal of eradication. To cure HIV-1 infection is exceptionally challenging and will likely require combining HAART with agents that can purge latent virus. The identification of 5HN not only expands the number of classes of latency-reversing agents but also demonstrates the possibility of utilizing pathway(s) further downstream of TCR stimulation to avoid global T cell activation. Although the toxicities of 5HN raise concerns for its clinical application, this is a proof of concept for this approach to finding novel strategies to reactivate latent HIV-1 without inducing global T cell activation.