T cell stimulation remodels the latently HIV-1 infected cell population by differential activation of proviral chromatin

The reservoir of latently HIV-1 infected cells is heterogeneous. To achieve an HIV-1 cure, the reservoir of activatable proviruses must be eliminated while permanently silenced proviruses may be tolerated. We have developed a method to assess the proviral nuclear micro-environment in single cells. In latently HIV-1 infected cells, a zinc finger protein tethered to the HIV-1 promoter produced a fluorescent signal as a protein of interest came in its proximity, such as the viral transactivator Tat when recruited to the nascent RNA. Tat is essential for viral replication. In these cells we assessed the proviral activation and chromatin composition. By linking Tat recruitment to proviral activity, we dissected the mechanisms of HIV-1 latency reversal and the consequences of HIV-1 production. A pulse of promoter-associated Tat was identified that contrasted to the continuous production of viral proteins. As expected, promoter H3K4me3 led to substantial expression of the provirus following T cell stimulation. However, the activation-induced cell cycle arrest and death led to a surviving cell fraction with proviruses encapsulated in repressive chromatin. Further, this cellular model was used to reveal mechanisms of action of small molecules. In a proof-of-concept study we determined the effect of modifying enhancer chromatin on HIV-1 latency reversal. Only proviruses resembling active enhancers, associated with H3K4me1 and H3K27ac and subsequentially recognized by BRD4, efficiently recruited Tat upon cell stimulation. Tat-independent HIV-1 latency reversal of unknown significance still occurred. We present a method for single cell assessment of the microenvironment of the latent HIV-1 proviruses, used here to reveal how T cell stimulation modulates the proviral activity and how the subsequent fate of the infected cell depends on the chromatin context. Upon infection with HIV-1, viral DNA is integrated into the cellular genome and adopts the surrounding chromatin structure. Integrated viral DNA can lead to production of new viral particles, or remain latently integrated in the cell. The chromatin environment strongly influences this state. Moreover, the viral protein Tat plays an important role in reversing latency and spreading the infection. In this work, we have developed a method to assess the microenvironment of the integrated HIV-1 promoter in single cells. Although continuous viral protein production occurred after T cell stimulation, we found that Tat was present only briefly at the viral promoter and the chromatin landscape around the HIV-1 locus was altered. This new method helps to understand the impact of specific chromatin modifications on the HIV-1 activation potential. Using small molecules, we also modified chromatin marks and analyzed their effect on HIV-1 latency. With this study, we provide new insights in the cellular mechanisms of HIV-1 latency and its reversal, which sheds light on the struggles to overcome to achieve a functional HIV-1 cure.

Automated summary

This study developed a method to assess the microenvironment of the integrated HIV-1 promoter in single cells, revealing how T cell stimulation modulates the proviral activity and the subsequent fate of the infected cell depends on the chromatin context. Using small molecules, they also analyzed the effect of modifying chromatin marks on HIV-1 latency.