Natural killer cells induce HIV-1 latency reversal after treatment with pan-caspase inhibitors.

The establishment of a latency reservoir is the major obstacle for a cure of HIV-1. The shock-and-kill strategy aims to reactivate HIV-1 replication in HIV -1 latently infected cells, exposing the HIV-1-infected cells to cytotoxic lymphocytes. However, none of the latency reversal agents (LRAs) tested so far have shown the desired effect in people living with HIV-1. We observed that NK cells stimulated with a pan-caspase inhibitor induced latency reversal in co-cultures with HIV-1 latently infected cells. Synergy in HIV-1 reactivation was observed with LRAs prostratin and JQ1. The supernatants of the pan-caspase inhibitor-treated NK cells activated the HIV-1 LTR promoter, indicating that a secreted factor by NK cells was responsible for the HIV-1 reactivation. Assessing changes in the secreted cytokine profile of pan-caspase inhibitor-treated NK cells revealed increased levels of the HIV-1 suppressor chemokines MIP1α (CCL3), MIP1ß (CCL4) and RANTES (CCL5). However, these cytokines individually or together did not induce LTR promoter activation, suggesting that CCL3-5 were not responsible for the observed HIV-1 reactivation. The cytokine profile did indicate that pan-caspase inhibitors induce NK cell activation. Altogether, our approach might be-in combination with other shock-and-kill strategies or LRAs-a strategy for reducing viral latency reservoirs and a step forward towards eradication of functionally active HIV-1 in infected individuals.

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 microenvironment 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.

Host-pathogen coevolution increases genetic variation in susceptibility to infection.

It is common to find considerable genetic variation in susceptibility to infection in natural populations. We have investigated whether natural selection increases this variation by testing whether host populations show more genetic variation in susceptibility to pathogens that they naturally encounter than novel pathogens. In a large cross-infection experiment involving four species of Drosophila and four host-specific viruses, we always found greater genetic variation in susceptibility to viruses that had coevolved with their host. We went on to examine the genetic architecture of resistance in one host species, finding that there are more major-effect genetic variants in coevolved host-pathogen interactions. We conclude that selection by pathogens has increased genetic variation in host susceptibility, and much of this effect is caused by the occurrence of major-effect resistance polymorphisms within populations.