Kinase Control Prevents HIV-1 Reactivation in Spite of High Levels of Induced NF-κB Activity

Abstract

Despite its clinical importance, the molecular biology of HIV-1 latency control is at best partially understood, and the literature remains conflicting. The most recent description that latent HIV-1 is integrated into actively expressed host genes has further confounded the situation. This lack of molecular understanding complicates our efforts to identify therapeutic compounds or strategies that could reactivate latent HIV-1 infection in patients, a prerequisite for the eradication of HIV-1 infection. Currently, many therapeutic development efforts operate under the assumption that a restrictive histone code could govern latent infection and that either dissipation of the histone-based restrictions or NF-B activation could be sufficient to trigger HIV-1 reactivation. We here present data that suggest an additional, higher level of molecular control. During a high-content drug screening effort, we identified AS601245 as a potent inhibitor of HIV-1 reactivation in latently infected primary T cells and T cell lines. In either system, AS601245 inhibited HIV-1 reactivation despite high levels of induced NF-B activation. This finding suggests the presence of a gatekeeper kinase activity that controls latent HIV-1 infection even in the presence of high levels of NF-B activity. Potential therapeutic stimuli that do not target this gatekeeper kinase will likely fail to trigger efficient system-wide HIV-1 reactivation.

Kinase Control of Latent HIV-1 Infection: PIM-1 Kinase as a Major Contributor to HIV-1 Reactivation

“…All T cell lines were maintained in RPMI 1640 medium supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 10% heat-inactivated fetal bovine serum (FBS). The latently HIV-1-infected CA5 and EF7 T cells were generated using an NL4-3-based green fluorescent protein (GFP) reporter virus (NLENG) (11,24). Each of these cell lines contains a single integration event within an actively expressed host gene.…”

“…PBMCs were generated as previously described (11). T cells were stimulated in the presence or absence of the respective inhibitors using 3 g/ml phytohemagglutinin L (PHA-L), and culture supernatants were harvested after 24 h. Cytokine expression was determined using MilliPlex kits for IL-2, IL-4, IL-6, IL-8, IL-17, and gamma interferon (IFN-␥) (Millipore).…”

“…We demonstrated the presence of a kinase function that acts as a master switch to control latent HIV-1 infection even in the presence of high levels of induced NF-B activity, which was present in latently infected T cell lines and primary CD4 T cells (11). Additional evidence for a role of specific transcription factors in latency control comes from our observation that naturally occurring variations of the AP-1 motif in the CD28-responsive element (CD28RE) of the HIV-1 long terminal repeat (LTR) influence the efficacy of latency establishment (12).…”

Novel Prospective Treatment Options

“…This led to the identification of a gatekeeper kinase which has to be concurrently activated along with NF-κB to facilitate viral replication. Other compounds such as prostratin and bryostatin-1 are also being evaluated for efficacy in the activation of NF-κB pathways [128]. Activators of elongation factor-b such as hexamethylene bisacetammide, disulfiram and certain quinolones are being evaluated as shocking agents.…”

“…The compound Jun N-terminal protein kinase inhibitor-5 which is a potent inhibitor of the gatekeeper kinase strongly prevents viral reactivation even upon strong stimulation of the NF-κB pathway under in vitro conditions. Inhibitors of the NF-κB pathway such as aloisine A and roscovitine also inhibit HIV reactivation to a lesser extent [128]. Various other molecules such as C-terminal truncated STAT5, Staf 50, prothymosin α, thioredoxin reductase, glucosamine and OKT-18 zinc finger protein have been identified to prolong the proviral repression.…”