Systems analysis of latent HIV reversal reveals altered stress kinase signaling and increased cell death in infected T cells

Linda E Fong,Kathryn Miller-Jensen,Endah S Sulistijo

doi:10.1038/s41598-017-15532-0

Abstract

Viral latency remains the most significant obstacle to HIV eradication. Clinical strategies aim to purge the latent CD4+ T cell reservoir by activating viral expression to induce death, but are undercut by the inability to target latently infected cells. Here we explored the acute signaling response of latent HIV-infected CD4+ T cells to identify dynamic phosphorylation signatures that could be targeted for therapy. Stimulation with CD3/CD28, PMA/ionomycin, or latency reversing agents prostratin and SAHA, yielded increased phosphorylation of IκBα, ERK, p38, and JNK in HIV-infected cells across two in vitro latency models. Both latent infection and viral protein expression contributed to changes in perturbation-induced signaling. Data-driven statistical models calculated from the phosphorylation signatures successfully classified infected and uninfected cells and further identified signals that were functionally important for regulating cell death. Specifically, the stress kinase pathways p38 and JNK were modified in latently infected cells, and activation of p38 and JNK signaling by anisomycin resulted in increased cell death independent of HIV reactivation. Our findings suggest that altered phosphorylation signatures in infected T cells provide a novel strategy to more selectively target the latent reservoir to enhance eradication efforts.

Highlights

Cellular reservoirs infected with latent human immunodeficiency virus-1 (HIV) are the primary obstacle to HIV eradication[1,2]
Kinase phosphorylation signatures following T cell activation are different between latent HIVinfected and uninfected primary TCM cells in vitro
We focused on T cell receptor (TCR) stimulation by CD3/CD28 and stimulation by phorbol 12-myristate 13-acetate/ionomycin (PMA/I), which elicit strong viral reactivation (Fig. 1d)

Summary

Introduction

Cellular reservoirs infected with latent human immunodeficiency virus-1 (HIV) are the primary obstacle to HIV eradication[1,2]. One reason biomarkers of latent HIV infection are difficult to identify is that biological changes which cause disease often do not produce clear differences in protein levels that can be observed in a basal state, but instead affect interactions between proteins[7] For this reason, stimulating diseased cells and following the dynamics of protein activation over time has proved to be a successful way to differentiate between healthy and diseased cells in cancer[8] and type 1 diabetes[9] and to therapeutically target the disease state[10]. There is evidence that latent HIV-infected T cells exhibit virus-induced changes, including chromatin-mediated transcriptional silencing and altered activities of select kinases[5,11,12], which might affect signaling in latently infected cells following stimulation in a manner similar to a disease state This raises the possibility–as yet untested–that T cell signaling networks are altered by latent HIV infection or by viral protein expression upon latency reversal, and that these differences could be targeted for HIV eradication. We propose that targeting modified systems-level signaling in latently infected cells provides a clinically promising strategy to improve LRA specificity and efficacy

Methods

Results

Conclusion