HIV integration and the establishment of latency in CCL19-treated resting CD4(+) T cells require activation of NF-κB.

Suha Saleh,Talia M Mota,David Harisson,Melissa Churchill,Anthony Jaworowski,Surekha Tennakoon,Dimitrios N Vatakis,Jingling Zhou,Johnson Mak,Sharon R Lewin,Georgina Sallmann,Karey Cheong ,Vanessa A Evans ,Andrew N Harman ,Thomas A Angelovich ,Anthony L Cunningham ,Jenny L Anderson ,Heidi E Drummer ,Lachlan Robert Gray ,Hao K Lu ,Paul Cameron

doi:10.1186/s12977-016-0284-7

Abstract

Background Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4+ T cells. We previously reported that HIV latency could be established in resting CD4+ T cells in the presence of the chemokine CCL19. To define how CCL19 facilitated the establishment of latent HIV infection, the role of chemokine receptor signalling was explored.ResultsIn resting CD4+ T cells, CCL19 induced phosphorylation of RAC-alpha serine/threonine-protein kinase (Akt), nuclear factor kappa B (NF-κB), extracellular-signal-regulated kinase (ERK) and p38. Inhibition of the phosphoinositol-3-kinase (PI3K) and Ras/Raf/Mitogen-activated protein kinase/ERK kinase (MEK)/ERK signalling pathways inhibited HIV integration, without significant reduction in HIV nuclear entry (measured by Alu-LTR and 2-LTR circle qPCR respectively). Inhibiting activation of MEK1/ERK1/2, c-Jun N-terminal kinase (JNK), activating protein-1 (AP-1) and NF-κB, but not p38, also inhibited HIV integration. We also show that HIV integrases interact with Pin1 in CCL19-treated CD4+ T cells and inhibition of JNK markedly reduced this interaction, suggesting that CCL19 treatment provided sufficient signals to protect HIV integrase from degradation via the proteasome pathway. Infection of CCL19-treated resting CD4+ T cells with mutant strains of HIV, lacking NF-κB binding sites in the HIV long terminal repeat (LTR) compared to infection with wild type virus, led to a significant reduction in integration by up to 40-fold (range 1–115.4, p = 0.03). This was in contrast to only a modest reduction of 5-fold (range 1.7–11, p > 0.05) in fully activated CD4+ T cells infected with the same mutants. Finally, we demonstrated significant differences in integration sites following HIV infection of unactivated, CCL19-treated, and fully activated CD4+ T cells.ConclusionsHIV integration in CCL19-treated resting CD4+ T cells depends on NF-κB signalling and increases the stability of HIV integrase, which allow subsequent integration and establishment of latency. These findings have implications for strategies needed to prevent the establishment, and potentially reverse, latent infection.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0284-7) contains supplementary material, which is available to authorized users.

Highlights

Eradication of HIV cannot be achieved with combination antiretroviral therapy because of the persistence of long-lived latently infected resting memory CD4+ T cells
HIV integration and latency is established in resting CD4+ T cells in vivo [4], in tonsil explants [5], or following co-culture with endothelial cells [6] or dendritic cells [7], or following culture with chemokines that bind to the chemokine receptors CCR7, CXCR3 and CCR6 expressed on resting CD4+ T cells [8, 9]
We demonstrated that activation of the NF-κB pathway is critical for efficient integration of HIV in CCL19-treated cells (CCL19)-treated resting CD4+ T cells and that the sites of HIV integration depended on the activation state of the cell at the time of infection

Summary

Introduction

Eradication of HIV cannot be achieved with combination antiretroviral therapy (cART) because of the persistence of long-lived latently infected resting memory CD4+ T cells. The chemokine CCR7 is expressed on naïve and central memory CD4+ T cells and culturing with the CCR7ligands CCL19 or CCL21 facilitates entry and integration of HIV into these resting CD4+ T cells with minimal virus production. In this model, resting CD4+ T cells 5 day post infection represent a stable latently infected population that respond to latency-reversing agents (LRA) in a similar pattern to CD4+ T cells from HIVinfected individuals on cART [10]. We believe this is an ideal model to study the early events required for establishing latency

Methods

Results

Discussion

Conclusion