Fighting HIV-1 Persistence: At the Crossroads of "Shoc-K and B-Lock"

Abstract

Despite the success of highly active antiretroviral therapy (HAART), integrated HIV-1 proviral DNA cannot be eradicated from an infected individual. HAART is not able to eliminate latently infected cells that remain invisible to the immune system. Viral sanctuaries in specific tissues and immune-privileged sites may cause residual viral replication that contributes to HIV-1 persistence. The "Shock or Kick, and Kill" approach uses latency reversing agents (LRAs) in the presence of HAART, followed by cell-killing due to viral cytopathic effects and immune-mediated clearance. Different LRAs may be required for the in vivo reactivation of HIV-1 in different CD4⁺ T cell reservoirs, leading to the activation of cellular transcription factors acting on the integrated proviral HIV-1 LTR. An important requirement for LRA drugs is the reactivation of viral transcription and replication without causing a generalized immune activation. Toll-like receptors, RIG-I like receptors, and STING agonists have emerged recently as a new class of LRAs that augment selective apoptosis in reactivated T lymphocytes. The challenge is to extend in vitro observations to HIV-1 positive patients. Further studies are also needed to overcome the mechanisms that protect latently infected cells from reactivation and/or elimination by the immune system. The Block and Lock alternative strategy aims at using latency promoting/inducing agents (LPAs/LIAs) to block the ability of latent proviruses to reactivate transcription in order to achieve a long term lock down of potential residual virus replication. The Shock and Kill and the Block and Lock approaches may not be only alternative to each other, but, if combined together (one after the other), or given all at once [namely "Shoc-K(kill) and B(block)-Lock"], they may represent a better approach to a functional cure.

Keywords: Block and Lock; CD4+ T cell reservoirs; HIV-1 latency; LPAs; LRAs; LTR enhancer; NF-κB; Shock and Kill; Tat; c-Jun; immunostimulatory agents; p50.

Figure 1

Schematic representation of the HIV-1 transcription elongation process in a generic CD4⁺T memory cell, latently infected. Upon transcription initiation mediated by transcription factors (TFs) binding the Long Terminal Repeats (LTR) enhancer, a proportion of the viral transcripts paused forming the TAR RNA structure. The elongation factor PTEF-b, made by CycT1 and CDK9, is hijacked in the 7SK snRNP/HEXIM1 inhibitory complex in resting CD4⁺ T memory cells. The treatment of these cells with Hexamethylenebisacetamide (HMBA) determines PTEF-b release from the inhibitory complex. PTEF-b can be bound to the Bromodomain-containing protein (BRD) 4, thus stimulating transcription elongation from cellular genes. As soon as the viral transactivator Tat accumulates, it displaces PTEF-b from BRD4 to delocalize it to the HIV-1 Trans-activation response element (TAR), thus strongly promoting HIV-1 transcription elongation and virus replication. Bryostatin-1 treatment increases the level of available CycT1 and CDK9 in resting memory CD4⁺ T cells, while the BRD4 inhibitor JQ1 prevents BRD4 from binding PTEF-b. Also indicated are the AFF1/4, ELL1/2, and AF9/ENL factors forming, together with PTEF-b and Tat, the multi-subunit complex named the “super elongation complex” (SEC). The negative elongation factor (NELF), and the 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) sensitivity inducing factor (DSIF), are phosphorylated by CDK9 determining NELF release and DSIF turned into a positive elongating factor. CDK9 also phosphorylates the largest subunit of RNA polymerase II (Pol II), thus allowing transcription elongation to proceed.

Figure 2

Schematic representation of the proposed molecular mechanisms acting in different subsets of CD4⁺ T central memory (_CM) cells driving HIV-1 transcription initiation. (A) Suggested molecular mechanisms acting in CD4⁺ T central memory (_CM) cells driving HIV-1 transcription initiation, following Bryostatin-1 plus Ionomycin or HMBA treatments. Bryostatin-1 drives protein kinase C (PKC) activation that, in turn, activates p38 kinase. Ionomycin and, to a lesser extent, Hexamethylenebisacetamide (HMBA), activate Calcineurin (CaN) phosphatase, that, together with p38 produce the accumulation of c-Jun transcription factor. Full c-Jun activation is obtained by its p38-mediated phosphorylation. Activated c-Jun binds the NF-κB inhibitory dimer p50/p50, bound to the long terminal repeat enhancer (LTR), determining initiation of HIV-1 transcription. The nucleosomes (Nuc 0) and Nuc1 -are also indicated. (B) Schematic representation of the latency reversing action of the combined treatments with Ingenol and Romidepsin, effective in almost all latently infected resting memory CD4⁺ T cell subtypes. The PKC agonist Ingenol activates PKC, which in turn stimulates the IκB Kinase (IKK) complex to phosphorylate IκB, determining its degradation. NF-κB is therefore free to accumulate in the nucleus and to bind the LTR enhancer. Full start of transcriptional activity is obtained by simultaneous treatment with the histone deacetylase inhibitor (HDI) Romidepsin, able to determine acetylation of nuc1 and of crucial NF-κB residues.

Figure 3

Schematic representation of LRAs mechanism of action. Histone Deacetylase Inhibitors (HDIs) induce histones hyperacetylation, which leads to a chromatin conformational change, activating transcription, thereby causing latency reversal; HDIs increase plasma viraemia in HIV-1 positive patients and, when administered together with HIV-specific CD8+ T-cells, induce selective killing of reactivated CD4+ T-Cells in vitro. TLRs agonists reactivate HIV-1 replication in a NF-κB, AP-1, and Type-I IFNs—dependent manner; TLRs stimulation also results in increased plasma viraemia in patients and in SIV-infected macaques, and triggers an HIV-specific adaptive response. Combination of TLRs agonists with therapeutic vaccines or broadly-neutralizing antibodies (bnAbs) protected SIV-infected monkeys and SHIV-infected macaques from viral rebound after ART cessation. RLRs agonist Acitretin stimulates latency reversal by activation of Histone Acetyl-Transferase p300 mediated by type-I IFN; STING agonists induce reactivation in a NF-κB dependent manner. Both RLRs and STING agonists induce selective apoptosis of reactivated cells. * Activated in apoptotic signaling.

Figure 4

Schematic representation of two different possibilities to combine the “Shock and Kill” and the “Block and Lock” approaches to reach a functional cure. (A) The sequential application of the “Shock and Kill” followed by the “Block and lock” could lead to an initial reduction/elimination of poorly latent reservoirs, easy to be reactivated by LRAs, followed by the blocking and locking of potential residual replication by LPAs. (B) “Shoc-K and B-Lock”. All-in-one cocktail of LRAs + LPAs could result in a tailored reactivation and killing of poorly latent reservoir and, at the same time, the long term locking of residual deeply latent proviruses.