The Intact Noninducible Latent HIV-1 Reservoir Is Established in an In Vitro Primary T _CM Cell Model of Latency

Abstract

The establishment of HIV-1 latency has hindered an HIV-1 cure. “Shock and Kill” strategies to target this reservoir aim to induce the latent provirus with latency reversing agents (LRAs). However, recent studies have shown that the majority of the intact HIV-1 viral reservoir found in ART-suppressed HIV infected individuals is not inducible. We sought to understand whether this non-inducible reservoir is established, and thus able to be studied, in an in vitro primary TCM model of latency. Furthermore, we wanted to expand this model system to include R5-tropic and non-B subtype viruses. To that end, we generated our TCM model of latency with an R5 subtype B virus, AD8 and an R5 subtype C virus, MJ4. Our results demonstrate that both intact and defective proviruses are generated in this model. Less than 50% of intact proviruses are inducible regardless of viral strain in the context of maximal stimulation through the TCR or with different clinically relevant LRAs including the HDAC inhibitors SAHA and MS-275, the PKC agonist Ingenol 3,20-dibenzoate or the SMAC mimetic AZD-5582. Our findings suggest that current LRA strategies are insufficient to effectively reactivate intact latent HIV-1 proviruses in primary CD4 TCM cells and that the mechanisms involved in the generation of the non-inducible HIV-1 reservoir can be studied using this primary in vitro model. Importance: HIV-1 establishes a latent reservoir that persists under antiretroviral therapy. Antiretroviral therapy is able to stop the spread of the virus and the progression of the disease but does not target this latent reservoir. If antiretroviral therapy is stopped, the virus is able to resume replication and the disease progresses. Recently, it has been demonstrated that most of the latent reservoir capable of generating replication competent virus cannot be induced in the laboratory setting. However, the mechanisms that influence the generation of this intact and non-inducible latent reservoir are still under investigation. Here we demonstrate the generation of defective, intact and intact non-inducible latent HIV-1 in a TCM model of latency using different HIV-1 strains. Thus, the mechanisms which control inducibility can be studied using this primary cell model of latency, which may accelerate our understanding of the latent reservoir and the development of curative strategies.

Knowledge From London and Berlin: Finding Threads to a Functional HIV Cure

“…Unfortunately, a recent study has shown that the majority of the intact HIV-1 viral reservoir is not inducible in ART-suppressed HIV-infected individuals (152). Among all the LRAs tested, none has wiped out HIV-1 latency or affected viral rebound persistently after the interruption of ART.…”

Targeting Epigenetics to Cure HIV-1: Lessons From (and for) Cancer Treatment

“…A detailed analysis of the reservoir reveals that most proviruses are defective (Ho et al, 2013;Imamichi et al, 2014;Imamichi et al, 2016;Bruner et al, 2016;Hiener et al, 2017;Pollack et al, 2017;Bruner et al, 2019). Even full length sequencing of intact proviruses overestimates the inducible reservoir as many of these proviruses are permanently silenced (de Verneuil et al, 2018;Jiang et al, 2020;Sarabia et al, 2021). The defective proviruses are unable to propagate HIV-1, even though they are able to produce truncated or chimeric proteins and trigger the immune response (Imamichi et al, 2016;Pollack et al, 2017).…”

“…Although accessible proviral chromatin is necessary for engagement of the transcription machinery, it is not sufficient for proviral latency reversal (Battivelli et al, 2018;Sarabia et al, 2021).…”

“…These drugs lead to a more open chromatin structure, suggesting a closed chromatin configuration prevents transcription in at least a subset of reservoir cells. Still, however, only a minority of infected cells also reactivate the provirus (Battivelli et al, 2018;Sarabia et al, 2021). An alternative strategy to achieve a functional cure is termed "lock-and-block".…”

“…Paralleling cancer therapy, to hinder propagation of HIV-1, all the infected cells that can regenerate new infectious HIV-1 particles have to be removed. However, cells with proviruses having incapacitating mutations or those that are permanently silenced may be left untouched (Jiang et al, 2020;Sarabia et al, 2021). Erasing the pathological phenotype can in both cases occur through cell death, or by rendering the cell incapable of producing diseasegenerating transcripts or proteins.…”