Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals

Morón-López, Sara; Telwatte, Sushama; Sarabia, Indra; Battivelli, Emilie; Montaño, Mauricio; Macedo, Amanda B.; Aran, Dvir; Butte, Atul J.; Jones, R. Brad; Bosque, Alberto; Verdin, Eric; Greene, Warner C.; Wong, Joseph K.; Yukl, Steven A.

doi:10.1371/journal.ppat.1009060

Cited by 21 publications

(29 citation statements)

References 48 publications

(84 reference statements)

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“…Factors that cause HIV-1 latency are incompletely understood and splicing dysregulation has been implicated as a possible mechanism [ 89 , 90 , 91 ]. One theory is that low levels of tat or rev mRNAs play a role in inducing latency [ 92 , 93 ]. If tat transcripts were insufficient to produce adequate Tat levels, transcription would abort, and the infected cell would become part of the latent reservoir.…”

Section: Hiv-1 Latency and Splicing—limited Prospects For A Cure Hmentioning

confidence: 99%

HIV-1: To Splice or Not to Splice, That Is the Question

Emery

Swanstrom

2021

Viruses

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The transcription of the HIV-1 provirus results in only one type of transcript—full length genomic RNA. To make the mRNA transcripts for the accessory proteins Tat and Rev, the genomic RNA must completely splice. The mRNA transcripts for Vif, Vpr, and Env must undergo splicing but not completely. Genomic RNA (which also functions as mRNA for the Gag and Gag/Pro/Pol precursor polyproteins) must not splice at all. HIV-1 can tolerate a surprising range in the relative abundance of individual transcript types, and a surprising amount of aberrant and even odd splicing; however, it must not over-splice, which results in the loss of full-length genomic RNA and has a dramatic fitness cost. Cells typically do not tolerate unspliced/incompletely spliced transcripts, so HIV-1 must circumvent this cell policing mechanism to allow some splicing while suppressing most. Splicing is controlled by RNA secondary structure, cis-acting regulatory sequences which bind splicing factors, and the viral protein Rev. There is still much work to be done to clarify the combinatorial effects of these splicing regulators. These control mechanisms represent attractive targets to induce over-splicing as an antiviral strategy. Finally, splicing has been implicated in latency, but to date there is little supporting evidence for such a mechanism. In this review we apply what is known of cellular splicing to understand splicing in HIV-1, and present data from our newer and more sensitive deep sequencing assays quantifying the different HIV-1 transcript types.

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Section: Hiv-1 Latency and Splicing—limited Prospects For A Cure Hmentioning

confidence: 99%

HIV-1: To Splice or Not to Splice, That Is the Question

Emery

Swanstrom

2021

Viruses

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“…Second, this model recapitulates similar integration patterns, including the generation of expanded sites (23). Third, this model recapitulates the blocks to transcription initiation, multiple splicing, and potentially elongation that is observed in CD4 T cells isolated from PLWH on long-term ART(26). These similarities are likely due to the use of replication competent viruses and primary cells, as their metabolism and abundance of host factors better mirrors primary CD4 T cells compared to tumoral cell lines.The main conclusions from this study are that the T CM latency model can indeed be expanded to use R5 and non-subtype B virus strains increasing the utility of this model for HIV-1 cure strategies as the majority of the worldwide HIV-1 infections are non-B(30).…”

mentioning

confidence: 72%

“…As both R5 viruses infected, replicated, and established latency in this model, it is a proof of concept that using more diverse R5-tropic viruses is possible within this system. Further, we recently showed that this model allows the generation of latently infected cells using primary viruses isolated from ARTsuppressed PLWH using the quantitative viral outgrowth assay (QVOA) (26).…”

Section: Discussionmentioning

confidence: 99%

“…This model has been extensively used to discover and evaluate latency-reversing agents (LRAs); to perform mechanistic studies examining pathways involved in the establishment and maintenance of latency; and has been extensively compared with latent cells isolated from PLWH including integration site analysis, clonal expansion and blocks in HIV-1 splicing (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29).…”

Section: Introductionmentioning

confidence: 99%

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

Sarabia

Huang

Ward

et al. 2021

J Virol

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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.

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“…On the other hand, the presence of multiply spliced RNA may be a more proximal surrogate of productive infection compared with unspliced RNA only (Pasternak and Berkhout, 2018). However, multiply spliced RNA is much less abundant than unspliced (Kaiser et al, 2007;Pasternak et al, 2020), due to both proviral genetic defects (as splicing requires the presence of several intact genomic regions) and latency blocks to completion of transcription and splicing (Yukl et al, 2018;Moron-Lopez et al, 2020). As a consequence, it is challenging to detect multiply spliced RNA in ART-treated individuals without ex vivo cellular stimulation, which explains why it has not yet been assessed as a potential predictor of the post-treatment remission.…”

Section: Predicting Post-treatment Hiv Remission: Time For a Comprehementioning

confidence: 99%

Predicting Post-treatment HIV Remission: Does Size of the Viral Reservoir Matter?

Pasternak

Psomas²,

Berkhout

2021

Front. Microbiol.

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Combination antiretroviral therapy (ART) suppresses human immunodeficiency virus (HIV) replication and improves immune function. However, due to the persistence of long-lived HIV reservoirs, therapy interruption almost inevitably leads to a fast viral rebound. A small percentage of individuals who are able to control HIV replication for extended periods after therapy interruption are of particular interest because they may represent a model of long-term HIV remission without ART. These individuals are characterized by a limited viral reservoir and low reservoir measures can predict post-treatment HIV remission. However, most individuals with a low reservoir still experience fast viral rebound. In this Perspective, we discuss the possible reasons behind this and propose to develop an integral profile, composed of viral and host biomarkers, that could allow the accurate prediction of post-treatment HIV remission. We also propose to incorporate information on the chromatin context of the proviral integration sites into the characterization of the HIV reservoir, as this likely influences the reactivation capacity of latent proviruses and, together with the actual number of intact proviruses, contributes to the replication competence of the reservoir.

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Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals

Cited by 21 publications

References 48 publications

HIV-1: To Splice or Not to Splice, That Is the Question

HIV-1: To Splice or Not to Splice, That Is the Question

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

Predicting Post-treatment HIV Remission: Does Size of the Viral Reservoir Matter?

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Human splice factors contribute to latent HIV infection in primary cell models and blood CD4+ T cells from ART-treated individuals

Cited by 21 publications

References 48 publications

HIV-1: To Splice or Not to Splice, That Is the Question

HIV-1: To Splice or Not to Splice, That Is the Question

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

Predicting Post-treatment HIV Remission: Does Size of the Viral Reservoir Matter?

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