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High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing
Published on: January 22, 2019
Selective reactivation of latent HIV using CyclinT1-Tat-containing virus-like particles
Thomas K Lavin1, Caroline O Tabler2, Thomas J Sweet2
1Department of Pathology, Case Western Reserve University, Cleveland, OH, 44106, USA; Medical Scientist Training Program, Case Western Reserve University School of Medicine, Cleveland, OH, USA.
Abstract:
The persistence of HIV reservoirs and their ability to create an active infection after anti-retroviral therapy cessation has prevented development of an HIV cure. Various chemical latency reversal agents (LRAs) have been investigated to promote HIV transcription as part of a kick and kill strategy, but many of these agents lack either potency or specificity and can cause widespread T cell activation and systemic toxicity. We report the development of novel virus-like particles (VLPs), based on HIV itself, that carry a CyclinT1-Tat fusion protein (CycTat) and reactivate HIV from latency both alone and synergistically with the two tested chemical LRAs; a bromodomain inhibitor and a protein kinase C agonist. CycTat resulted in higher reactivation than Tat, although Tat and CycTat delivery were equivalent in some cell lines after co-stimulation with LRAs thought to increase cellular P-TEFb levels. Targeted mutations disrupting key residues in Tat and CycT1 interactions dampened reactivation, suggesting the particles work mechanistically as anticipated. Fusion of VLPs with target cells was required for HIV reactivation, demonstrating that CycTat proteins do not non-specifically cross cell membranes when packaged into VLPs. Additionally, we addressed safety concerns by testing high doses of VLPs on primary CD4+ T cells, which resulted in minimal T cell activation. This serves as proof-of-concept for specific reactivation of HIV by delivery of Tat protein by VLPs and shows that additional components, here a truncated CyclinT1, can be engineered into particles to enhance viral reactivation.
Insights
Novel virus-like particles (VLPs) carrying a CyclinT1-Tat fusion protein (CycTat) effectively reactivate latent HIV. This targeted approach shows promise for HIV cure strategies with minimal T cell activation.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Persistent HIV reservoirs prevent a cure, necessitating strategies like "kick and kill."
- Chemical latency reversal agents (LRAs) show limitations in potency, specificity, and safety.
- Novel delivery systems are needed to enhance HIV reactivation and therapeutic efficacy.
Purpose of the Study:
- To develop and characterize novel HIV-based virus-like particles (VLPs) engineered to deliver a CyclinT1-Tat fusion protein (CycTat).
- To evaluate the efficacy of CycTat-carrying VLPs in reactivating latent HIV, both alone and in synergy with chemical LRAs.
- To assess the safety and specificity of these engineered VLPs for potential HIV cure applications.
Main Methods:
- Construction and characterization of HIV-based VLPs encapsulating a CyclinT1-Tat fusion protein (CycTat).
- Assessment of HIV reactivation from latency in cell models using CycTat-VLPs, Tat, and combinations with chemical LRAs (bromodomain inhibitor, PKC agonist).
- In vitro safety evaluation of CycTat-VLPs on primary CD4+ T cells at high doses.
Main Results:
- CycTat-VLPs demonstrated potent HIV reactivation, outperforming Tat alone in some conditions.
- Synergistic reactivation was observed when combining CycTat-VLPs with tested chemical LRAs.
- Targeted mutations affecting Tat-CycT1 interactions reduced reactivation, confirming mechanism.
- VLP fusion with target cells was essential for reactivation, indicating specific delivery.
- High-dose VLP administration showed minimal activation of primary CD4+ T cells, suggesting a favorable safety profile.
Conclusions:
- Engineered HIV-based VLPs carrying CycTat represent a promising platform for targeted HIV reactivation.
- The CycTat fusion protein enhances HIV reactivation compared to Tat alone.
- VLPs offer specific delivery and improved safety profiles for latency-reversal therapies.
- This approach provides a proof-of-concept for enhancing HIV cure strategies through targeted protein delivery via VLPs.
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