High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

Georges Khoury; Damian F.J. Purcell

doi:10.3791/58753

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

High Throughput In Vitro Assessment of Latency Reversing Agents on HIV Transcription and Splicing

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07:18 min

January 22, 2019

DOI: 10.3791/58753-v

Georges Khoury¹, Damian F.J. Purcell¹

¹Department of Microbiology and Immunology,University of Melbourne

Overview

This article describes a high throughput protocol for assessing the functional impact of latency reversing agents (LRAs) on HIV transcription and splicing. The method allows for simultaneous evaluation of LRA effects on HIV mRNA processing, providing insights into virus reactivation and clearance of latent proviruses.

Key Study Components

Area of Science

Neuroscience
Virology
Molecular Biology

Background

HIV latency poses a significant challenge in treatment.
Latency reversing agents are being explored for their potential to reactivate latent HIV.
Understanding HIV transcription and splicing is crucial for developing effective therapies.
This study aims to provide a reliable method for assessing LRA effects.

Purpose of Study

To evaluate the impact of LRAs on HIV mRNA processing.
To assess the ability of LRAs to induce virus reactivation.
To provide a high throughput method for functional assessment.

Methods Used

Cell cultivation in a 96-well plate format.
Use of a dual color reporter construct for monitoring transcription.
Assessment of LRA effects on LTR-driven transcription and splicing.
Analysis of results to determine the efficacy of LRAs.

Main Results

Demonstrated the ability of LRAs to enhance HIV transcription.
Provided evidence of LRA effects on splicing mechanisms.
Showed potential for clearing latent HIV reservoirs.
Results support further investigation into LRA applications.

Conclusions

The protocol offers a reliable method for assessing LRA efficacy.
Findings contribute to understanding HIV latency and reactivation.
Future studies may expand on these results to improve HIV treatment strategies.

Frequently Asked Questions

What are latency reversing agents?

Latency reversing agents are compounds that aim to reactivate latent HIV, allowing for its clearance from the body.

How does the protocol assess HIV transcription?

The protocol uses a dual color reporter construct to monitor LTR-driven transcription in response to LRAs.

What is the significance of splicing in HIV?

Splicing is crucial for the production of different HIV proteins and influences the virus's ability to replicate.

Can this method be applied to other viruses?

While this method is designed for HIV, similar approaches may be adapted for other viruses with latency.

What are the next steps after this study?

Further research will focus on optimizing LRA combinations and understanding their mechanisms of action.

A high throughput protocol for functional assessment of HIV efficient reactivation and clearance of latent proviruses is described and applied by testing the impact of interventions on HIV transcription and splicing. Representative results of the effect of latency reversing agents on LTR-driven transcription and splicing are provided.

The overall goal of this procedure is to assess in vitro, the impact of latency reversing agents on HIV mRNA processing. The protocol provide a simple efficient and reliable method for assessing, simultaneously the effect of LRA on HIV transcription and splicing. This technique provided an insight on the ability of LRAs to induce virus reactivation and clearance of the latent reservoir.

After cultivating cells, place 20, 000 of them in 100 microliters of DMEM medium, supplemented with 10%FBS, and without antibiotics in the wells of a 96-well flat bottom plate for 24 hours. The next day dilute the dual color reporter construct. Tat and Rev DNA in 50 microliters of serum free medium, and mix gently.

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