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

Georges Khoury; Damian F.J. Purcell

doi:10.3791/58753

대기 시간 반전 HIV 전사 및 접합에 에이전트의 체 외 평가에서 높은 처리량

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

대기 시간 반전 HIV 전사 및 접합에 에이전트의 체 외 평가에서 높은 처리량

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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

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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.

에이즈 효율적인 재 활성화의 기능 평가 및 잠재 proviruses의 높은 처리량 프로토콜 설명 이며 HIV 전사에 개입의 영향을 테스트 하 고 접합 하 여 적용 합니다. 대기 시간 반전 LTR 기반 전사에 에이전트와 접합의 효과의 대표적인 결과 제공 됩니다.

이 절차의 전반적인 목표는 시험관 내, HIV mRNA 처리에 대기 시간 반전 에이전트의 충격을 평가하는 것입니다. 이 프로토콜은 LRA가 HIV 전사 및 접합에 미치는 영향을 동시에 평가하기 위한 간단효율적이고 신뢰할 수 있는 방법을 제공합니다. 이 기술은 LA가 잠복 저장소의 바이러스 재활성화 및 허가를 유도하는 능력에 대한 통찰력을 제공했습니다.

세포를 재배한 후, DMEM 배지 100마이크로리터에 20, 000을 배치하고, 10%의 FBS로 보충하고, 96웰의 평평한 바닥 판의 우물에 항생제없이 24시간 동안 배치한다. 다음날 이중색 기자 구도가 희석됩니다. Tat와 Rev DNA는 무혈제 배지 50 마이크로리터로 부드럽게 섞습니다.

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