A Restriction Enzyme Based Cloning Method to Assess the <em>In vitro</em> Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

Daniel T. Claiborne; Jessica L. Prince; Eric Hunter

doi:10.3791/51506

A Enzima de Restrição método baseado Clonagem para avaliar a In vitro Replicação Capacid...

JoVE Journal
Immunology and Infection

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JoVE Journal Immunology and Infection

A Restriction Enzyme Based Cloning Method to Assess the In vitro Replication Capacity of HIV-1 Subtype C Gag-MJ4 Chimeric Viruses

A Enzima de Restrição método baseado Clonagem para avaliar a In vitro Replicação Capacidade de HIV-1 do subtipo C gag-MJ4 quiméricos Vírus

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14:23 min

August 31, 2014

DOI: 10.3791/51506-v

Daniel T. Claiborne*¹, Jessica L. Prince*¹, Eric Hunter^1,2

¹Emory Vaccine Center at Yerkes National Primate Research Center,Emory University, ²Department of Pathology and Laboratory Medicine,Emory University

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Overview

This study presents a method to evaluate the impact of gag gene sequence variation on HIV-1 replication capacity in vitro. By incorporating patient-derived gag genes into a replication competent HIV-1 plasmid, the research aims to enhance understanding of HIV-1 pathogenesis.

Key Study Components

Area of Science

Virology
HIV Research
Genetic Analysis

Background

HIV-1 pathogenesis involves viral characteristics and host genetics.
Understanding gag gene variations is crucial for HIV research.
In vitro studies help assess viral replication capacity.
Robust methods are needed for reproducible measurements.

Purpose of Study

To assess the effect of gag sequence polymorphisms on HIV-1 replication capacity.
To develop a method for incorporating patient-derived gag genes into HIV-1 plasmids.
To quantify virion production and visualize viral replication kinetics.

Methods Used

Amplification of the gag gene from patient plasma.
Cloning of the gag sequence into the infectious molecular clone MJ4.
Transfection of the chimera plasmid to generate infectious viral stocks.
Measurement of replication capacity using a radio labeled reverse transcriptase assay.

Main Results

Successful incorporation of patient-derived gag genes into HIV-1 plasmids.
Quantification of virion production over time.
Visualization of viral replication kinetics in vitro.
Demonstration of the impact of gag sequence variations on replication capacity.

Conclusions

The method provides a reliable approach to study HIV-1 replication.
Insights gained can inform future HIV research and treatment strategies.
Understanding gag gene variations is essential for comprehending HIV-1 pathogenesis.

Frequently Asked Questions

What is the significance of the gag gene in HIV-1?

The gag gene is crucial for the structural proteins of the virus and influences its replication capacity.

How does this study contribute to HIV research?

It provides a method to assess the impact of genetic variations on HIV-1 replication, enhancing understanding of the virus.

What techniques are used to measure viral replication?

A radio labeled reverse transcriptase assay is employed to quantify virion production over time.

Why is it important to study patient-derived gag genes?

Studying these genes helps reveal how genetic variations affect the virus's behavior and replication.

What are the potential implications of this research?

Findings could lead to improved strategies for HIV treatment and vaccine development.

What cell line is used to assess infectivity?

The GXR25 T-cell line is used to evaluate the infectivity of the generated viral stocks.

A patogênese do HIV-1 é definida tanto pelas características virais quanto pelos fatores genéticos do hospedeiro. Aqui descrevemos um método robusto que permite medições reprodutíveis para avaliar o impacto da variação da sequência do gene gag na capacidade de replicação in vitro do vírus.

O objetivo geral deste procedimento é incorporar genes gag derivados do subtipo CHIV um indivíduos infectados em um plasmídeo HIV um competente para replicação. Para avaliar o efeito dos polimorfismos da sequência gag na capacidade de replicação do HIV um in vitro, isso é feito primeiro amplificando o gene gag do plasma do paciente. O segundo passo é clonar a sequência gag derivada do paciente no clone molecular infeccioso competente para replicação MJ quatro.

Em seguida, o plasmídeo quimera gag MJ quatro é transfectado para gerar estoques virais infecciosos e titulado para avaliar a infectividade em uma linha celular indicadora. A etapa final é medir a capacidade de replicação in vitro na linhagem de células T G XR 25. Em última análise, um ensaio de transcriptase reversa marcado com rádio é usado para quantificar a produção de vírion ao longo do tempo, a fim de visualizar a cinética de replicação viral.

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Doenças Infecciosas Issue 90 HIV-1 da mordaça a replicação viral a capacidade de replicação replicação viral MJ4 CEM GXR25