Damaging the Integrated HIV Proviral DNA with TALENs.

Christy L Strong,Nervik Roy,Horacio P Guerra,Lacy R Simpson,Martin R Schiller,Kiran R Mathew,David Harrich

doi:10.1371/journal.pone.0125652

Christy L Strong, Nervik Roy + Show 5 more

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https://doi.org/10.1371/journal.pone.0125652

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Journal: PloS one	Publication Date: May 6, 2015
Citations: 104	License type: CC BY 4.0

Affiliation: University of Nevada, Las Vegas

Abstract

HIV-1 integrates its proviral DNA genome into the host genome, presenting barriers for virus eradication. Several new gene-editing technologies have emerged that could potentially be used to damage integrated proviral DNA. In this study, we use transcription activator-like effector nucleases (TALENs) to target a highly conserved sequence in the transactivation response element (TAR) of the HIV-1 proviral DNA. We demonstrated that TALENs cleave a DNA template with the HIV-1 proviral target site in vitro. A GFP reporter, under control of HIV-1 TAR, was efficiently inactivated by mutations introduced by transfection of TALEN plasmids. When infected cells containing the full-length integrated HIV-1 proviral DNA were transfected with TALENs, the TAR region accumulated indels. When one of these mutants was tested, the mutated HIV-1 proviral DNA was incapable of producing detectable Gag expression. TALEN variants engineered for degenerate recognition of select nucleotide positions also cleaved proviral DNA in vitro and the full-length integrated proviral DNA genome in living cells. These results suggest a possible design strategy for the therapeutic considerations of incomplete target sequence conservation and acquired resistance mutations. We have established a new strategy for damaging integrated HIV proviral DNA that may have future potential for HIV-1 proviral DNA eradication.

Highlights

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), is a pathogenic retrovirus that integrates a proviral DNA copy of its genome into the genome of host cells
We built and tested a NS-transcription activator-like effector nucleases (TALENs) designed with some degenerate recognition to accommodate escape mutations in regions where viral genome mutations have been previously observed. We report that both TALEN pairs can be used to damage the integrated HIV-1 proviral DNA in cultured cells infected with HIV-1
Previously latent HIV-1 infected memory CD4+ T cells and other cell types, which are not targeted by Highly Active Antiretroviral Therapy (HAART) therapy, can reseed viral infection [51,52]

Summary

Introduction

Human Immunodeficiency Virus (HIV), the causative agent of Acquired Immunodeficiency Syndrome (AIDS), is a pathogenic retrovirus that integrates a proviral DNA copy of its genome into the genome of host cells. Even with chronic HAART treatment, an integrated copy of proviral HIV DNA remains in latent cells, which can re-establish viral production and cause a rebound, producing plasma viremia [2]. Paired, permeant endonuclease excision” authored by MRS and CLS This intellectual property is owned by The Board of Reagents of the Nevada System of Higher Education on Behalf of the University of Nevada, Las Vegas. This does not alter the authors’ adherence to all PLOS ONE policies on sharing data and materials

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