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Damaging the Integrated HIV Proviral DNA with TALENs.

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.

SUBMITTER: Strong CL

PROVIDER: S-EPMC4422436 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Damaging the Integrated HIV Proviral DNA with TALENs.

Strong Christy L CL Guerra Horacio P HP Mathew Kiran R KR Roy Nervik N Simpson Lacy R LR Schiller Martin R MR

PloS one 20150506 5

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 i ...[more]

PMID: 25946221

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Project description:BackgroundUsing HIV proviral DNA as a template may be suitable for initial detection of transmitted drug resistance mutations (TDRMs) as it is easy to handle and less expensive compared to RNA. However, existing literatures which are mainly focused on HIV-1B subtypes DNA extracted from PBMCs revealed controversial findings ranging from the detection of significantly lower or higher mutations in proviral DNA compared to historic viral RNA. Thus, to verify whether viral RNA or proviral DNA has improved sensitivity in detecting transmitted genotypic drug resistance mutations paired viral RNA and proviral DNA (which is directly extracted from stored whole blood) samples were tested from Ethiopian antiretroviral naive HIV-1C infected subjects.MethodsIn the present comparative study the frequency of TDR mutations was assessed in paired samples of viral RNA and proviral DNA (extracted directly from stored whole blood) of HIV-1C infected treatment naïve patients and interpreted using the 2009 WHO drug resistance surveillance mutation lists, Stanford University drug resistance data base and International Antiviral Society-USA mutation lists.ResultsHigh agreement in rate of TDR between the two compartments was observed using the WHO mutation lists. While mutations G190A and E138A were concurrently found in both compartments, others such as G73S on PR and A62V, M184I, M230I on RT were identified in proviral DNA only. All signature mutations seen in viral RNA were not missed in proviral DNA.ConclusionsThe concordance of major genotype drug resistance mutation between RNA and proviral DNA in treatment naïve patients suggests that proviral DNA might be an alternative approaches for an initial assessment of drug resistance prior to initiation of antiretroviral therapy using the WHO mutations lists in resource-limited countries. However, the clinical importance of TDRMs observed only in proviral DNA in terms of being a risk factor for virologic failure and whether they limit future treatment options needs additional investigation using more sensitive sequencing approaches such as Next Generation Sequencing (NGS).

Dataset Information

Damaging the Integrated HIV Proviral DNA with TALENs.

Publications

Damaging the Integrated HIV Proviral DNA with TALENs.

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