title: 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.
authors.affiliation: Nevada Institute of Personalized Medicine and School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States of America.
authors.forename: Christy L
authors.initials: CL
authors.lastname: Strong
authors.affiliation: Nevada Institute of Personalized Medicine and School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States of America.
authors.forename: Horacio P
authors.initials: HP
authors.lastname: Guerra
authors.affiliation: Nevada Institute of Personalized Medicine and School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States of America.
authors.forename: Kiran R
authors.initials: KR
authors.lastname: Mathew
authors.affiliation: Nevada Institute of Personalized Medicine and School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States of America.
authors.forename: Nervik
authors.initials: N
authors.lastname: Roy
authors.affiliation: Nevada Institute of Personalized Medicine and School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States of America.
authors.forename: Lacy R
authors.initials: LR
authors.lastname: Simpson
authors.affiliation: Nevada Institute of Personalized Medicine and School of Life Sciences, University of Nevada Las Vegas, Las Vegas, NV, United States of America.
authors.forename: Martin R
authors.initials: MR
authors.lastname: Schiller
mesh_terms: D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D017124:Conserved Sequence / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects; D004262:DNA Restriction Enzymes / Q000494:pharmacology*; D004279:DNA, Viral / Q000235:genetics*; D015658:HIV Infections / Q000235:genetics; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D011533:Proviruses / Q000235:genetics*; D020218:Response Elements / Q000235:genetics*; D016662:Virus Integration
chemical_list: D004279:DNA, Viral; D004262:DNA Restriction Enzymes