title: Detection of treatment-resistant infectious HIV after genome-directed antiviral endonuclease therapy.
abstract: Incurable chronic viral infections are a major cause of morbidity and mortality worldwide. One potential approach to cure persistent viral infections is via the use of targeted endonucleases. Nevertheless, a potential concern for endonuclease-based antiviral therapies is the emergence of treatment resistance. Here we detect for the first time an endonuclease-resistant infectious virus that is found with high frequency after antiviral endonuclease therapy. While testing the activity of HIV pol-specific zinc finger nucleases (ZFNs) alone or in combination with three prime repair exonuclease 2 (Trex2), we identified a treatment-resistant and infectious mutant virus that was derived from a ZFN-mediated disruption of reverse transcriptase (RT). Although gene disruption of HIV protease, RT and integrase could inhibit viral replication, a chance single amino acid insertion within the thumb domain of RT produced a virus that could actively replicate. The endonuclease-resistant virus could replicate in primary CD4(+) T cells, but remained susceptible to treatment with antiretroviral RT inhibitors. When secondary ZFN-derived mutations were introduced into the mutant virus's RT or integrase domains, replication could be abolished. Our observations suggest that caution should be exercised during endonuclease-based antiviral therapies; however, combination endonuclease therapies may prevent the emergence of resistance.
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Electronic address: hdesilva@fredhutch.org.
authors.forename: Harshana S
authors.initials: HS
authors.lastname: De Silva Feelixge
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Electronic address: dstone2@fredhutch.org.
authors.forename: Daniel
authors.initials: D
authors.lastname: Stone
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Microbiology, University of Washington, Seattle, WA, USA; Department of Biochemistry, University of Washington, Seattle, WA, USA. Electronic address: harlan.pietz@nih.gov.
authors.forename: Harlan L
authors.initials: HL
authors.lastname: Pietz
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Electronic address: proychou@fredhutch.org.
authors.forename: Pavitra
authors.initials: P
authors.lastname: Roychoudhury
authors.affiliation: Department of Laboratory Medicine, University of Washington, Seattle, WA, USA. Electronic address: agrening@uw.edu.
authors.forename: Alex L
authors.initials: AL
authors.lastname: Greninger
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA. Electronic address: jschiffe@fredhutch.org.
authors.forename: Joshua T
authors.initials: JT
authors.lastname: Schiffer
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA. Electronic address: maubert@fredhutch.org.
authors.forename: Martine
authors.initials: M
authors.lastname: Aubert
authors.affiliation: Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Laboratory Medicine, University of Washington, Seattle, WA, USA; Department of Microbiology, University of Washington, Seattle, WA, USA. Electronic address: kjerome@fredhutch.org.
authors.forename: Keith R
authors.initials: KR
authors.lastname: Jerome
mesh_terms: D019380:Anti-HIV Agents / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D004279:DNA, Viral / Q000235:genetics; D024882:Drug Resistance, Viral; D004720:Endonucleases / Q000378:metabolism; D005090:Exodeoxyribonucleases / Q000378:metabolism / Q000494:pharmacology; D015684:Gene Products, pol / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D015658:HIV Infections / Q000188:drug therapy / Q000235:genetics / Q000628:therapy / Q000821:virology*; D016333:HIV Protease / Q000235:genetics / Q000378:metabolism; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism / Q000494:pharmacology; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology*; D014161:Transduction, Genetic; D014779:Virus Replication / Q000187:drug effects; D016335:Zinc Fingers
chemical_list: D019380:Anti-HIV Agents; D004279:DNA, Viral; D015684:Gene Products, pol; D010750:Phosphoproteins; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase; D004720:Endonucleases; D005090:Exodeoxyribonucleases; C486104:TREX2 protein, human; D016333:HIV Protease
keywords: Endonuclease; Resistance; Reverse transcriptase; Zinc finger nuclease