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Development of Resistance to 4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine (EFdA) by WT and Nucleoside Reverse Transcriptase Inhibitor Resistant Human Immunodeficiency Virus Type 1

View ORCID ProfileMaria E. Cilento, View ORCID ProfileEleftherios Michailidis, View ORCID ProfileTatiana V. Ilina, Eva Nagy, Hiroaki Mitsuya, Michael A. Parniak, View ORCID ProfilePhilip R. Tedbury, View ORCID ProfileStefan G. Sarafianos
doi: https://doi.org/10.1101/2020.09.04.281485
Maria E. Cilento
aLaboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
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Eleftherios Michailidis
bLaboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY
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Tatiana V. Ilina
cDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
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Eva Nagy
cDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
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Hiroaki Mitsuya
dDepartment of Refractory Viral Infections, National Center for Global Health & Medicine Research Institute, Tokyo, Japan; Experimental Retrovirology Section, HIV and AIDS Malignancy Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA; Department of Clinical Sciences, Kumamoto University Hospital, Kumamoto, Japan
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Michael A. Parniak
cDepartment of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
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Philip R. Tedbury
aLaboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
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Stefan G. Sarafianos
aLaboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine, Atlanta, GA, USA
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  • For correspondence: ssarafi@emory.edu
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Abstract

4’-ethynyl-2-fluoro-2’-deoxyadenosine (EFdA, MK-8591, islatravir) is a nucleoside reverse transcriptase translocation inhibitor (NRTTI) with exceptional potency against WT and drug-resistant HIV strains. However, HIV resistance to EFdA is not well characterized. We therefore developed resistance to EFdA by serial passages using progressively increasing concentrations of EFdA. The starting virus was either WT or clinically relevant NRTI-resistant viruses K65R, M184V, and D67N/K70R/T215F/K219Q). In all cases, the selected mutations included M184V. Additional mutations in the RT connection domain (R358K and E399K) and one mutation in the RNase H domain (A502V) were noted. Site-specific mutagenesis validated the role for M184V as the primary determinant for resistance to EFdA; none of the connection domain mutations contributed significantly to phenotypic resistance to EFdA. A novel EFdA resistance mutation was also observed in the background of M184V. The A114S/M184V combination of mutations imparted higher resistance to EFdA (~24-fold) than M184V (−8-fold) or A114S (~2-fold) alone. Virus fitness data suggested that A114S affects HIV fitness by itself and in the presence of M184V. This is consistent with biochemical experiments that showed decreases in the enzymatic efficiency (kcat/Km) of WT RT vs. A114S (2.1-fold) and A114S/M184V/502V (6.5-fold), whereas there was no significant effect of A502V on RT or virus fitness. The observed EFdA resistance of M184V by itself and in combination with A114S combined with the strong published in vitro and in vivo data, confirm that EFdA is an excellent candidate as a potential HIV therapeutic.

Competing Interest Statement

The authors declare that H.M. is a coinventor of EFdA.

Footnotes

  • ↵† Deceased

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Posted September 04, 2020.
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Development of Resistance to 4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine (EFdA) by WT and Nucleoside Reverse Transcriptase Inhibitor Resistant Human Immunodeficiency Virus Type 1
Maria E. Cilento, Eleftherios Michailidis, Tatiana V. Ilina, Eva Nagy, Hiroaki Mitsuya, Michael A. Parniak, Philip R. Tedbury, Stefan G. Sarafianos
bioRxiv 2020.09.04.281485; doi: https://doi.org/10.1101/2020.09.04.281485
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Development of Resistance to 4’-Ethynyl-2-Fluoro-2’-Deoxyadenosine (EFdA) by WT and Nucleoside Reverse Transcriptase Inhibitor Resistant Human Immunodeficiency Virus Type 1
Maria E. Cilento, Eleftherios Michailidis, Tatiana V. Ilina, Eva Nagy, Hiroaki Mitsuya, Michael A. Parniak, Philip R. Tedbury, Stefan G. Sarafianos
bioRxiv 2020.09.04.281485; doi: https://doi.org/10.1101/2020.09.04.281485

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