title: Reduced dNTP binding affinity of 3TC-resistant M184I HIV-1 reverse transcriptase variants responsible for viral infection failure in macrophage.
abstract: We characterized HIV-1 reverse transcriptase (RT) variants either with or without the (-)-2',3'-deoxy-3'-thiacytidine-resistant M184I mutation isolated from a single HIV-1 infected patient. First, unlike variants with wild-type M184, M184I RT variants displayed significantly reduced DNA polymerase activity at low dNTP concentrations, which is indicative of reduced dNTP binding affinity. Second, the M184I variant displayed a approximately 10- to 13-fold reduction in dNTP binding affinity, compared with the Met-184 variant. However, the k(pol) values of these two RTs were similar. Third, unlike HIV-1 vectors with wild-type RT, the HIV-1 vector harboring M184I RT failed to transduce cell types containing low dNTP concentrations, such as human macrophage, likely due to the reduced DNA polymerization activity of the M184I RT under low cellular dNTP concentration conditions. Finally, we compared the binary complex structures of wild-type and M184I RTs. The Ile mutation at position 184 with a longer and more rigid beta-branched side chain, which was previously known to alter the RT-template interaction, also appears to deform the shape of the dNTP binding pocket. This can restrict ground state dNTP binding and lead to inefficient DNA synthesis particularly at low dNTP concentrations, ultimately contributing to viral replication failure in macrophage and instability in vivo of the M184I mutation.
authors.affiliation: Department of Microbiology and Immunology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY 14642, USA.
authors.forename: Varuni K
authors.initials: VK
authors.lastname: Jamburuthugoda
authors.forename: Jose M
authors.initials: JM
authors.lastname: Santos-Velazquez
authors.forename: Mark
authors.initials: M
authors.lastname: Skasko
authors.forename: Darwin J
authors.initials: DJ
authors.lastname: Operario
authors.forename: Vandana
authors.initials: V
authors.lastname: Purohit
authors.forename: Pauline
authors.initials: P
authors.lastname: Chugh
authors.forename: Erika A
authors.initials: EA
authors.lastname: Szymanski
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Wedekind
authors.forename: Robert A
authors.initials: RA
authors.lastname: Bambara
authors.forename: Baek
authors.initials: B
authors.lastname: Kim
mesh_terms: D019943:Amino Acid Substitution; D002460:Cell Line; D004279:DNA, Viral / Q000096:biosynthesis / Q000235:genetics; D003854:Deoxyribonucleotides / Q000378:metabolism*; D024882:Drug Resistance, Viral / Q000235:genetics; D015658:HIV Infections / Q000201:enzymology* / Q000235:genetics; D054303:HIV Reverse Transcriptase / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology* / Q000235:genetics; D006801:Humans; D019259:Lamivudine / Q000494:pharmacology; D008264:Macrophages / Q000378:metabolism* / Q000821:virology; D020125:Mutation, Missense; D020836:Protein Structure, Quaternary / Q000235:genetics; D017433:Protein Structure, Secondary / Q000235:genetics; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology
chemical_list: D004279:DNA, Viral; D003854:Deoxyribonucleotides; D018894:Reverse Transcriptase Inhibitors; D019259:Lamivudine; C514824:reverse transcriptase, Human immunodeficiency virus 1; D054303:HIV Reverse Transcriptase