title: Characterization of novel non-nucleoside reverse transcriptase (RT) inhibitor resistance mutations at residues 132 and 135 in the 51 kDa subunit of HIV-1 RT.
abstract: Several rare and novel NNRTI [non-nucleoside reverse transcriptase (RT) inhibitor] resistance mutations were recently detected at codons 132 and 135 in RTs from clinical isolates using the yeast-based chimaeric TyHRT (Ty1/HIV-1 RT) phenotypic assay. Ile132 and Ile135 form part of the beta7-beta8 loop of HIV-1 RT (residues 132-140). To elucidate the contribution of these residues in RT structure-function and drug resistance, we constructed twelve recombinant enzymes harbouring mutations at codons 132 and 135-140. Several of the mutant enzymes exhibited reduced DNA polymerase activities. Using the yeast two-hybrid assay for HIV-1 RT dimerization we show that in some instances this decrease in enzyme activity could be attributed to the mutations, in the context of the 51 kDa subunit of HIV-1 RT, disrupting the subunit-subunit interactions of the enzyme. Drug resistance analyses using purified RT, the TyHRT assay and antiviral assays demonstrated that the I132M mutation conferred high-level resistance (>10-fold) to nevirapine and delavirdine and low-level resistance (approximately 2-3-fold) to efavirenz. The I135A and I135M mutations also conferred low level NNRTI resistance (approximately 2-fold). Subunit selective mutagenesis studies again demonstrated that resistance was conferred via the p51 subunit of HIV-1 RT. Taken together, our results highlight a specific role of residues 132 and 135 in NNRTI resistance and a general role for residues in the beta7-beta8 loop in the stability of HIV-1 RT.
authors.affiliation: Basic Research Program, SAIC-Frederick, Inc., NCI-Frederick, Frederick, Maryland 21702, USA.
authors.forename: Dwight V
authors.initials: DV
authors.lastname: Nissley
authors.forename: Jessica
authors.initials: J
authors.lastname: Radzio
authors.forename: Zandrea
authors.initials: Z
authors.lastname: Ambrose
authors.forename: Chih-Wei
authors.initials: CW
authors.lastname: Sheen
authors.forename: Noureddine
authors.initials: N
authors.lastname: Hamamouch
authors.forename: Katie L
authors.initials: KL
authors.lastname: Moore
authors.forename: Gilda
authors.initials: G
authors.lastname: Tachedjian
authors.forename: Nicolas
authors.initials: N
authors.lastname: Sluis-Cremer
mesh_terms: D002942:Circular Dichroism; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000096:biosynthesis; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000201:enzymology; D008958:Models, Molecular; D008968:Molecular Conformation; D011487:Protein Conformation; D021122:Protein Subunits / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D018894:Reverse Transcriptase Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D012441:Saccharomyces cerevisiae / Q000235:genetics
chemical_list: D004279:DNA, Viral; D021122:Protein Subunits; D012367:RNA, Viral; D011994:Recombinant Proteins; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase