Augmentation of human immunodeficiency virus type 1 subtype E (CRF01_AE) multiple-drug resistance by insertion of a foreign 11-amino-acid fragment into the reverse transcriptase.
A human immunodeficiency virus type 1 (HIV-1) subtype E (CRF01_AE) variant (99JP-NH3-II) possessing an in-frame 33-nucleotide insertion mutation in the beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT inhibitors. This virus exhibited an extremely high level of multiple nucleoside analog resistance (MNR). Neighbor-joining tree analysis of the pol sequences indicated that the 99JP-NH3-II variant had originated from the swarm of drug-sensitive predecessors in the patient. Population-based sequence analyses of 82 independently cloned RT segments from the patient suggested that the variants with the insertion, three or four 3'-azido-3'-deoxythymidine resistance mutations, and a T69I mutation in combination had strong selective advantages during chemotherapy. Consistently, in vitro mutagenesis of a drug-sensitive predecessor virus clone demonstrated that this mutation set functions cooperatively to confer a high level of MNR without deleterious effects on viral replication capability. Homology modeling of the parental RT and its MNR mutant showed that extension of the beta3-beta4 loop by an insertion caused reductions in the distances between the loop and the other subdomains, narrowing the template-primer binding cleft and deoxynucleoside triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsuccessful. Taken together, these data suggest that (i) HIV-1 tolerates in vivo insertions as long as 33 nucleotides into the highly conserved enzyme gene to survive multiple anti-HIV-1 inhibitors and (ii) the insertion mutation augments multiple-drug resistance, possibly by reducing the biochemical inaccuracy of substrate-enzyme interactions in the active center.
Sato
H
H
AIDS Research Center, National Institute of Infectious Diseases, Japan. hirosato@nih.go.jp
Tomita
Y
Y
Ebisawa
K
K
Hachiya
A
A
Shibamura
K
K
Shiino
T
T
Yang
R
R
Tatsumi
M
M
Gushi
K
K
Umeyama
H
H
Oka
S
S
Takebe
Y
Y
Nagai
Y
Y
D000328:Adult; D000595:Amino Acid Sequence; D019380:Anti-HIV Agents / Q000494:pharmacology; D001483:Base Sequence; D002648:Child; D004352:Drug Resistance, Microbial; D018432:Drug Resistance, Multiple; D019143:Evolution, Molecular; D005260:Female; D015684:Gene Products, pol / Q000235:genetics; D015658:HIV Infections / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D008297:Male; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010802:Phylogeny; D018894:Reverse Transcriptase Inhibitors / Q000494:pharmacology
D019380:Anti-HIV Agents; D015684:Gene Products, pol; D018894:Reverse Transcriptase Inhibitors; D054303:HIV Reverse Transcriptase