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 th e beta3-beta4 loop coding region of the reverse transcriptase (RT) gene was isolated from a patient who had not responded to nucleoside analogue RT in hibitors. This virus exhibited an extremely high level of multiple nucleosi de analog resistance (MNR). Neighbor-joining tree analysis of the pal seque nces indicated that the 99JP-NH3-II variant had originated from the swarm o f drug-sensitive predecessors in the patient. Population-based sequence ana lyses of 82 independently cloned RT segments from the patient suggested tha t the variants with the insertion, three or four 3 ' -azido-3 ' -deoxythymi dine resistance mutations, and a T69I mutation in combination had strong se lective 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 deleteri ous effects on viral replication capability. Homology modeling of the paren tal 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 ot her subdomains, narrowing the template-primer binding cleft and deoxynucleo side triphosphate-binding pocket in a highly flexible manner. The origin of the insert is elusive, as every effort to find a homologue has been unsucc essful. 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 ina ccuracy of substrate-enzyme interactions in the active center.