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.