N88D facilitates the co-occurrence of D30N and L90M and the development of multidrug resistance in HIV type 1 protease following nelfinavir treatment failure

Abstract

Nelfinavir was once one of the most commonly used protease inhibitors (PIs). To investigate the genetic mechanisms of multidrug resistance in protease isolates with the primary nelfinavir resistance mutation D30N, we analyzed patterns of protease mutations in 582 viruses with D30N from 460 persons undergoing HIV-1 genotypic resistance testing at Stanford University Hospital from 1997 to 2005. Three patterns of mutational associations were identified. First, D30N was positively associated with N88D but negatively associated with N88S. Second, D30N and L90M were negatively associated except in the presence of N88D, which facilitated the co-occurrence of D30N and L90M. Third, D30N+N88D+L90M formed a stable genetic backbone for the accumulation of additional protease inhibitor (PI) resistance mutations. In 16 patients having isolates with more than one combination of mutations at positions 30, 88, and 90, all exhibited one of the steps in the following progression: D30N-->D30N+N88D-->D30N+N88D+L90M-->D30N+N88D+L90M+(L33F+/-I84V or M46I/L+/-I54V). Although nelfinavir is now used less frequently than other PIs, the well-delineated mutational pathway we describe is likely to influence patterns of cross-resistance in viruses from persons who experience virologic failure while receiving this PI.

FIG. 1

Patterns of drug resistance mutations in 43 isolates containing the mutations D30N, N88D, and L90M. Each of these isolates was obtained from a different person. Positions with electrophoretic mixtures are underlined.

FIG. 2

Sequences of molecular clones of HIV protease from four isolates that contained the protease mutations D30N, N88D, and L90M by direct PCR sequencing. The one-letter amino acid code is used to indicate differences from consensus B. The residue at positions 30, 88, and 90 are shown in bold.

FIG. 3

Structural relationship among protease residues 30, 88, and 90. (A) Superimposition of the three-dimensional structures of mutant viruses reported by Mahalingam et al. containing D30N alone (2F80, blue ribbon), N88D alone (1FG 8, green ribbon), and I90M alone (2F8, red ribbon). (B) van der Waals surfaces of wild-type residues D30, N88, and L90 deduced from wild-type protease complexed with the PI TMC114 (1T7J).