Interference between D30N and L90M in selection and development of protease inhibitor-resistant human immunodeficiency virus type 1

Abstract

We studied the evolutionary relationships between the two protease inhibitor (PI) resistance mutations, D30N and L90M, of human immunodeficiency virus type 1 (HIV-1). The former is highly specific for nelfinavir resistance, while the latter is associated with resistance to several PIs, including nelfinavir. Among patients with nelfinavir treatment failure, we found that D30N acquisition was strongly suppressed when L90M preexisted. Thus, D30N/L90M double mutations not only were detected in a very limited number of patients but also accounted for a minor fraction within each patient. In the disease course, the D30N and L90M clones readily evolved independently of each other, and later the D30N/L90M double mutants emerged. The double mutants appeared to originate from the D30N lineage but not from the L90M lineage, or were strongly associated with the former. However, their evolutionary pathways appeared to be highly complex and to still have something in common, as they always contained several additional polymorphisms, including L63P and N88D, as common signatures. These results suggest that D30N and L90M are mutually exclusive during the evolutionary process. Supporting this notion, the D30N/L90M mutation was also quite rare in a large clinical database. Recombinant viruses with the relevant mutations were generated and compared for the ability to process p55gag and p160pol precursor proteins as well as for their infectivity. L90M caused little impairment of the cleavage activities, but D30N was detrimental, although significant residual activity was observed. In contrast, D30N/L90M demonstrated severe impairment. Thus, the concept of mutual antagonism of the two mutations was substantiated biochemically and functionally.

FIG. 1.

(a and c) Clinical courses of JPR-1 (a) and JPR-3 (c). Treatment protocols and durations are demonstrated with arrows. AZT, zidovudine; ddI, dideoxyinosine; IDV, idinavir; NFV, nelfinavir; SQV, saquinavir; RTV, ritonavir; ddC, dideoxycytosine. (b and d) Development of clones with various mutations, and their phylogenetic relationships, of JPR-1(b) and JPR-3(d). A to H (JPR-1) and a to f (JPR-3) indicate the sampling time points and are referred to the respective clone names with additional serial numbers. Codon 30 and 90 amino acid patterns of the clones are indicated.

FIG. 2.

Analyses of Gag-Pol precursor protein processing of the wild type (HXB2cv) and various mutant viruses by Western blotting. Conditions and viruses used are indicated above each lane. The top part of the membrane (a) was probed with anti-gp120 C-terminal polyclonal antibody, and the bottom part (b) was probed with anti-HIV-1-positive human serum. Positions of molecular markers are indicated on the left, and positions of mature proteins, partially processed intermediate products, and unprocessed precursors are indicated on the right. nfv, nelfinavir.