Drug resistance during indinavir therapy is caused by mutations in the protease gene and in its Gag substrate cleavage sites

Abstract

Two different responses to the therapy were observed in a group of patients receiving the protease inhibitor indinavir. In one, suppression of virus replication occurred and has persisted for 90 weeks (bDNA, < 500 human immunodeficiency virus type 1 [HIV-1] RNA copies/ml). In the second group, a rebound in virus levels in plasma followed the initial sharp decline observed at the start of therapy. This was associated with the emergence of drug-resistant variants. Sequence analysis of the protease gene during the course of therapy revealed that in this second group there was a sequential acquisition of protease mutations at amino acids 46, 82, 54, 71, 89, and 90. In the six patients in this group, there was also an identical mutation in the gag p7/p1 gag protease cleavage site. In three of the patients, this change was seen as early as 6 to 10 weeks after the start of therapy. In one patient, a second mutation occurred at the gag p1/p6 cleavage site, but it appeared 18 weeks after the time of appearance of the p7/p1 mutation. Recombinant HIV-1 variants containing two or three mutations in the protease gene were constructed either with mutations at the p7/p1 cleavage site or with wild-type (WT) gag sequences. When recombinant HIV-1-containing protease mutations at 46 and 82 was grown in MT2 cells, there was a 68% reduction in its rate of replication compared to the WT virus. Introduction of an additional mutation at the gag p7/p1 protease cleavage site compensated for the partially defective protease gene. Similarly, rates of replication of viruses with mutations M46L/I, I54V, and V82A in protease were enhanced both in the presence and in the absence of Indinavir when combined with mutations in the gag p7/p1 and the gag p1/p6 cleavage sites. Optimal rates of virus replication require protease cleavage of precursor polyproteins. A mutation in the cleavage site that enhanced the availability of a protein that was rate limiting for virus maturation would confer on that virus a significant growth advantage and may explain the uniform emergence of viruses with alterations at the p7/p1 cleavage site. This is the first report of the emergence of mutations in the gag p7/p1 protease cleavage sites in patients receiving protease therapy and identifies this change as an important determinant of HIV-1 resistance to protease inhibitors in patient populations.

Molecular basis for substrate recognition and drug resistance from 1.1 to 1.6 Å resolution crystal structures of HIV‐1 protease mutants with substrate analogs

“…Cleavage of p6 pol ‐PR is essential for the release of mature active protease [15]. Mutations in the NC‐p1 and p1‐p6 sites contribute to drug resistance both in vitro and in vivo [16–18]. These two cleavage sites show significant sequence polymorphism [19,20], and the specificity of cleavage has been studied with PR and several mutants [21].…”

Effect of sequence polymorphism and drug resistance on two HIV‐1 Gag processing sites

“…The development of high levels of resistance to PR inhibitors, possibly requiring multiple mutations in the PR, was therefore expected to be limited by the functional constraints of the enzyme, which must cleave all precursor cleavage sites during viral replication. Subsequently, a second locus was found to be involved in drug resistance to HIV PR inhibitors, both in vitro and in vivo , at the nucleocapsid (NC)/p1 and p1/p6 cleavage sites [9–14]. Evolution of PR cleavage sites other than NC/p1 and p1/p6 in the internal (P2‐P2′) positions is limited, and mutations are rarely observed even upon drug treatment [12].…”

Selection of HIV-1 for Resistance to Fifth Generation Protease Inhibitors Reveals Two Independent Pathways to High-Level Resistance

“…Protease cleavage site mutations are known to coevolve with protease inhibitor 513 resistance within the protease itself REF. The two resistance pathways were associated with 514 distinctive patterns of evolution within the NC/SP2 and SP2/p6 cleavage sites (Doyon et al, 515 1996, Zhang et al, 1997, Mammano et al, 1998. The NC/SP2 site has a suboptimal alanine at 516 the P2 site in the cleavage site sequence, with the resistance associated mutation placing a more 517 favorable valine or isoleucine in that position to make the cleavage site sequence more 518 favorable (Potempa et al, 2018).…”