Functional Correlation between a Novel Amino Acid Insertion at Codon 19 in the Protease of Human Immunodeficiency Virus Type 1 and Polymorphism in the p1/p6 Gag Cleavage Site in Drug Resistance and Replication Fitness

Abstract

ABSTRACT Population-based sequence analysis revealed the presence of a variant of human immunodeficiency virus type 1 (HIV-1) containing an insertion of amino acid Ile in the protease gene at codon 19 (19I) and amino acid substitutions in the protease at codons 21 (E21D) and 22 (A22V) along with multiple mutations associated with drug resistance, M46I/P63L/A71V/I84V/I93L, in a patient who had failed protease inhibitor (PI) therapy. Longitudinal analysis revealed that the P63L/A71V/I93L changes were present prior to PI therapy. Polymorphisms in the Gag sequence were only seen in the p1/p6 cleavage site at the P1′ position (Leu to Pro) and the P5′ position (Pro to Leu). To characterize the role of these mutations in drug susceptibility and replication capacity, a chimeric HIV-1 strain containing the 19I/E21D/A22V mutations with the M46I/P63L/A71V/I84V/I93L and p1/p6 mutations was constructed. The chimera displayed high-level resistance to multiple PIs, but not to lopinavir, and grew to 30% of that of the wild type. To determine the relative contribution of each mutation to the phenotypic characteristic of the virus, a series of mutants was constructed using site-directed mutagenesis. A high level of resistance was only seen in mutants containing the 19I/A22V and p1/p6 mutations. The E21D mutation enhanced viral replication. These results suggest that the combination of the 19I/E21D/A22V mutations may emerge and lead to high-level resistance to multiple PIs. The combination of the 19I/A22V mutations may be associated with PI resistance; however, the drug resistance may be caused by the presence of a unique set of mutations in the p1/p6 mutations. The E21D mutation contributes to replication fitness rather than drug resistance.