Second Locus Involved in Human Immunodeficiency Virus Type 1 Resistance to Protease Inhibitors

Abstract

Protease inhibitors are potent antiviral agents against human immunodeficiency virus type 1. As with reverse transcriptase inhibitors, however, resistance to protease inhibitors can develop and is attributed to the appearance of mutations in the protease gene. With the substrate analog protease inhibitors BILA 1906 BS and BILA 2185 BS, 350-to 1,500-fold-resistant variants have been selected in vitro and were found not only to contain mutations in the protease gene but also to contain mutations in Gag precursor p1/p6 and/or NC (p7)/p1 cleavage sites. Mutations in cleavage sites give rise to better peptide substrates for the protease in vitro and to improved processing of p15 precursors in drug-resistant clones. Importantly, removal of cleavage site mutations in resistant clones leads to a decrease or even an absence of viral growth, confirming their role in viral fitness. Therefore, these second-locus mutations indicate that cleavage of p15 is a rate-limiting step in polyprotein processing in highly resistant viruses. The functional constraint of p15 processing also suggests that additional selective pressure could further compromise viral fitness and maintain the benefits of antiviral therapies. Human immunodeficiency virus type 1 (HIV-1) encodes a small homodimeric aspartic protease required for the matura-tion of the structural proteins p17, p24, p2, p7, p1, and p6 and the viral enzymes protease, reverse transcriptase (RT), RNase, and integrase (reviewed in reference 26). To exert its function, the protease must first recognize and then cleave nine different cleavage sites in Gag and Gag-Pol precursor polyproteins (26). In HIV-1 polyproteins, only limited sequence similarity between the nine cleavage sites, giving rise to qualitatively different substrates for the protease, is observed. By using pep-tides corresponding to natural cleavage sites, it was determined that p2/p7 and TF/PR are the most rapidly processed cleavage sites, while p7/p1 and p1/p6 are the most slowly cleaved (6, 34, 38). The protease plays an essential role in HIV-1 replication, since point mutations or deletions that abolish its activity do not give rise to infectious particles (15, 25). Inhibitors of this enzyme have therefore been developed and have proven to be potent antiviral agents against HIV-1 (5, 37). As observed with RT inhibitors, however, inhibitors of the protease have been met with the emergence of resistant variants due to the high replication rate of the virus and the low fidelity of the RT enzyme (10, 30, 36). Moderate resistance (2-to 100-fold) to protease inhibitors has indeed been obtained both in vivo (4, 18) and in vitro (18) and has been attributed to the appearance of mutations in the protease gene. Not surprisingly, since many of these mutations are located in the active site of the enzyme, drug resistance mutations also have considerable impact on protease activity. This is reflected by impaired processing of Gag precursors in protease-mutated virions and by decreased in vitro catalytic efficiency of the protease towards peptides representing natural cleavage sites (16, 17, 20, 24, 32). The development of high levels of resistance to protease inhibitors, possibly requiring multiple mutations in the active site of the protease, was therefore expected to be limited by the functional constraints of the enzyme, which must cleave all precursor cleavage sites. BILA 1906 BS and BILA 2185 BS are substrate analog protease inhibitors that have potent antiviral activity with limited cytotoxicity (7). With these inhibitors, highly resistant HIV-1 variants were obtained in vitro. These variants contain multiple mutations in the protease gene and also contain mutations in one or two cleavage sites. These results not only involve a second locus in the development of high-level resistance to protease inhibitors but suggest a limit to the mutability of the protease. MATERIALS AND METHODS Cells and viruses. Cell lines C8166 (obtained from J. Sullivan), 293 (American Type Culture Collection), and H-9 (chronically infected with HIV-1 strain IIIB) (28, 29, 31) (AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases; obtained from Robert Gallo) were used. All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum, 10 5 M-mercaptoethanol, 2 mM L-glutamine, and 10 g of gentamicin per ml. Viral stocks were prepared from chronically infected H-9 cells. Proviral DNA pNL4.3 (1) was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, from Malcolm Martin. Protease inhibitors. BILA 1906 BS and BILA 2185 BS are pipecolinic acid derivatives whose chemical structures and HIV-2 protease-bound conformations have been described previously (33). BILA 1906 BS and BILA 2185 BS correspond to inhibitors 1 and 4, respectively, described in reference 33. Selection of HIV-1 protease inhibitor-resistant variants. Variants were obtained by infecting 10 6 C8166 T cells with HIV-1 strain IIIB (multiplicity of infection of 1) in the presence of challenging concentrations of BILA 1906 BS or BILA 2185 BS. At each passage (3 to 4 days), the cell-free culture supernatant was used to infect fresh cells with a greater concentration of the drug if a cytopathic effect persisted. The final drug concentrations were 2 M for BILA 1906 BS passage 33 and 1.6 and 7 M for BILA 2185 BS passages 37 and 58, respectively. The drug concentration giving 50% inhibition of viral replication (50% effective concentration [EC 50 ]) was determined by extracellular p24 anti-gen (Ag) production (Coulter HIV-1 p24 Antigen Assay) in acutely infected C8166 cells. Protease and cleavage site sequence determination. For sequence determination , genomic DNA of HIV-1 variant-infected C8166 cells was amplified by PCR with one of five sets of primers. Primer set 1 (5-CCCCATATGCCTCAGGTC ACTCTTTGG-3 and 5-CCCGGATCCTCAAAAATTTAAAGTGCAACC-3