Non-Cleavage Site Gag Mutations in Amprenavir-Resistant Human Immunodeficiency Virus Type 1 (HIV-1) Predispose HIV-1 to Rapid Acquisition of Amprenavir Resistance but Delay Development of Resistance to Other Protease Inhibitors

Abstract

In an attempt to determine whether mutations in Gag in human immunodeficiency virus type 1 (HIV-1) variants selected with a protease inhibitor (PI) affect the development of resistance to the same or a different PI(s), we generated multiple infectious HIV-1 clones carrying mutated Gag and/or mutated protease proteins that were identified in amprenavir (

C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir

“…Variants resistant to GRL-015, -085, and -097 were generated as previously described (21,30,33) and cultured in the presence of each compound at an initial concentration equivalent to the EC 50 . On the last day of each passage (weeks 1 to 3), 1.5 ml of the cell-free supernatant was harvested and transferred to a culture of fresh uninfected MT-4 cells in the presence of increased concentrations of the drug for the following round of culture.…”

Loss of Protease Dimerization Inhibition Activity of Darunavir Is Associated with the Acquisition of Resistance to Darunavir by HIV-1

“…MT-4 cells (10 5 ) were exposed to each infectious HIV-PR YFP clone (100 ng of p24 Gag protein/ml) for 6 h, washed twice with phosphate-buffered saline (PBS), and cultured in 7 ml of complete medium with some modification as described previously (3,13). Culture supernatants (50 l) were harvested every other day, and virus replication was monitored by the amounts of p24 Gag produced in the culture supernatants.…”

Loss of the Protease Dimerization Inhibition Activity of Tipranavir (TPV) and Its Association with the Acquisition of Resistance to TPV by HIV-1

“…When the replication of HIV-1 in the culture was confirmed by substantial Gag protein production (greater than 200 ng/ml), the highest drug concentration among the three concentrations was used to continue the selection (for the next round of culture). This protocol was repetitively used until the drug concentration reached the targeted concentration (2,19). Proviral DNA samples obtained from the lysates of infected cells were subjected to nucleotide sequencing.…”

“…Molecular cloning and determination of the nucleotide sequences of HIV-1 strains passaged in the presence of TPV were performed as previously described (2,40). In brief, high-molecular-weight DNA was extracted from HIV-1-infected MT-4 cells by using the InstaGene matrix (Bio-Rad Laboratories, Hercules, CA) and was subjected to molecular cloning, followed by sequence determination.…”

“…The PCR products obtained as described above were digested with two of the three enzymes BssHII, ApaI, and XmaI, and the obtained fragments were introduced into pHIV-1 NLSma , designed to have a SmaI site by changing two nucleotides (2590 and 2593) of pHIV-1 NL4-3 (2,11). To generate HIV-1 clones carrying the mutations, site-directed mutagenesis was performed using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), and the mutation-containing genomic fragments were introduced into pHIV-1 NLSma .…”