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 (APV)-selected HIV-1 variants and examined their virological characteristics. In an HIV-1 preparation selected with APV (33 passages, yielding HIV(APVp33)), we identified six mutations in protease and six apparently critical mutations at cleavage and non-cleavage sites in Gag. An infectious recombinant clone carrying the six protease mutations but no Gag mutations failed to replicate, indicating that the Gag mutations were required for the replication of HIV(APVp33). An infectious recombinant clone that carried wild-type protease and a set of five Gag mutations (rHIV(WTpro)(12/75/219/390/409gag)) replicated comparably to wild-type HIV-1; however, when exposed to APV, rHIV(WTpro)(12/75/219/390/409gag) rapidly acquired APV resistance. In contrast, the five Gag mutations significantly delayed the acquisition of HIV-1 resistance to ritonavir and nelfinavir (NFV). Recombinant HIV-1 clones containing NFV resistance-associated mutations, such as D30N and N88S, had increased susceptibilities to APV, suggesting that antiretroviral regimens including both APV and NFV may bring about favorable antiviral efficacy. The present data suggest that the preexistence of certain Gag mutations related to PI resistance can accelerate the emergence of resistance to the PI and delay the acquisition of HIV resistance to other PIs, and these findings should have clinical relevance in the therapy of HIV-1 infection with PI-including regimens.

FIG. 1.

Amino acid sequences deduced from the nucleotide sequences of protease (A)- and Gag (B)-encoding regions of proviral DNA isolated at the indicated passages (p3, p12, and p33) from HIV-1_NL4-3 variants selected in the presence of APV. The amino acid sequences of the protease and Gag proteins of wild-type HIV-1_NL4-3 are shown at the top as a reference. Identity to the sequence at individual amino acid positions is indicated by dots. The numbers of clones with the given amino acid substitutions among a total of 10 clones are listed.

FIG. 2.

Replication kinetics of Gag mutant clones with or without protease mutations. MT-2 cells (A and C) and MT-4 cells (B and D) were exposed to Gag mutant clones with (A and B) or without (C and D) protease mutations. Virus replication was monitored by the amounts of p24 Gag produced in the culture supernatants. The results shown are representative of results from three independent experiments. HIV_APVp33 variants had six mutations (L10F, V32I, M46I, I54M, A71V, and I84V) in the viral protease.

FIG. 3.

In vitro selection of APV-resistant variants using HIV-1 carrying Gag mutations. HIV_WT (▪) and three infectious HIV clones, rHIV_WTpro^75/219gag (▵), rHIV_WTpro^219/409gag (⋄), and rHIV_WTpro^{12/75/219/390/409gag} (○), were propagated in the presence of increasing concentrations of APV (starting at 0.03 μM) in MT-2 cells (A) or MT-4 cells (B). The selection was carried out in a cell-free manner for a total of 14 to 29 passages. The results of statistical evaluation of the selection profiles are as follows: panel A, HIV_WT versus rHIV_WTpro^75/219gag, P = 0.53; HIV_WT versus rHIV_WTpro^{12/75/219/390/409gag}, P = 0.0080; HIV_WT versus rHIV_WTpro^219/409gag, P = 0.22; rHIV_WTpro^75/219gag versus rHIV_WTpro^{12/75/219/390/409gag}, P = 0.0065; rHIV_WTpro^75/219gag versus rHIV_WTpro^219/409gag, P = 0.15; and rHIV_WTpro^{12/75/219/390/409gag} versus rHIV_WTpro^219/409gag, P = 0.0018, and panel B, HIV_WT versus rHIV_WTpro^75/219gag, P = 0.65; HIV_WT versus rHIV_WTpro^{12/75/219/390/409gag}, P < 0.0001; HIV_WT versus rHIV_WTpro^219/409gag, P < 0.0001; rHIV_WTpro^75/219gag versus rHIV_WTpro^{12/75/219/390/409gag}, P < 0.0001; rHIV_WTpro^75/219gag versus rHIV_WTpro^219/409gag, P < 0.0001; and rHIV_WTpro^{12/75/219/390/409gag} versus rHIV_WTpro^219/409gag, P = 0.088.

FIG. 4.

Number of amino acid substitutions corresponding to the protease-encoding region of each infectious HIV-1 clone selected in the presence of APV. Nucleotide sequences of proviral DNA of HIV_WT (A and E) and three infectious HIV-1 clones, rHIV_WTpro^75/219gag (B and F), rHIV_WTpro^219/409gag (C and G), and rHIV_WTpro^{12/75/219/390/409gag} (D and H), were determined using lysates of HIV-1-infected MT-2 cells (A to D) and MT-4 cells (E to H) at the termination of each passage and compared to the nucleotide sequence of HIV-1_NL4-3. The number within each symbol represents the number of mutations identified in the protease when each infectious HIV-1 clone was selected in the presence of APV.

FIG. 5.

In vitro selection of PI-resistant variants using HIV-1 carrying Gag mutations. HIV_WT (▪ and ⧫) and rHIV_WTpro^{12/75/219/390/409gag} (○ and ▵) were propagated in MT-4 cells in the presence of increasing concentrations of SQV (A), IDV (B), RTV (C), or NFV (D). The initial drug concentrations used were 0.01 μM for SQV, 0.03 μM for IDV and NFV, and 0.06 μM for RTV, and each virus was selected by up to a 5 μM concentration of each PI. The selection was carried out in a cell-free manner for a total of 13 to 32 passages. NFV selection was performed twice. Data from the first selection are shown with a solid line; the second selection was started using the HIV-1 from passage 10 of the first selection (with NFV at 0.7 μM), and the data are shown with a dashed line. The results of statistical evaluation of the selection profiles are as follows: panel A, P = 0.80; panel B, P = 0.22; panel C, P = 0.0001; and panel D, first selection, P < 0.0001, and second selection, P = 0.0016.

FIG. 6.

Results from the CHRA for HIV_WT and rHIV_WTpro^{12/75/219/390/409gag} in the absence or presence of each drug. Replication profiles of HIV_WT (▪) and rHIV_WTpro^{12/75/219/390/409gag} (○) in the absence (A) or presence of 0.03 μM APV (B), 0.02 μM SQV (C), 0.03 μM IDV (D), 0.03 μM RTV (E), or 0.03 μM NFV (F) were examined by the CHRA. The cell-free supernatant was transferred to fresh MT-4 cells every 7 days. High-molecular-weight DNA extracted from infected cells at the end of each passage was subjected to nucleotide sequencing, and the proportions of Arg and Lys at position 409 in Gag were determined.