In Vitro Selection and Characterization of HIV-1 Variants with Increased Resistance to Sifuvirtide, a Novel HIV-1 Fusion Inhibitor

Zhonghua Liu,Mei Shan,Li Li,Lu Lu,Shu Meng,Cheng Chen,Yuxian He,Shibo Jiang,Linqi Zhang

doi:10.1074/jbc.m110.199323

Abstract

Sifuvirtide, a novel fusion inhibitor against human immunodeficiency virus type I (HIV-1), which is more potent than enfuvirtide (T20) in cell culture, is currently under clinical investigation for the treatment of HIV-1 infection. We now report that in vitro selection of HIV-1 variants resistant to sifuvirtide in the presence of increasing concentrations of sifuvirtide has led to several specific mutations in the gp41 region that had not been previously reported. Many of these substitutions were confined to the N-terminal heptad repeat region at positions 37, 38, 41, and 43, either singly or in combination. A downstream substitution at position 126 (N126K) in the C-terminal heptad repeat region was also found. Site-directed mutagenesis studies have further identified the critical amino acid substitutions and combinations thereof in conferring the resistant genotypes. Furthermore, the mutant viruses demonstrated variable degrees of cross-resistance to enfuvirtide, some of which are preferentially more resistant to sifuvirtide. Impaired infectivity was also found for many of the mutant viruses. Biophysical and structural analyses of the key substitutions have revealed several potential novel mechanisms against sifuvirtide. Our results may help to predict potential resistant patterns in vivo and facilitate the further clinical development and therapeutic utility of sifuvirtide.

Highlights

The envelope glycoprotein (Env) of HIV-1 is critical in mediating viral entry into the target cells, and it represents a major target for the development of novel antiretroviral therapeutics
Many of the mutant viruses revealed variable and diminished infectivity, and the most profound impact came from substitutions, such as Q41K, Q41R, or I37T/V38A
It needs to be stressed that mutant viruses bearing additional substitutions on top of Q41K or Q41R resulted in defective viruses that failed to infect the target cells (Table 2)

Summary

EXPERIMENTAL PROCEDURES

Selection of Sifuvirtide-resistant HIV-1 Variants—MT4 cells were seeded at 3 ϫ 105/ml in RPMI 1640 medium containing 10% fetal bovine serum on a 96-well plate. Transfection and p24 Determination—The virus stock for wild type and mutant viruses containing various mutations were produced by transfecting 293T cells using FuGENE௡6 transfection reagent (Roche Applied Science). IC50 and Infectivity Determination—We used TZM-bl cells (NIH AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, National Institutes of Health) to study viral infectivity. These cells are genetically engineered to stably express high levels of CD4 and HIV-1 co-receptors CCR5 and CXCR4 and to contain the luciferase and ␤-galactosidase genes under the control of the HIV-1 long terminal repeat promoter. The 6-HB for each mutant peptide was constructed through symmetry operation in alignment mode using the automated protein modeling program on the SWISSMODEL protein-modeling server [35]

RESULTS

Mutant viruses

DISCUSSION