Membrane-anchored peptide inhibits human immunodeficiency virus entry

Abstract

Peptides derived from the heptad repeats of human immunodeficiency virus (HIV) gp41 envelope glycoprotein, such as T20, can efficiently inhibit HIV type 1 (HIV-1) entry. In this study, replication of HIV-1 was inhibited more than 100-fold in a T-helper cell line transduced with a retrovirus vector expressing membrane-anchored T20 on the cell surface. Inhibition was independent of coreceptor usage.

FIG. 1

(A) Maps of retroviral vectors. The SP and the MSD are derived from the LNGFR. The hinge is derived from the IgG heavy chain. (B) Amino acid sequence of the membrane-anchored T20 expressed by M87. (C) Amino acid sequence of the SP and the T20 region in the mutated proteins expressed from M87m1 through M87m3.

FIG. 2

Expression of membrane-anchored T20. PM-1 cells were transduced with the retroviral vectors indicated and selected. The bulk cultures were stained either with a control human monoclonal antibody directed against gp120 (2G12) or with a human monoclonal antibody directed against a sequence within T20 (2F5) followed by a PE-conjugated anti-human IgG goat serum. Shaded curve, PM-1/MPIN; solid line, PM-1 transduced with the vector indicated.

FIG. 3

Inhibition of HIV-1 replication by membrane-anchored T20. (A) PM-1 cells were transduced with different retroviral vectors encoding T20 and then selected with G418. These bulk cultures were infected with NL4–3 at a multiplicity of infection of 0.1. On day 5, medium was replaced and the concentration of p24 antigen was determined in supernatants collected on day 6. ∗, detection limit. (B) In parallel, the selected PM-1 cultures were infected with NL4–3AGFP and the spread of virus was monitored by flow cytometry. × M85; □ M86; ▴ M87; ○ MPIN. (C) Selected cultures were infected with NL4–3AGFP, and the spread of virus was monitored by flow cytometry. ▴ M87; ○ MPIN; □ M87m1; ▪ M87m2; □ M87m3. (D) Inhibition of a single round of infection by membrane-anchored T20. PM-1 transduced with the control vector MPIN or the vector M87 expressing the membrane-anchored T20 was infected with fivefold dilutions of a replication-deficient virus NL4–3env⁻GFP pseudotyped either with VSV G protein or an HIV-1 envelope glycoprotein from one of the HIV strains depicted (solid bars). Cells were also transduced with an MLV vector with EGFP as a marker gene pseudotyed with the envelope glycoprotein of HIV-1 strain BH10 (shaded bar). Titers of different pseudotypes were determined on day 3 by flow cytometry for each of the PM-1 cultures. Titers on PM-1/M87 relative to the titer on PM-1/MPN are given.