Coreceptor-dependent inhibition of the cell fusion activity of simian immunodeficiency virus Env proteins

Abstract

The cytoplasmic tail (R peptide) sequence is able to regulate the fusion activity of the murine leukemia virus (MuLV) envelope (Env) protein. We have previously shown that this sequence exerts a profound inhibitory effect on the fusion activity of simian immunodeficiency virus (SIV)-MuLV chimeric Env proteins which contain the extracellular and transmembrane domains of the SIV Env protein. Recent studies have shown that SIV can utilize several alternative cellular coreceptors for its fusion and entry into the cell. We have investigated the fusion activity of SIV and SIV-MuLV chimeric Env proteins using cells that express different coreceptors. HeLa cells were transfected with plasmid constructs that carry the SIV or SIV-MuLV chimeric Env protein genes and were overlaid with either CEMx174 cells or Ghost Gpr15 cells, which express the Gpr15 coreceptor for SIV, or Ghost CCR5 cells, which express CCR5, an alternate coreceptor for SIV. The R-peptide sequence in the SIV-MuLV chimeric proteins was found to inhibit the fusion with CEMx174 cells or Ghost Gpr15 cells. However, a significant level of fusion was still observed when HeLa cells expressing the chimeric Env proteins were cocultivated with Ghost CCR5 cells. These results show that the R-peptide sequence exerts differential effects on the fusion activity of SIV Env proteins using target cells that express alternative coreceptors.

FIG. 1

(A) Schematic diagram of the transmembrane region of Env protein constructs used in this study. Black boxes represent the transmembrane domains of the SIV or HIV Env protein, hatched boxes represent cytoplasmic sequences of MuLV origin, gray boxes represent the MuLV R-peptide sequence, and white boxes represent the SU or cytoplasmic domain sequences of SIV or HIV origin (the SU and full-length cytoplasmic domains of SIV and HIV Env proteins are not drawn to scale). Construction of genes encoding the chimeric Env proteins was carried out by following standard cloning procedures described previously (42). (B) Expression of SIV and SIV-MuLV chimeric Env proteins. Lanes 1, mock transfection; lanes 2, full-length SIV Env; lanes 3, S-Menv; lanes 4, S-MenvR-; lanes 5, SIVenv733T+R; lanes 6, SIVenv733T. (C) Expression of HIV and HIV-MuLV chimeric Env proteins. Lanes 1, mock transfection; lanes 2, full-length HIV Env; lanes 3, H-Menv; lanes 4, H-MenvR-. Proteins were expressed in HeLa cells using the vaccinia virus T7 expression system (18). Surface biotinylation and immunoprecipitation were carried out as described previously (42).

FIG. 2

Fusion activity of SIV and SIV-MuLV chimeric Env proteins in cells expressing the coreceptor CCR5. Env proteins were expressed in HeLa cells using the vaccinia virus T7 expression system. HeLa cells were infected by recombinant virus VTF7-3 (a recombinant vaccinia virus expressing the T7 polymerase, provided by B. Moss) for 1 h and then transfected with DNA constructs encoding the Env proteins. At 12 h posttransfection, HeLa cells were overlaid with Ghost cells (obtained from the NIH AIDS Research and Reference Reagent Program) which express CD4 and CCR5. Cell fusion pictures were taken at 8 h after overlay of Ghost CCR5 cells under a Nikon microscope. (a) Mock transfection; (b) full-length SIV Env; (c) S-Menv; (d) S-MenvR-; (e) SIVenv733T+R; (f) SIVenv733T.

FIG. 3

Fusion activity of SIV and SIV-MuLV chimeric Env proteins in cells expressing the coreceptor Gpr15. Env proteins were expressed in HeLa cells using the vaccinia virus T7 expression system. HeLa cells were infected by recombinant virus VTF7-3 for 1 h and then transfected with DNA constructs encoding the Env proteins. At 12 h posttransfection, HeLa cells were overlaid with Ghost cells (obtained from the NIH AIDS Research and Reference Reagent Program) which express CD4 and Gpr15. Cell fusion pictures were taken at 8 h after overlay of Ghost Gpr15 cells under a Nikon microscope. (a) Mock transfection; (b) SIVenv; (c) S-Menv; (d) S-MenvR-; (e) SIVenv733T+R; (f) SIVenv733T.

FIG. 4

Fusion activity of HIV and HIV-MuLV chimeric Env proteins. Env proteins were expressed in HeLa cells using the vaccinia virus T7 expression system. HeLa cells were infected by recombinant virus VTF7-3 for 1 h and then transfected with DNA constructs encoding the Env proteins. At 12 h posttransfection, HeLa cells were overlaid with HeLa T4 cells (obtained from the NIH AIDS Research and Reference Reagents Program) which express CD4 and fusin. Cell fusion pictures were taken at 8 h after overlay of HeLa cells under a Nikon microscope. (a) Mock transfection; (b) full-length HIV Env; (c) H-Menv; (d) H-MenvR-.