Porcine endogenous retroviruses infect cells lacking cognate receptors by an alternative pathway: implications for retrovirus evolution and xenotransplantation

Abstract

A PHQ motif near the amino termini of gammaretroviral envelope glycoprotein surface (SU) subunits is important for infectivity but not for incorporation into virions or binding to cognate receptors. The H residue of this motif is most critical, with all substitutions we tested being inactive. Interestingly, porcine endogenous retroviruses (PERVs) of all three host-range groups, A, B, and C, lack full PHQ motifs, but most members have an H residue at position 10. H10A PERV mutants are noninfectious but were efficiently transactivated by adding to the assays a PHQ-containing SU or receptor-binding subdomain (RBD) derived from a gibbon ape leukemia virus (GALV). A requirement of this transactivation was a functional GALV receptor on the cells. In contrast to this heterologous transactivation, PERV RBDs and SUs were inactive in all tested cells, including porcine ST-IOWA cells. Surprisingly, transactivation by GALV RBD enabled wild-type or H10A mutant PERVs of all three host-range groups to efficiently infect cells from humans and rodents that lack functional PERV receptors and it substantially enhanced infectivities of wild-type PERVs, even for cells with PERV receptors. Thus, PERVs can suboptimally infect cells that contain cognate receptors or they can employ a transactivation pathway to more efficiently infect all cells. This ability to infect cells lacking cognate receptors was previously demonstrated only for nontransmissible variant gammaretroviruses with recombinant and mutant envelope glycoproteins. We conclude that some endogenously inherited mammalian retroviruses also have a receptor-independent means for overcoming host-range and interference barriers, implying a need for caution in xenotransplantation, especially of porcine tissues.

FIG. 1.

Amino acid sequence aligment of the amino termini of processed gammaretroviral SU glycoproteins. The amino acids that are the most conserved are boxed. The histidine of the SPHQV motif is the only amino acid in the SPHQV motif that is common to all of these envelope glycoproteins.

FIG. 2.

Mutagenic analysis of the conserved amino-terminal SPHQV motif of the FeLV-B envelope glycoprotein. (A) Each amino acid of the SPHQV motif was replaced individually by an alanine. (B) The histidine of this motif was deleted (ΔH) or replaced with a basic (K or R) or aromatic amino acid (F or W). The bar graphs show infectivities of the wild-type and mutant viruses in CE-Pit1 cells in the absence or presence of FeLV-B RBD (Fe-RBD) or of the Friend MuLV RBD (Fr-RBD). The titers are averages of four independent experiments ± standard errors of the means. The middle data are Western immunoblots of the wild-type and mutant pseudotyped virus pellets, showing the SU glycoproteins and the viral capsid (CA) proteins. The bottom data show FACS analyses of the binding of the wild-type and mutant envelope glycoproteins onto MDTF/FePit1 cells. The background fluorescence (black lines) was measured when the cells were incubated with media from nontransfected TELCeb6 cells. The assays were done three times. wt, wild type.

FIG. 3.

Efficient rescue of fusion-defective PERV-A and PERV-B by GALV RBD. (A and B) 293T/mCAT1 cells expressing different soluble RBDs or SUs were infected with LacZ-carrying viruses pseudotyped with wild-type or H10A mutant PERV-A or PERV-B envelope glycoproteins. (C) TE671 cells were infected with these viruses or with GALV or ΔH GALV in the presence or absence of soluble GALV RBD or PERV RBDs and SUs. Infectivities are the averages of four independent experiments ± standard errors of the means. Ga, GALV; GadelH, ΔH GALV; Fe, FeLV-B; Fr, Friend MuLV; MO, Mo-MuLV; A, amphotropic strain 4070A MuLV.

FIG. 4.

Characterization of RBD and SU envelope glycoproteins. (A) 293T/mCAT1 cells that had been stably transfected with RBD and SU expression vectors were used for analysis of the cell-associated envelope glycoproteins by Western immunoblotting as previously described (28, 31, 34) using the anti-RGS(H)₄-specifc mouse antibody. (B) Conditioned medium from negative control TE671 cells or cells making RBDs or SUs were incubated with EDTA-detached 293T/mCAT1 cells for 1 h at 37°C before washing the cells and incubating them with the anti-RGS(H)₄ antibody. The data show FACS analyses. (C) The conditioned media used for the latter binding assays were analyzed directly by Western immunoblotting with the anti-RGS(H)₄ antibody. The RBDs were shed into the culture media in similar amounts, although they bound to the 293T/mCAT1 cells to different extents. Ga, GALV; Fe, FeLV-B; Fr, Friend MuLV; MO, Mo-MuLV; A, amphotropic strain 4070A MuLV.

FIG. 5.

PERV RBDs and SUs do not rescue infections of fusion-defective H10A mutant PERV-A, -B, or -C in porcine ST-IOWA cells. (A) ST-IOWA cells were infected with wild-type or fusion-defective H10A PERVs, with GALV (Ga) or ΔH GALV (GadelH), and with FeLV-B (Fe) or ΔH FeLV-B (FedelH), in the presence or absence of soluble GALV or FeLV-B RBDs or PERV RBDs and SUs. (B) Conditioned medium from negative control TE671 cells (open curve) or cells making RBDs (filled black) were incubated with ST-IOWA cells for 1 h at 37°C before washing the cells and incubating them with the anti-RGS(H)₄ antibody. Fluorescence of 10⁴ living cells (negative for propidium iodide) were analyzed by FACS. Experiments were repeated three times.

FIG. 6.

GALV RBD transactivates PERV-A and PERV-B infections of cells that lack functional PERV receptors. (A) Rat XC and MDTF/FePit1 cells are completely resistant to PERV-A (open bars, see arrows) but become susceptible in the presence of GALV RBD (black stipled bars) or Friend MuLV RBD (striped bars). (B) FEA and HeLa cells are completely resistant to PERV-B but become susceptible in the presence of a transactivating GALV RBD. The data are the averages of four experiments ± standard errors of the means. Ga, GALV; GadelH, ΔH GALV; Fe, FeLV-B; MO, Mo-MuLV; ModelH, ΔH Mo-MuLV. The arrows represent assays that indicated there was no infection.

FIG. 7.

The presence of the receptor for the transactivating RBD is necessary to confer susceptibility to PERV-A, -B, and -C viruses on nonpermissive hamster cells. CHO cells (control) or the CHO cell derivative (CE-hPit1) that expresses the GALV receptor hPit1 and the ecotropic MuLV receptor mCAT1 were infected by wild-type and fusion-defective mutant LacZ viruses in the absence or presence of GALV RBD or Friend MuLV RBD. The hamster cells were completely resistant to PERVs unless RBDs were present that could bind to receptors on the target cells. The data are representative of three independent experiments. Ga, GALV; GadelH, ΔH GALV; MO, Mo-MuLV; ModelH, ΔH Mo-MuLV; Fr, Friend MuLV.