Identification of the hASCT2-binding domain of the Env ERVWE1/syncytin-1 fusogenic glycoprotein
© Cheynet et al; licensee BioMed Central Ltd. 2006
Received: 28 February 2006
Accepted: 04 July 2006
Published: 04 July 2006
The cellular HERV-W envelope/syncytin-1 protein, encoded by the envelope gene of the ERVWE1 proviral locus is a fusogenic glycoprotein probably involved in the formation of the placental syncytiotrophoblast layer. Syncytin-1-induced in vitro cell-cell fusion is dependent on the interaction with hASCT2. As no receptor binding domain has been clearly defined in the SU of neither the HERV-W Env nor the retroviruses of the same interference group, we designed an in vitro binding assay to evaluate the interaction of the HERV-W envelope with the hASCT2 receptor. Using truncated HERV-W SU subunits, a region consisting of the N-terminal 124 amino acids of the mature SU glycoprotein was determined as the minimal receptor-binding domain. This domain contains several sub-domains which are poorly conserved among retroviruses of this interference group but a region of 18 residus containing the SDGGGX2DX2R conserved motif was proved to be essential for syncytin-1-hASCT2 interaction.
The cellular HERV-W envelope protein, also named syncytin-1, is a fusogenic glycoprotein probably involved in the formation of the placental syncytiotrophoblast layer [1–3]. This protein, encoded by the envelope gene of the ERVWE1 proviral locus  is synthesized as a gPr73 precursor and is specifically cleaved into two mature proteins, a gp50 surface subunit (SU) and a gp24 transmembrane subunit (TM) . The HERV-W Env glycoprotein is phylogenetically related to an interference group of retroviruses  that includes feline endogenous virus (RD114), baboon endogenous virus (BaEV), simian retroviruses (SRV-1, SRV-2), avian reticuloendotheliosis viruses (REV-A) and spleen necrosis virus (SNV) . All retroviruses of this group share a common cell surface receptor, the human sodium-dependent neutral amino acid transporter type 2 (hASCT2) [8, 9]. Accordingly, syncytin-1-induced in vitro cell-cell fusion is dependent upon interaction with hASCT2  and also with the amino acid transporter hASCT1 . Moreover, HERV-W Env confers host cell resistance to infection by SNV . To date, no RBD has been clearly defined in the SU of the HERV-W Env interference group. Within the type C mammalian retroviruses, the SU subunit harbors three contiguous regions consisting of the SU amino-terminal receptor binding globular domain [12–14], a proline-rich region (PRR) and the TM-interacting SU carboxy-terminal domain. A similar RBD has been also delineated in the SU N-terminal region of HTLV1, HTLV2 [15, 16] and predicted in Mammary Tumor Virus  and Bovine Leukemia virus envelopes .
In this study, we have designed an in vitro binding assay to evaluate the interaction of HERV-W envelope-derived soluble SU domains with the hASCT2 receptor. We have identified a 124 residue region of the mature SU as the minimal domain that interacts with the hASCT2 receptor.
In order to verify that the soluble SU protein behaves similarly to the SU subunit within the context of the native syncytin-1, we first examined the interaction of SU protein with hASCT2 receptor using parental and receptor-blocked TE671 human cells as target cells (Figure 1B). Indeed, TE671 cells were originally used to demonstrate the fusogenic properties of the native syncytin-1 protein. The target cells were TE671 subclones stably expressing the envelope glycoproteins derived from the GALV and RD114 retroviruses that respectively recognize the PiT-1  and hASCT2 (also named RDR for type D mammalian retrovirus receptor) [8, 9] receptors, thus blocking the accessibility of exogenously presented retroviral envelopes to these receptors, as previously described . As expected, the soluble SU protein bound to TE671 parental cells, which express the receptors for several retrovirus groups, to PiT1-blocked cells, but not to hASCT2-blocked cells. These data confirmed that soluble SU protein, similarly to syncytin-1, recognizes the type D mammalian retrovirus receptor expressed on human cells and thus behaves like retroviral glycoproteins of the interference group.
To infer the sensitivity and specificity of the cell surface binding assay, soluble SU subunit was bound to a panel of target cell types expressing different combinations of hASCT1 and hASCT2 receptors: TE671 cells which contain hASCT1 and hASCT2 , HeLa cells which contain hASCT2 but not hASCT1 , HEK293T cells which contain small amounts of both hASCT1 and hASCT2 , and XC rat cell clones stably expressing either hASCT2 or hASCT1 human receptors and derived from XC cells by transfection of the pCD3.1VHR16/14  and pCDNA3.1-hASCT1  expression plasmids, respectively (Figure 1C). In this assay, binding to HeLa cells was slightly higher than to TE671 cells, demonstrating the different expression and/or distribution of the hASCT2 receptor on these target cells. In contrast, the HEK293T cells weakly bound SU protein. Using XC-hASCT1 and XC-hASCT2 cells (Figure 1C), we showed that the SU subunit could efficiently bind both to hASCT1 and hASCT2 receptors, as observed for both cell-cell fusion and infection assays .
Finally to exclude any potential binding of soluble SU onto non-human ASCT-related receptor expressed by parental XC cells, we compare the binding efficiency of SU between parental XC cells and XC-hASCT2 or XC-hASCT1 cells (Figure 1D). As expected, the SU protein bound to XChASCT2 and XChASCT1 cells but not XC parental cells .
The absence of binding in cell surface assays using Env71, Env95 and notably Env117 protein (Figure 2C) suggested the loss of at least one receptor-binding determinant within the 117–144 region. This region contains the epitope of an anti-SU polyclonal antibody (anti-SU raised against residues 112–129 of EnvW) . This antibody was used to detect native syncytin-1 in primary cytotrophoblasts  and in b30BeWo carcinoma cells . Hence, the Env144 protein was pre-incubated with the anti-SU antibody. The binding of the protein-antibody complex onto the receptor was significantly altered in the presence of the specific anti-SU antibody but not in the presence of a rabbit anti-TM antibody (Figure 3B). This result indicates that this domain is essential for hASCT2 receptor binding.
In conclusion, we have used HERV-W SU soluble subunits to determine that the 124 N-terminal amino-acids of the HERV-W mature SU protein are sufficient to interact with the hASCT2 and hASCT1 amino-acid transporters (Figure 3C). Although still not elucidated, the envelope-receptor interaction seems to some extent to be tolerant of the RBD amino acid content. Thus, 17 positions out of the 124 residues of the RBD were shown to be variable in ERVWE1 envelopes of hominoids and humans, without altering the hASCT2-dependent fusogenic properties of each envelope variant . In addition, a weak sequence conservation exists between retroviral members of the interference group with the notable exception of three cysteine-containing motifs (PCXC, CYX5C and CX8–9CW) and a SDGGGX2DX2R motif. Interestingly, it was shown previously that introduction of 5 residues downstream from serine 51 (see Figure 3C) preserves the fusogenicity of the syncytin-1 protein . This suggests that the first 30 aa of the RBD, that include the conserved PCXC motif, constitute an actual sub-domain. It should be noted that regions of variable sizes are observed between the CYX5C and CX8–9CW motifs of the retroviruses of the interference group, which suggests a certain flexibility of the interdomain. In addition, the conserved SDGGGX2DX2R motif seems to be directly involved in receptor interaction, as supported by receptor-binding inhibition with a regio-selective antibody and receptor recognition alteration due to aspartic acid mutated residues in SNV-related retrovirus . Lastly, the carboxy-terminal end of the RBD presents a conserved predicted alpha-helix located immediately upstream of the SDGGGX2D sequence. Taken together, these results suggest, for all the retroviruses of the interference group, the existence of 5 structurally or sequence-conserved sub-domains. Crystallography studies will be required to confirm that these sub-domains interact. Finally, as the syncytin-1 belongs to the HERV-W family, many natural HERV-W truncated envelope ORFs probably contain the RBD. When reactivated, these partial envelopes could interfere with the hASCT receptors, which suggests putative physiopathological functions for such truncated envelopes.
- HTLV 1 and 2:
Human T-cell leukemia virus 1–2
human endogenous retrovirus W
type D mammalian retrovirus receptor
Epidermal growth factor
We are grateful to Bertrand Bonnaud for assistance with preparing the manuscript, and Robin Buckland for critical reading of the manuscript. We also thank Dimitri Lavillette for discussions and David Kabat for providing the pCD3.1VHR16/14 and pCDNA3.1-hASCT1 expression plasmids.
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