The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors - PubMed (original) (raw)

The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors

Dimitri Lavillette et al. J Virol. 2002 Jul.

Abstract

The human endogenous retrovirus type W (HERV-W) family includes proviruses with intact protein-coding regions that appear to be under selection pressure, suggesting that some HERV-W proviruses may remain active in higher primates. The envelope glycoprotein (Env) encoded by HERV-W is highly fusogenic, is naturally expressed in human placental syncytiatrophoblasts, and has been reported to function as a superantigen in lymphocyte cultures. Recent evidence suggested that HERV-W Env can mediate syncytium formation by interacting with the human sodium-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) (J.-L. Blond, D. Lavillette, V. Cheynet, O. Bouton, G. Oriol, S. Chapel-Fernandez, B. Mandrand, F. Mallet, and F.-L. Cosset, J. Virol. 74:3321-3329, 2000) and that it can pseudotype human immunodeficiency virus cores (D. S. An, Y. Xie, and I. S. Y. Chen, J. Virol. 75:3488-3489, 2001). By using cell-cell fusion and pseudotype virion infection assays, we found that HERV-W Env efficiently uses both hASCT2 and the related transporter hASCT1 (gene name, SLC1A4) as receptors. In addition, although HERV-W Env mediates only slight syncytium formation or infection of mouse cells, it utilizes the mouse transporters mASCT1 and mASCT2 when their sites for N-linked glycosylation are eliminated by mutagenesis. Consistent with their role as a battlefield in host-virus coevolution, the viral recognition regions in ASCT1 and ASCT2 of humans and mice are highly divergent compared with other regions of these proteins, and their ratios of nonsynonymous to synonymous nucleotide sequence changes are extremely large. The recognition of ASCT1 and ASCT2 despite this divergence of their sequences strongly suggests that the use of both receptors has been highly advantageous for survival and evolution of the HERV-W family of retroviruses.

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Figures

FIG. 1.

HERV-W glycoprotein envelope can use both hASCT2 and hASCT1 to induce cell-cell fusion. CHO cells expressing either hASCT1 or hASCT2 and human cell line TE671 were transfected with RD-Rless, BaEV-Rless, HERV-W, or LacZ (control) expression vectors. The determination of the fusion activity of the transfected envelope glycoproteins was performed at 24 h posttransfection. (A) The cultures were fixed and stained with May-Grunwald and Giemsa solutions. Magnification, ×100. (B) Histogram showing the mean fusion index of each combination of cell-cell fusion assay (error bars are standard deviations; n = 4). The fusion index represents the percentage of fusion events in a cell population and is defined as [(N − S)/_T_] × 100, where N is the number of nuclei in syncytia, S is the number of syncytia, and T is the total number of nuclei counted (2).

FIG. 2.

Analysis of HERV-W and HERV-Wcyt16 envelope glycoproteins in HIV particles. (A) Schematic representation of the carboxy-terminal extremity of the HERV-W envelope. The arrow indicates the stop codon insertion for HERV-Wcyt16. SU, surface subunit; TM, transmembrane subunit; ect, tm, and cyt, ectodomain, transmembrane domain, and cytoplasmic domain of the TM, respectively. (B) Electrophoretic immunoblot of sedimented virus samples probed with monoclonal antibody (Ab) 6A2B2 against HERV-W TM. HEK293 cells were transfected with plasmids expressing HIV Gag-Pol-Rev (pCMVdR8.74), a GFP reporter gene (pRRLsin18.cPPT.CMV.eGFP.WPRE), and a plasmid expressing either no envelope or the HERV-W or HERV-Wcyt16 envelopes. Thirty-six hours later, the supernatants were centrifuged through a 25% sucrose cushion and the pelleted virus samples were analyzed by Western immunoblotting. Normalization of samples was done using an anti-HIV capsid (CA) antibody. Tri, trimer; Δ, truncated form of HERV-W TM.

FIG. 3.

Mediation of infections by ASCT1 and ASCT2 receptors. Infectivity assays were done using CHO cell clones that constitutively express ASCT1 or ASCT2 receptors. The titers are averages from three independent experiments (error bars are standard errors). (A) Infections of cells that express hASCT1 and hASCT2 receptors; (B) infections of cells that express mASCT1, mASCT2, or their N-deglycosylated mutants.

FIG. 4.

Fusion assays suggest that the HERV-W envelope glycoprotein functionally interacts with mASCT1 and with N-deglycosylated forms of mASCT1 and mASCT2. CHO cells expressing either wild-type or deglycosylated forms of mASCT1 or mASCT2 were transfected with plasmids expressing the RD-Rless, the BaEV-Rless, or the HERV-Wcyt16 envelopes or the LacZ protein (control). The fusion activities of the transfected envelope glycoproteins were analyzed at 24 h posttransfection. Results are expressed as percent fusion indices (error bars are standard deviations; n = 3) as described for Fig. 1.

FIG. 5.

Identification of mouse ASCT proteins on the surfaces of CHO cells. CHO cells stably expressing _myc_-tagged mASCT1, mASCT2, and their N-deglycosylated mutant proteins were surface biotinylated as described in Materials and Methods. Samples of the total protein cell lysates were also used in a parallel analysis. The biotinylated proteins were then purified by affinity chromatography. The samples were analyzed by Western immunoblotting with anti-myc tag monoclonal antibody 9E10 (Sigma). (A) Total cellular _myc_-tagged mASCT1, mASCT2, and their N-deglycosylated mutants proteins; (B) cell surface _myc_-tagged mASCT1, mASCT2, and their N-deglycosylated mutants proteins that were biotinylated on cell surfaces and then affinity purified prior to immunoblot analysis. Numbers on the left are molecular weights in thousands.

FIG. 6.

Amino acid sequence comparison of putative ECL2 of hASCT1, mASCT1, mASCT2, and hASCT2 indicates 27% sequence identity. Numbers at the left of the sequences correspond to the first and last amino acids shown. Common amino acids are shaded. Deletions in sequences are indicated by dashes. N-glycosylation sites are indicated by asterisks. Region C is hypervariable and contains N-linked oligosaccharides that negatively interfere with viral receptor function, whereas region B is highly conserved (see text).

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The envelope glycoprotein of human endogenous retrovirus type W uses a divergent family of amino acid transporters/cell surface receptors - PubMed (original) (raw)