N-linked glycosylation and sequence changes in a critical negative control region of the ASCT1 and ASCT2 neutral amino acid transporters determine their retroviral receptor functions - PubMed (original) (raw)

N-linked glycosylation and sequence changes in a critical negative control region of the ASCT1 and ASCT2 neutral amino acid transporters determine their retroviral receptor functions

Mariana Marin et al. J Virol. 2003 Mar.

Abstract

A widely dispersed interference group of retroviruses that includes the feline endogenous virus (RD114), baboon endogenous virus (BaEV), human endogenous virus type W (HERV-W), and type D primate retroviruses uses the human Na(+)-dependent neutral amino acid transporter type 2 (hASCT2; gene name, SLC1A5) as a common cell surface receptor. Although hamster cells are fully resistant to these viruses and murine cells are susceptible only to BaEV and HERV-W pseudotype viruses, these rodent cells both become highly susceptible to all of the viruses after treatment with tunicamycin, an inhibitor of protein N-linked glycosylation. A partial explanation for these results was recently provided by findings that the orthologous murine transporter mASCT2 is inactive as a viral receptor, that a related (ca. 55% identity) murine paralog (mASCT1; gene name, SLC1A4) mediates infections specifically of BaEV and HERV-W, and that N-deglycosylation of mASCT1 activates it as a receptor for all viruses of this interference group. Because the only two N-linked oligosaccharides in mASCT1 occur in the carboxyl-terminal region of extracellular loop 2 (ECL2), it was inferred that this region contributes in an inhibitory manner to infections by RD114 and type D primate viruses. To directly and more thoroughly investigate the receptor active sites, we constructed and analyzed a series of hASCT2/mASCT2 chimeras and site-directed mutants. Our results suggest that a hypervariable sequence of 21 amino acids in the carboxyl-terminal portion of ECL2 plays a critical role in determining the receptor properties of ASCT2 proteins for all viruses in this interference group. In addition, we analyzed the tunicamycin-dependent viral susceptibility of hamster cells. In contrast to mASCT1, which contains two N-linked oligosaccharides that partially restrict viral infections, hamster ASCT1 contains an additional N-linked oligosaccharide clustered close to the others in the carboxyl-terminal region of ECL2. Removal of this N-linked oligosaccharide by mutagenesis enabled hamster ASCT1 to function as a receptor for all viruses of this interference group. These results strongly suggest that combinations of amino acid sequence changes and N-linked oligosaccharides in a critical carboxyl-terminal region of ECL2 control retroviral utilization of both the ASCT1 and ASCT2 receptors.

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Figures

FIG. 1.

Construction of hASCT2/mASCT2 chimeras. (A) Amino acid sequence comparison of the putative ECL regions of hASCT2 and mASCT2. Numbers at the left of the sequences correspond to the positions of the first and last amino acids shown. Dots indicate amino acid identity. Deletions in sequences are indicated by dashes. N-glycosylation sites are indicated by asterisks. Numbers at the right of the sequences indicate the percent amino acid identity. (B) The topologies and the nomenclatures of the wild-type and chimeric ASCT2 proteins. The representation on the top shows the putative topology of the hASCT2 and mASCT2 cell surface receptors; the putative ECLs are numbered 1 through 5.

FIG. 2.

Mediation of infections by hASCT2, mASCT2, and chimeric receptors. Infectivity assays were done with CHO cells that had been transiently transfected with expression vectors for the receptors. Infections with LacZ(RD114) and LacZ(BaEV) pseudotype viruses were initiated 24 h after the transfections were begun. The titers are averages of three independent experiments ± standard errors. RD114 titers were zero for some of the receptors, and in these cases the bars are not shown.

FIG. 3.

Amino acid sequence comparison of the putative ECL2 of mASCT1 and hamster ASCT1 (CHO.ASCT1) indicates 87% sequence identity. Numbers at the left of the sequences correspond to the positions of the first and last amino acids shown. Dots indicate amino acid identity. N-linked glycosylation sites are indicated by asterisks.

FIG. 4.

Surface expression and viral receptor properties of wild-type and mutant hamster ASCT1 proteins. (A) Titers of infection of LacZ pseudotype viruses in CHO cells that were transiently expressing CHO.ASCT1 and its N-glycosylation mutants. Infections with LacZ pseudotype viruses were initiated 24 h after the transfections were begun. The titers of infection are averages of three independent experiments ± standard errors. With several of the receptors, the titers of the RD114 or SRV-2 viruses were zero, in which cases the bars are not shown. (B) Expression of wild-type and mutant hamster ASCT1 proteins on the surface of HEK293T cells and analysis of the N-linked oligosaccharides. HEK293T cells transiently expressing Myc-tagged CHO.ASCT1 and its N-deglycosylation mutants were surface biotinylated as described in Materials and Methods. The biotinylated proteins were purified by affinity chromatography. The samples that were untreated (−) or treated (+) with PNGaseF were analyzed by Western immunoblotting with anti-Myc tag monoclonal antibody 9E10 (Sigma).

FIG. 5.

N-linked glycosylation of hamster ASCT1 (CHO.ASCT1) blocks infectivity and membrane fusion mediated by the HERV-W envelope glycoprotein. (A) Mediation of HIV/HERV-W pseudotype virus infections by CHO.ASCT1 and its N-deglycosylated mutants. Infectivity assays were done with CHO cell clones that constitutively express the proteins. The titers are averages of three independent infection experiments ± standard errors. (B) Histogram showing the mean fusion index of each virus-receptor combination in the cell-cell fusion assay (error bars are standard deviations; n = 3). The fusion index represents the percent 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).

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N-linked glycosylation and sequence changes in a critical negative control region of the ASCT1 and ASCT2 neutral amino acid transporters determine their retroviral receptor functions - PubMed (original) (raw)