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.
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.
Marin
Mariana
M
Department of Biochemistry and Molecular Biology, Oregon Health & Science University, Portland, Oregon 97201-3098, USA.
Lavillette
Dimitri
D
Kelly
Sean M
SM
Kabat
David
D
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D026962:Amino Acid Transport System ASC / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D005786:Gene Expression Regulation; D006031:Glycosylation; D006801:Humans; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011991:Receptors, Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000378:metabolism* / Q000472:pathogenicity; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology
D026962:Amino Acid Transport System ASC; D000998:Antiviral Agents; D015778:Minor Histocompatibility Antigens; D011991:Receptors, Virus; C498848:SLC1A4 protein, human; C420506:SLC1A5 protein, human; D014415:Tunicamycin