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
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
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DOI：
10.1128/jvi.77.5.2936-2945.2003
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发表时间：
2003-03-01
影响因子：
5.4
通讯作者：
Kabat, D
中科院分区：
文献类型：
--
作者：
Marin, M;Lavillette, D;Kabat, D
广泛分布的干扰组逆转录病毒（包括猫内源性病毒（RD 114）、狒狒内源性病毒（BaEV）、人内源性病毒W型（HERV-W）和D型灵长类逆转录病毒）使用人Na+依赖性中性氨基酸转运蛋白2型（hASCT 2;基因名称，SLC 1A 5）作为常见的细胞表面受体。尽管仓鼠细胞对这些病毒完全耐药，鼠细胞仅对BaEV和HERV-W假型病毒敏感，但这些啮齿动物细胞在用衣霉素（蛋白质N-连接糖基化的抑制剂）处理后对所有病毒都变得高度敏感。这些结果的部分解释最近通过以下发现提供，即正链鼠转运蛋白mASCT 2作为病毒受体是无活性的，相关的（ca. 55%的同一性）鼠副伤寒沙门氏菌（mASCT 1，基因名称，SLC 1A 4）特异性介导BaEV和HERV-W的感染，并且mASCT 1的N-去糖基化激活其作为该干扰组的所有病毒的受体。由于mASCT 1中仅有的两个N-连接寡糖发生在细胞外环2（ECL 2）的羧基末端区域，因此推断该区域以抑制方式对RD 114和D型灵长类病毒的感染起作用。为了直接和更彻底地研究受体活性位点，我们构建并分析了一系列hASCT 2/mASCT 2嵌合体和定点突变体。我们的研究结果表明，在ECL 2的羧基末端部分的21个氨基酸的高变序列起着关键作用，在确定ASCT 2蛋白的受体特性的所有病毒在这个干扰组。此外，我们分析了仓鼠细胞的衣霉素依赖性病毒易感性。与mASCT 1（包含两个部分限制病毒感染的N-连接寡糖）相反，仓鼠ASCT 1包含一个额外的N-连接寡糖，该寡糖在ECL 2的羧基末端区域中与其他寡糖靠近。通过诱变去除该N-连接寡糖使仓鼠ASCT 1能够作为该干扰组的所有病毒的受体发挥作用。这些结果强烈表明，氨基酸序列的变化和N-连接的寡糖在ECL 2的一个关键的羧基末端区域的组合控制逆转录病毒利用ASCT 1和ASCT 2受体。
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.