Department of Viral Encephalitis, Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, and State Key Laboratory for Infectious Disease Prevention and Control (SKLID), Beijing, China.
PLoS One. 2010 Mar 11;5(3):e9656. doi: 10.1371/journal.pone.0009656.
Cell culture-adapted strains of Sindbis virus (SINV) initially attach to cells by the ability to interact with heparan sulfate (HS) through selective mutation for positively charged amino acid (aa) scattered in E2 glycoprotein (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72: 7357-7366, 1998). Here we have further confirmed that interaction of E2 protein with HS is crucial for cellular infection of SINV based on the reverse genetic system of XJ-160 virus, a Sindbis-like virus (SINLV). Both SINV YN87448 and SINLV XJ-160 displayed similar infectivity on BHK-21, Vero, or C6/36 cells, but XJ-160 failed to infect mouse embryonic fibroblast (MEF) cells. The molecular mechanisms underlying the selective infectivity of XJ-160 were approached by substituting the E1, E2, or both genes of XJ-160 with that of YN87448, and the chimeric virus was denominated as XJ-160/E1, XJ-160/E2, or XJ-160/E1E2, respectively. In contrast to the parental XJ-160, all chimeric viruses became infectious to wild-type MEF cells (MEF-wt). While MEF-Ext(-/-) cells, producing shortened HS chains, were resistant not only to XJ-160, but also to YN87448 as well as the chimeric viruses, indicating that the inability of XJ-160 to infect MEF-wt cells likely due to its incompetent discrimination of cellular HS. Treatment with heparin or HS-degrading enzyme resulted in a substantial decrease in plaque formation by YN87448, XJ-160/E2, and XJ-160/E1E2, but had marginal effect on XJ-160 and XJ-160/E1, suggesting that E2 glycoprotein from YN87448 plays a more important role than does E1 in mediating cellular HS-related cell infection. In addition, the peptide containing 145-150 aa from E2 gene of YN87448 specifically bound to heparin, while the corresponding peptide from the E2 gene of XJ-160 essentially showed no binding to heparin. As a new dataset, these results clearly confirm an essential role of E2 glycoprotein, especially the domain of 145-150 aa, in SINV cellular infection through the interaction with HS.
细胞培养适应的辛德毕斯病毒（SINV）株最初通过 E2 糖蛋白（E2）中带正电荷的氨基酸（aa）的选择性突变与肝素硫酸（HS）相互作用而附着在细胞上（W. B. Klimstra、K. D. Ryman 和 R. E. Johnston，J. Virol. 72: 7357-7366，1998）。在这里，我们通过辛德毕斯样病毒（SINLV）XJ-160 的反向遗传系统进一步证实了 E2 蛋白与 HS 的相互作用对于 SINV 的细胞感染至关重要。SINV YN87448 和 SINLV XJ-160 在 BHK-21、Vero 或 C6/36 细胞上表现出相似的感染力，但 XJ-160 不能感染小鼠胚胎成纤维细胞（MEF）细胞。通过用 YN87448 的 E1、E2 或两者取代 XJ-160 的 E1、E2 或两者，研究了 XJ-160 选择性感染的分子机制，并用相应的嵌合病毒分别命名为 XJ-160/E1、XJ-160/E2 或 XJ-160/E1E2。与亲本 XJ-160 相比，所有嵌合病毒都能感染野生型 MEF 细胞（MEF-wt）。而 MEF-Ext(-/-) 细胞产生缩短的 HS 链，不仅对 XJ-160 而且对 YN87448 以及嵌合病毒都具有抗性，表明 XJ-160 不能感染 MEF-wt 细胞可能是由于其不能区分细胞 HS。肝素或 HS 降解酶处理会导致 YN87448、XJ-160/E2 和 XJ-160/E1E2 的蚀斑形成显著减少，但对 XJ-160 和 XJ-160/E1 的影响很小，表明 YN87448 的 E2 糖蛋白比 E1 更重要在介导细胞 HS 相关细胞感染中起作用。此外，YN87448 的 E2 基因中的 145-150 aa 肽特异性结合肝素，而 XJ-160 的 E2 基因的相应肽基本上不结合肝素。作为新的数据集，这些结果清楚地证实了 E2 糖蛋白，特别是 145-150 aa 结构域，通过与 HS 的相互作用在 SINV 细胞感染中的重要作用。
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