Chu Justin Jang-Hann, Ng Mah-Lee
Flavivirology Laboratory, Department of Microbiology, 5 Science Drive 2, National University of Singapore, Singapore 117597.
J Biol Chem. 2004 Dec 24;279(52):54533-41. doi: 10.1074/jbc.M410208200. Epub 2004 Oct 7.
The functional receptor for the flavivirus West Nile (WNV) infection has been characterized in this study with a combination of biochemical and molecular approaches. A 105-kDa protease-sensitive glycoprotein that binds WNV was isolated from the plasma membrane of cells permissive to WNV infection. The protein was subjected to peptide sequencing, and this glycoprotein was identified as a member of the integrin superfamily. Infection of WNV was shown to be markedly inhibited in Vero cells pretreated with blocking antibodies against alpha(v)beta(3) integrin and its subunits by receptor competition assay. It was also noted that cells pretreated with antibodies against alpha(v)beta(3) integrin can effectively inhibit flavivirus Japanese encephalitis but to a lesser extent flavivirus dengue infections. West Nile virus entry is independent of divalent cations and is not highly blocked by arginine-glycine-aspartic acid (RGD) peptides, suggesting that the interaction between the virus and alpha(v)beta(3) integrin is not highly dependent on the classical RGD binding motif. In addition, gene silencing of the beta(3) integrin subunit in cells has resulted in cells largely resistant to WNV infection. In contrast, expression of recombinant human beta(3) integrin substantially increased the permissiveness of CS-1 melanoma cells for WNV infection. Soluble alpha(v)beta(3) integrin can also effectively block WNV infection in a dose-dependent manner. Furthermore, WNV infection also triggered the outside-in signaling pathway via the activation of integrin-associated focal adhesion kinase. The identification of alpha(v)beta(3) integrin as a receptor for WNV provides insight into virus-receptor interaction, hence creating opportunities in the development of anti-viral strategies against WNV infection.
在本研究中，通过生物化学和分子方法相结合的方式，对西尼罗河病毒（WNV）感染的功能性受体进行了表征。从允许WNV感染的细胞的质膜中分离出一种与WNV结合的105 kDa蛋白酶敏感糖蛋白。对该蛋白进行了肽测序，并将这种糖蛋白鉴定为整合素超家族的成员。通过受体竞争试验表明，用针对α(v)β(3)整合素及其亚基的阻断抗体预处理的Vero细胞中，WNV感染受到显著抑制。还注意到，用针对α(v)β(3)整合素的抗体预处理的细胞可以有效抑制黄病毒日本脑炎，但对黄病毒登革热感染的抑制程度较小。西尼罗河病毒的进入不依赖于二价阳离子，也不会被精氨酸-甘氨酸-天冬氨酸（RGD）肽高度阻断，这表明病毒与α(v)β(3)整合素之间的相互作用并非高度依赖于经典的RGD结合基序。此外，细胞中β(3)整合素亚基的基因沉默导致细胞对WNV感染具有很大的抗性。相反，重组人β(3)整合素的表达显著增加了CS-1黑色素瘤细胞对WNV感染的易感性。可溶性α(v)β(3)整合素也可以以剂量依赖性方式有效阻断WNV感染。此外，WNV感染还通过激活整合素相关的粘着斑激酶触发了由外向内信号通路。将α(v)β(3)整合素鉴定为WNV的受体，为病毒-受体相互作用提供了深入了解，从而为开发针对WNV感染的抗病毒策略创造了机会。
