K亚群禽白血病病毒细胞受体的鉴定

Identification of cellular receptor mediating subgroup K avian leukosis virus binding and entry its host cell

禽白血病病毒(ALV)进入细胞启动感染依赖于其囊膜表面蛋白gp85与细胞受体的结合。K亚群ALV(ALV-K)是2012年从我国本地芦花鸡中分离到的一个ALV新亚群。为了鉴定ALV-K的细胞受体,本研究利用真核细胞表达了A和K亚群ALV gp85蛋白,并利用实验室前期拯救的重组病毒RCAS-A-GFP和RCAS-K-GFP进行病毒受体的交叉干扰试验,结果显示A亚群ALV(ALV-A)gp85蛋白能够抑制ALV-K的感染,同样ALV-Kgp85蛋白也能够抑制ALV-A的感染,存在受体交叉干扰现象,因而推测二者可能共用细胞受体。已有研究表明Tva是ALV-A的细胞受体,本研究利用Co-IP和Pull-down试验检测了ALV-Kgp85与Tva的互作,结果显示ALV-Kgp85蛋白能与可溶性的Tva(sTva)相互作用。进一步的流式细胞术结果显示ALV-Kgp85能够高效结合Tva,结合率在90%以上。为进一步验证Tva是否能介导ALV-K进入细胞,利用ALV的非易感细胞HEK293T进行了Tva受体重建试验,结果显示RCAS-K-GFP能够进入表达Tva的293T细胞并复制产生较强的绿色荧光。以上结果充分表明Tva也是ALV-K的细胞受体,能够介导ALV-K进入宿主细胞,启动其感染。该研究为阐明ALV-K侵入宿主细胞机制及抗病毒靶点筛选奠定了基础。

To enter host cells and initiate infection, avian leukosis virus depends on that viral envelope surface unit(gp85) binds to corresponding cellular receptor. Subgroup K avian leukosis virus(ALV-K) is a novel subgroup isolated from Chinese indigenous breed ’luhua chicken’ in 2012. In order to identify the cellular receptorof ALV-K, the gp85 proteins of ALV-A and ALV-K were expressed by eukaryotic cell. This result of cross-interference of ALV-A and K recombination viruses indicated gp85 protein of subgroup A ALV(ALV-A) could inhibit the infection by ALV-K. Similarly, the gp85 protein of ALV-K could also inhibit ALV-A infection. Therefore, it is speculated that ALV-K and ALV-A share the same cellular receptor. Previous studies have shown that Tva has been a cell receptor for ALV-A. In this study, the interaction between ALV-K gp85 and Tva were tested by Co-IP and Pull-down.The results showed that ALV-K gp85 protein could interact with soluble Tva(sTva). Further flow cytometry results indicated that ALV-K gp85 could efficiently bind Tva with a binding rate of over 90%. To further verify whether Tva could mediate the entry of ALV-K into cells, a receptor reconstruction experiment was performed using ALV’s non-susceptible cell HEK293 T. The results showed that RCAS-K-GFP could enter Tva-expressing 293 T cells and replicate to produce stronger green fluorescence. The above results fully indicate that Tva is also a cellular receptor for ALV-K, which can mediate ALV-K into host cells and initiate its infection.This study laid the foundation for elucidating the mechanism of ALV-K invasion into host cells and screening antiviral targets.