Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion
Bimolecular complementation reveals that glycoproteins gB and gH/gL of herpes simplex virus interact with each other during cell fusion
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DOI：
10.1073/pnas.0707452104
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发表时间：
2007-11-20
影响因子：
11.1
通讯作者：
Eisenberg, Roselyn J.
中科院分区：
文献类型：
--
作者：
Atanasiu, Doina;Whitbeck, J. Charles;Eisenberg, Roselyn J.
单纯疱疹病毒进入细胞需要四种糖蛋白，gB、gD、gH和gL。gD与其受体之一的结合触发了需要核心融合蛋白gB以及gH/gL异二聚体的步骤。有证据表明gH/gL启动细胞的半融合，但该复合物是否与gB发生物理相互作用以导致完全融合尚不清楚。我们利用增强型黄色荧光蛋白（EYFP）的双分子互补（BiMC）来检测细胞 - 细胞融合过程中的糖蛋白相互作用。EYFP的N端或C端半段与gD、gB和gH的C末端融合，形成六种嵌合蛋白（Dn、Dc、Bn、Bc、Hn和Hc）。通过共聚焦显微镜检测BiMC。用Dn和Bc或Dn、Hc以及无标签的gL共转染的带有受体的（C10）细胞显示出EYFP荧光，这表明gD与gB以及gD与gH/gL之间存在相互作用。在转染了gL、Bc和Hn的细胞中未发生EYFP互补。然而，当gD与其他三种蛋白共表达时，发生了细胞 - 细胞融合，并且合胞体显示出明亮的EYFP荧光。为了将糖蛋白表达与融合分开，我们用gL、Bc和Hn转染C10细胞20小时，然后加入可溶性gD以触发融合。我们在10分钟内检测到荧光合胞体，并且其数量和大小随着gD暴露时间的增加而增加。因此，当gD结合其受体时，核心融合机制被触发，形成一种多蛋白复合物，作为融合以及可能的病毒进入的一个步骤。
Herpes simplex virus entry into cells requires four glycoproteins, gB, gD, gH, and gL. Binding of gD to one of its receptors triggers steps requiring the core fusion proteins, gB and the gH/gL heterodimer. There is evidence that gH/gL initiates hemifusion of cells, but whether this complex interacts physically with gB to cause complete fusion is unknown. We used bimolecular complementation (BiMC) of enhanced yellow fluorescent protein (EYFP) to detect glycoprotein interactions during cell-cell fusion. The N- or C-terminal half of EYFP was fused to the C terminus of gD, gB, and gH to form six chimeric proteins (Dn, Dc, Bn, Bc, Hn, and Hc). BiMC was detected by confocal microscopy. Receptor-bearing (C10) cells cotransfected with Dn and Bc or Din, Hc, and untagged gL exhibited EYFP fluorescence, indicative of interactions between gD and gB and between gD and gH/gL. EYFP complementation did not occur in cells transfected with gL, Bc, and Hn. However, when gD was coexpressed with these other three proteins, cell-cell fusion occurred and the syncytia exhibited bright EYFP fluorescence. To separate glycoprotein expression from fusion, we transfected C10 cells with gL, Bc, and Hn for 20 h and then added soluble gD to trigger fusion. We detected fluorescent syncytia within 10min,and both their number and size increased with exposure time to gD. Thus, when gD binds its receptor, the core fusion machinery is triggered to form a multiprotein complex as a step in fusion and possibly virus entry.