Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors.
Development and characterization of a Rift Valley fever virus cell-cell fusion assay using alphavirus replicon vectors.
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DOI：
10.1016/j.virol.2006.07.035
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发表时间：
2006-12-05
期刊：
影响因子：
3.7
通讯作者：
Bertolotti-Ciarlet A
中科院分区：
文献类型：
--
作者：
Filone CM;Heise M;Doms RW;Bertolotti-Ciarlet A
裂谷热病毒（RVFV）是布尼亚病毒科中白蛉病毒属的成员，由蚊子传播，感染人类和家畜，特别是牛和羊。由于原代RVFV毒株必须在BSL-3+或BSL-4设施中处理，因此RVFV细胞-细胞融合试验将有助于在BSL-2条件下研究RVFV糖蛋白功能。至于布尼亚病毒科的其他成员，RVFV糖蛋白靶向病毒出芽的高尔基体，并且不能有效地递送到细胞表面。然而，使用甲病毒复制子载体过表达RVFV糖蛋白导致糖蛋白在多种细胞类型的表面上表达。用弱酸性培养基（pH 6.2及以下）短暂处理RVFV糖蛋白表达细胞导致快速有效的合胞体形成，我们通过β-半乳糖苷酶α-互补对其进行定量。在几种细胞类型中观察到融合，表明RVFV的受体广泛表达，或者这种酸依赖性病毒不需要特异性受体来介导细胞-细胞融合。融合发生在一个广泛的温度范围内，正如预期的病毒与蚊子和哺乳动物宿主。与VSV-G糖蛋白介导的细胞融合相反，RVFV糖蛋白依赖性细胞融合可以通过用胰蛋白酶处理靶细胞来防止，这表明宿主细胞表面上的一种或多种蛋白质（或蛋白质相关的碳水化合物）需要支持膜融合。本文报道的细胞-细胞融合试验将使研究RVFV糖蛋白的高通量形式的膜融合活性和筛选具有阻断病毒特异性膜融合能力的小分子抑制剂成为可能。
Rift Valley fever virus (RVFV), a member of the Phlebovirus genus in the Bunyaviridae family, is transmitted by mosquitoes and infects both humans and domestic animals, particularly cattle and sheep. Since primary RVFV strains must be handled in BSL-3+ or BSL-4 facilities, a RVFV cell–cell fusion assay will facilitate the investigation of RVFV glycoprotein function under BSL-2 conditions. As for other members of the Bunyaviridae family, RVFV glycoproteins are targeted to the Golgi, where the virus buds, and are not efficiently delivered to the cell surface. However, overexpression of RVFV glycoproteins using an alphavirus replicon vector resulted in the expression of the glycoproteins on the surface of multiple cell types. Brief treatment of RVFV glycoprotein expressing cells with mildly acidic media (pH 6.2 and below) resulted in rapid and efficient syncytia formation, which we quantified by β-galactosidase α-complementation. Fusion was observed with several cell types, suggesting that the receptor(s) for RVFV is widely expressed or that this acid-dependent virus does not require a specific receptor to mediate cell–cell fusion. Fusion occurred over a broad temperature range, as expected for a virus with both mosquito and mammalian hosts. In contrast to cell fusion mediated by the VSV-G glycoprotein, RVFV glycoprotein-dependent cell fusion could be prevented by treating target cells with trypsin, indicating that one or more proteins (or protein-associated carbohydrate) on the host cell surface are needed to support membrane fusion. The cell–cell fusion assay reported here will make it possible to study the membrane fusion activity of RVFV glycoproteins in a high-throughput format and to screen small molecule inhibitors for the ability to block virus-specific membrane fusion.
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影响因子：
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