Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without
Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without
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DOI：
10.1128/jvi.72.6.5323-5327.1998
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发表时间：
1998-06-01
影响因子：
5.4
通讯作者：
Greenberg, HB
中科院分区：
文献类型：
--
作者：
Gilbert, JM;Greenberg, HB
我们最近描述了我们的发现，重组杆状病毒产生的病毒样颗粒(VLP)可以诱导细胞-细胞融合，类似于我们在病毒进入组织培养细胞的实验中所诱导的完整轮状病毒(J.M.Gilbert和H.B.Greenberg，J.Virol)。71：3555-4563,1997)。合胞体形成所需的条件类似于病毒在感染过程中穿透质膜的条件。这种VLP介导的融合活性依赖于外层蛋白VP4和VP7的存在，以及VP4的胰酶作用。融合活性只与允许轮状病毒感染的细胞发生。在这里，我们开始剖析VP4在轮状病毒进入中的作用，通过检测VP4的精确胰酶裂解和与病毒进入相关的VP4功能的激活的重要性。我们提出的证据表明，通过特定的定点突变消除VP4的三个胰酶敏感的精氨酸残基可以防止合胞体的形成。VP4的三个精氨酸残基中有两个对于合胞体的形成是不必要的，只有247位的精氨酸残基似乎是激活VP4功能和细胞-细胞融合所必需的。在我们的合胞体实验中使用重组VLP将有助于理解VP4中发生的构象变化，这些变化涉及轮状病毒对宿主细胞的渗透。
We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:3555-4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.