Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication
Identification of the Capsid Binding Site in the Herpes Simplex Virus 1 Nuclear Egress Complex and Its Role in Viral Primary Envelopment and Replication
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DOI：
10.1128/jvi.01290-19
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发表时间：
2019-11-01
影响因子：
5.4
通讯作者：
Kawaguchi, Yasushi
中科院分区：
文献类型：
--
作者：
Takeshima, Kosuke;Arii, Jun;Kawaguchi, Yasushi
在新生的后代疱疹病毒核衣壳的核外泄过程中，核衣壳通过感染细胞的内核膜萌发进入内外核膜之间的核周空间，从而获得一个初级包膜。单纯疱疹病毒1型、U(L)34和U(L)31蛋白形成核出口复合体，在这一萌发过程中发挥关键作用，称为初级囊膜。为了阐明NEC与子代核衣壳结合在HSV-1初级囊膜中的作用，我们建立了HSV-1 NEC与核衣壳和衣壳蛋白结合的体外检测体系。结果表明，单纯疱疹病毒1NEC与核衣壳和衣壳蛋白U(L)25结合，而与其他衣壳蛋白(如VP5、VP23和U(L)17)不结合；单纯疱疹病毒1NEC与核衣壳蛋白的结合是由NEC与U(L)25相互作用介导的。U(L)31个氨基酸残基需要精氨酸-281(R281)和天冬氨酸-282(D282)残基才能与核衣壳蛋白和U(L)25个氨基酸残基有效结合。我们还发现，U(L)31、R281和D282的丙氨酸替代降低了HSV-1的复制，导致核内衣壳的异常积累，并诱导了在大小和形态上与核周空间的初级囊膜相似的空泡的积累。这些结果表明，NEC通过U(L)31R281和D282与核衣壳结合，并可能通过与核衣壳中的U(L)25结合，在HSV-1复制过程中可能通过促进核衣壳进入囊泡而在复制中发挥重要作用。然而，到目前为止，还没有直接支持这一假设的数据报道。在本研究中，我们提供的数据表明，HSV-1 NEC膜远端的两个氨基酸实际上是NEC与核衣壳有效结合和在一次包膜期间将核衣壳有效地掺入囊泡所必需的，其中包含基于所解析的NEC结构的推测的衣壳结合部位。这是首次报道NEC与核衣壳结合和疱疹病毒初级包膜期间核衣壳掺入囊泡的增加之间的直接联系。
During nuclear egress of nascent progeny herpesvirus nucleocapsids, the nucleocapsids acquire a primary envelope by budding through the inner nuclear membrane of infected cells into the perinuclear space between the inner and outer nuclear membranes. Herpes simplex virus 1 (HSV-1) U(L)34 and U(L)31 proteins form a nuclear egress complex (NEC) and play critical roles in this budding process, designated primary envelopment. To clarify the role of NEC binding to progeny nucleocapsids in HSV-1 primary envelopment, we established an assay system for HSV-1 NEC binding to nucleocapsids and capsid proteins in vitro. Using this assay system, we showed that HSV-1 NEC bound to nucleocapsids and to capsid protein U(L)25 but not to the other capsid proteins tested (i.e., VP5, VP23, and U(L)17) and that HSV-1 NEC binding of nucleocapsids was mediated by the interaction of NEC with U(L)25. U(L)31 residues arginine-281 (R281) and aspartic acid-282 (D282) were required for efficient NEC binding to nucleocapsids and U(L)25. We also showed that alanine substitution of U(L)31 R281 and D282 reduced HSV-1 replication, caused aberrant accumulation of capsids in the nucleus, and induced an accumulation of empty vesicles that were similar in size and morphology to primary envelopes in the perinuclear space. These results suggested that NEC binding via U(L)31 R281 and D282 to nucleocapsids, and probably to U(L)25 in the nucleocapsids, has an important role in HSV-1 replication by promoting the incorporation of nucleocapsids into vesicles during primary envelopment.IMPORTANCE Binding of HSV-1 NEC to nucleocapsids has been thought to promote nucleocapsid budding at the inner nuclear membrane and subsequent incorporation of nucleocapsids into vesicles during nuclear egress of nucleocapsids. However, data to directly support this hypothesis have not been reported thus far. In this study, we have present data showing that two amino acids in the membrane-distal face of the HSV-1 NEC, which contains the putative capsid binding site based on the solved NEC structure, were in fact required for efficient NEC binding to nucleocapsids and for efficient incorporation of nucleocapsids into vesicles during primary envelopment. This is the first report showing direct linkage between NEC binding to nucleocapsids and an increase in nucleocapsid incorporation into vesicles during herpesvirus primary envelopment.