HVEM and nectin-1 are the major mediators of herpes simplex virus 1 (HSV-1) entry into human conjunctival epithelium
HVEM and nectin-1 are the major mediators of herpes simplex virus 1 (HSV-1) entry into human conjunctival epithelium
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DOI：
10.1167/iovs.08-1807
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发表时间：
2008-09-01
影响因子：
4.4
通讯作者：
Shukla, Deepak
中科院分区：
文献类型：
--
作者：
Akhtar, Jihan;Tiwari, Vaibhav;Shukla, Deepak
目的。人类结膜是单纯疱疹病毒(HSV)-1感染的天然靶点。本研究的目的是探讨HSV-1进入人结膜上皮细胞的细胞和分子机制。进入细胞的特征包括病毒与细胞的初始结合方法、pH依赖性以及特定HSV-1进入受体的表达和使用。方法：通过活细胞成像观察HSV-1感染的HCjE细胞的初始结合。用表达β-半乳糖苷酶的HSV-1病毒粒子检测野生型HSV-1(Kos)和突变型HSV-1(Kos)Rid1进入HCjE细胞。空斑试验检测HSV-1在HCjE细胞中的复制。溶酶促进剂用于确定病毒进入是否是pH依赖的。用逆转录聚合酶链式反应、流式细胞仪和免疫组织化学方法检测受体的表达。结果：HSV-1病毒粒子附着在存在于HCjE细胞上的丝状足上，并利用它们到达细胞体进行进入。培养的HCjE细胞表现出对HSV-1进入的敏感性，并形成证实病毒复制的斑块。阻断囊泡酸化显著减少进入，这意味着一种依赖于pH的进入模式。多种检测证实进入受体Nectin-1、Hvem和3-O-硫酸乙酰肝素(3-OS HS)在HCjE细胞膜上的表达。结论：HSV-1进入HCjE细胞是一个pH依赖的过程，该过程依赖于靶向病毒在丝状足上的传播。HCjE细胞表达三种主要的进入受体，其中Nectin-1和Hvem在介导进入过程中起主要作用。
PURPOSE. The human conjunctiva is a natural target for herpes simplex virus (HSV)-1 infection. The goals of this study were to investigate the cellular and molecular mechanisms of HSV-1 entry into human conjunctival epithelial (HCjE) cells. Specific features of entry studied included the method of initial viral binding to cells, pH dependency, and expression and usage of specific HSV-1 entry receptors.METHODS. To observe HSV-1 initial binding, live cell imaging was performed on HSV-1-infected HCjE cells. Reporter HSV-1 virions expressing beta-galactosidase were used to determine entry of wild-type HSV-1(KOS) and a mutant, HSV-1(KOS) Rid1, into HCjE cells. HSV-1 replication in HCjE cells was determined by plaque assays. Lysosomotropic agents were used to determine whether viral entry was pH dependent. Reverse transcription (RT)-PCR, flow cytometry, and immunohistochemistry were used to determine the expression of receptors. Receptorspecific siRNAs were used to define the role of individual entry receptors.RESULTS. HSV-1 virions attach to filopodia present on HCjE cells and use them to reach the cell body for entry. Cultured HCjE cells demonstrate susceptibility to HSV-1 entry and form plaques confirming viral replication. Blocking vesicular acidification significantly reduces entry, implicating a pH-dependent mode of entry. Multiple assays confirm the expression of entry receptors nectin-1, HVEM, and 3-O-sulfated heparan sulfate (3-OS HS) on the HCjE cell membrane. Knocking down of gD receptors by siRNAs interference implicates nectin-1 and HVEM as the major mediators of entry.CONCLUSIONS. HSV-1 entry into HCjE cells is a pH-dependent process that is aided by targeted virus travel on filopodia. HCjE cells express all three major entry receptors, with nectin-1 and HVEM playing the predominant role in mediating entry.