Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1.
Cell-to-cell transmission of HSV1 in human keratinocytes in the absence of the major entry receptor, nectin1.
复制标题
DOI：
10.1371/journal.ppat.1009631
复制
发表时间：
2021-09
期刊：
影响因子：
6.7
通讯作者：
Elliott G
中科院分区：
文献类型：
--
作者：
Kite J;Russell T;Jones J;Elliott G
单纯疱疹病毒1（HSV 1）感染表皮、口腔或生殖器粘膜的复层上皮，其中主要细胞类型是角质形成细胞。在这里，我们使用nTERT人角质形成细胞来产生主要候选HSV 1受体nectin 1的CRISPR-Cas9敲除（KO），从而产生对HSV 1进入难治的细胞系。尽管如此，一小群KO细胞能够支持感染，其不被nectin 1抗体阻断，因此不是残余nectin 1表达的结果。令人惊讶的是，在后来的时间，原来对HSV 1感染有抵抗力的细胞群体也被感染了。通过抑制病毒基因组复制或感染衣壳向细胞质输出有缺陷的Δ UL 34病毒，可以阻止后一群体的出现。此外，在初始感染细胞周围的细胞中检测到新形成的GFP标记的衣壳，表明病毒在原始易感细胞中复制后传播。额外的siRNA耗尽的第二个主要的HSV 1受体HVEM，或PTP 1B，细胞因子在其他地方显示参与细胞间的传输，在nectin 1的情况下，对病毒传播没有影响。中和人血清也未能阻断nectin 1 KO细胞中的病毒传播，这依赖于受体结合蛋白糖蛋白D和细胞间传播糖蛋白gI和gE，表明病毒通过直接细胞间传播传播。与这些结果一致，HSV 1和HSV 2均在nectin 1 KO细胞上形成噬斑，尽管滴度降低，证实一旦原始细胞群被感染，病毒就可以扩散到单层中的所有其他细胞中。我们的结论是，虽然nectin 1是所需的细胞外进入到大多数的人角质形成细胞，它是直接的细胞到细胞的传输。单纯疱疹病毒1型（HSV 1）感染表皮、口腔或生殖器粘膜的上皮细胞，引起唇疱疹、生殖器疱疹或更严重的后果，如角膜炎和新生儿疱疹。像许多病毒一样，HSV 1可以通过细胞外环境或直接细胞间传播传播，后者的机制对于避免宿主中的抗体反应很重要。在这里，我们通过产生主要候选病毒受体nectin 1的CRISPR-Cas9敲除，研究了HSV 1在人角质形成细胞（靶上皮细胞中的主要细胞类型）中的进入和传播。虽然HSV 1不能感染大多数nectin 1敲除的角质形成细胞，但这些nectin 1 KO细胞的一小部分仍然容易受到病毒进入，并且一旦感染，病毒就能够扩散到单层的其余部分。在存在中和血清的情况下，这种传播继续进行，中和血清可阻断细胞外病毒，并需要糖蛋白D（主要的病毒受体结合蛋白）和糖蛋白gE和gI（已知参与细胞间传播）。因此，虽然nectin 1是病毒进入大多数人角质形成细胞所必需的，但它是病毒细胞间传播的关键。这些数据对HSV 1上皮扩散和发病机制具有重要意义。
Herpes simplex virus 1 (HSV1) infects the stratified epithelia of the epidermis, oral or genital mucosa, where the main cell type is the keratinocyte. Here we have used nTERT human keratinocytes to generate a CRISPR-Cas9 knockout (KO) of the primary candidate HSV1 receptor, nectin1, resulting in a cell line that is refractory to HSV1 entry. Nonetheless, a small population of KO cells was able to support infection which was not blocked by a nectin1 antibody and hence was not a consequence of residual nectin1 expression. Strikingly at later times, the population of cells originally resistant to HSV1 infection had also become infected. Appearance of this later population was blocked by inhibition of virus genome replication, or infection with a ΔUL34 virus defective in capsid export to the cytoplasm. Moreover, newly formed GFP-tagged capsids were detected in cells surrounding the initial infected cell, suggesting that virus was spreading following replication in the original susceptible cells. Additional siRNA depletion of the second major HSV1 receptor HVEM, or PTP1B, a cellular factor shown elsewhere to be involved in cell-to-cell transmission, had no effect on virus spread in the absence of nectin1. Neutralizing human serum also failed to block virus transmission in nectin1 KO cells, which was dependent on the receptor binding protein glycoprotein D and the cell-to-cell spread glycoproteins gI and gE, indicating that virus was spreading by direct cell-to-cell transmission. In line with these results, both HSV1 and HSV2 formed plaques on nectin1 KO cells, albeit at a reduced titre, confirming that once the original cell population was infected, the virus could spread into all other cells in the monolayer. We conclude that although nectin1 is required for extracellular entry in to the majority of human keratinocytes, it is dispensable for direct cell-to-cell transmission. Herpes simplex virus 1 (HSV1) infects the epithelia of the epidermis, oral or genital mucosa to cause cold sores, genital herpes, or more serious outcomes such as keratitis and neonatal herpes. Like many viruses, HSV1 can spread through the extracellular environment or by direct cell-to-cell transmission, with the latter mechanism being important for avoiding antibody responses in the host. Here we have studied HSV1 entry and transmission in the human keratinocyte, the main cell type in the target epithelia, by generating a CRISPR-Cas9 knockout of the primary candidate virus receptor, nectin1. While HSV1 was unable to infect the majority of nectin1 knockout keratinocytes, a small population of these nectin1 KO cells remained susceptible to virus entry, and once infected, the virus was able to spread into the rest of the monolayer. This spread continued in the presence of neutralising serum which blocks extracellular virus, and required glycoprotein D, the main virus receptor-binding protein, and glycoproteins gE and gI which are known to be involved in cell-to-cell spread. Hence, while nectin1 is required for virus entry into the majority of human keratinocyte cells, it is dispensable for cell-to-cell transmission of the virus. These data have implications for the mechanism of HSV1 epithelial spread and pathogenesis.
登录
查看更多内容
影响因子：
3.8
作者：
Jones, Juliet;Depledge, Daniel Pearce;Elliott, Gillian
通讯作者：
Elliott, Gillian
影响因子：
5.4
作者：
DINGWELL, KS;DOERING, LC;JOHNSON, DC
通讯作者：
JOHNSON, DC
DOI：
10.5423/ppj.rw.09.2016.0198
发表时间：
2017-06
期刊：
The plant pathology journal
影响因子：
--
作者：
Hong JS;Ju HJ
通讯作者：
Ju HJ
影响因子：
5.4
作者：
Lawrence, DMP;Patterson, CE;Rall, GF
通讯作者：
Rall, GF
影响因子：
5.4
作者：
Huber, MT;Wisner, TW;Johnson, DC
通讯作者：
Johnson, DC