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
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在缺乏主要进入受体 nectin1 的情况下，HSV1 在人角质形成细胞中的细胞间传播
DOI：
10.1101/2021.05.13.443971
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发表时间：
2021
期刊：
影响因子：
--
通讯作者：
Kite J
中科院分区：
文献类型：
--
作者：
Kite J
单纯疱疹病毒 1 (HSV1) 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 人类角质形成细胞对主要候选 HSV1 受体 nectin1 进行 CRISPR-Cas9 敲除 (KO)，从而产生 HSV1 难以进入的细胞系。尽管如此，一小群 KO 细胞能够支持感染，这种感染未被 nectin1 抗体阻断，因此不是残留 nectin1 表达的结果。令人惊讶的是，后来，最初对 HSV1 感染具有抵抗力的细胞群也被感染了。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 ΔUL34 病毒，可以阻止该后来群体的出现。此外，在初始感染细胞周围的细胞中检测到新形成的带有 GFP 标签的衣壳，表明病毒在原始易感细胞中复制后正在传播。额外的 siRNA 敲除第二个主要 HSV1 受体 HVEM（或 PTP1B）（一种在其他地方显示参与细胞间传播的细胞因子）在缺乏 nectin1 的情况下对病毒传播没有影响。中和人血清也未能阻止病毒在nectin1 KO细胞中的传播，这种传播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，表明病毒是通过直接细胞间传播传播的。与这些结果一致，HSV1和HSV2都在nectin1 KO细胞上形成噬斑，尽管滴度降低，这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。我们得出的结论是，虽然 nectin1 是细胞外进入大多数人类角质形成细胞所必需的，但它对于细胞间的直接传播是可有可无的。
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.
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