Department of Medicine, McMaster Universitygrid.25073.33, Michael G. DeGroote Center for Learning and Discovery, Hamilton, Ontario, Canada.
McMaster Immunology Research Center, McMaster Universitygrid.25073.33, Michael G. DeGroote Center for Learning and Discovery, Hamilton, Ontario, Canada.
J Virol. 2022 Dec 14;96(23):e0155322. doi: 10.1128/jvi.01553-22. Epub 2022 Nov 9.
Herpes simplex virus 2 (HSV-2) is a lifelong sexually transmitted virus that disproportionately infects women through heterosexual transmission in the vaginal tract. The vaginal epithelium is known to be highly susceptible to HSV-2 infection; however, the cellular mechanism of HSV-2 uptake and replication in vaginal epithelium has not been extensively studied. Previously, we observed that lysosomal-associated membrane protein-3 (LAMP3/CD63) was among the highly upregulated genes during HSV-2 infection of human vaginal epithelial cell line VK2, leading us to posit that LAMP3/CD63 may play a role in HSV-2 infection. Consequently, we generated two gene-altered VK2-derived cell lines, a LAMP3-overexpressed (OE) line and a LAMP3 knockout (KO) line. The wild-type VK2 and the LAMP3 OE and KO cell lines were grown in air-liquid interface (ALI) cultures for 7 days and infected with HSV-2. Twenty-four hours postinfection, LAMP3 OE cells produced and released significantly higher numbers of HSV-2 virions than wild-type VK2 cells, while virus production was greatly attenuated in LAMP3 KO cells, indicating a functional association between LAMP3/CD63 expression and HSV-2 replication. Fluorescence microscopy of HSV-2-infected cells revealed that HSV-2 colocalized with LAMP3 in both early endosomes and lysosomal compartments. In addition, blocking endosomal maturation or late endosomal/lysosomal fusion using specific inhibitors resulted in reduced HSV-2 replication in VK2 cells. Similarly, LAMP3 KO cells exhibited very low viral entry and association with endosomes, while LAMP3 OE cells demonstrated large amounts of virus that colocalized with LAMP3/CD63 in endosomes and lysosomes. Collectively, these results showed that HSV-2 is taken up by human vaginal epithelial cells through an endosomal-lysosomal pathway in association with LAMP3, which plays a crucial role in the enhancement of HSV-2 replication. These findings provide the basis for the future design of antiviral agents for prophylactic measures against HSV-2 infection.
单纯疱疹病毒 2 (HSV-2) 是一种终生性的性传播病毒，通过异性传播在阴道内不成比例地感染女性。阴道上皮易受 HSV-2 感染；然而，HSV-2 在阴道上皮细胞中的摄取和复制的细胞机制尚未得到广泛研究。此前，我们观察到溶酶体相关膜蛋白-3 (LAMP3/CD63) 是 HSV-2 感染人阴道上皮细胞系 VK2 时高度上调的基因之一，这使我们推测 LAMP3/CD63 可能在 HSV-2 感染中发挥作用。因此，我们生成了两种基因改变的 VK2 衍生细胞系，一种 LAMP3 过表达 (OE) 系和一种 LAMP3 敲除 (KO) 系。野生型 VK2 和 LAMP3 OE 和 KO 细胞系在气液界面 (ALI) 培养物中培养 7 天，然后感染 HSV-2。感染后 24 小时，LAMP3 OE 细胞产生和释放的 HSV-2 病毒粒子数量明显高于野生型 VK2 细胞，而 LAMP3 KO 细胞中的病毒产生大大减弱，表明 LAMP3/CD63 表达与 HSV-2 复制之间存在功能关联。HSV-2 感染细胞的荧光显微镜观察显示，HSV-2 在早期内体和溶酶体区室中与 LAMP3 共定位。此外，使用特异性抑制剂阻断内体成熟或晚期内体/溶酶体融合会导致 VK2 细胞中的 HSV-2 复制减少。同样，LAMP3 KO 细胞表现出极低的病毒进入和与内体的关联，而 LAMP3 OE 细胞则表现出大量与 LAMP3/CD63 在内体和溶酶体中共定位的病毒。总之，这些结果表明，HSV-2 通过与 LAMP3 相关的内体-溶酶体途径被人阴道上皮细胞摄取，LAMP3 在增强 HSV-2 复制中起关键作用。这些发现为针对 HSV-2 感染的预防性措施设计抗病毒药物提供了依据。
