LAMP3/CD63 Expression in Early and Late Endosomes in Human Vaginal Epithelial Cells Is Associated with Enhancement of HSV-2 Infection

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. IMPORTANCE 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.

Keywords: CD63; HSV-2; LAMP3; endosome; epithelial cells; viral replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Line
  • Endosomes / metabolism
  • Epithelial Cells
  • Female
  • Herpes Simplex* / metabolism
  • Herpesvirus 2, Human* / genetics
  • Humans
  • Lysosome-Associated Membrane Glycoproteins / genetics
  • Lysosome-Associated Membrane Glycoproteins / metabolism
  • Neoplasm Proteins / metabolism
  • Tetraspanin 30 / genetics
  • Tetraspanin 30 / metabolism
  • Virus Replication


  • LAMP3 protein, human
  • Neoplasm Proteins
  • Lysosome-Associated Membrane Glycoproteins
  • CD63 protein, human
  • Tetraspanin 30