Cell-to-cell transmission of HSV-1 in differentiated keratinocytes promotes multinucleated giant cell formation

Abstract

Background: Herpes simplex virus (HSV) infection in the skin causes small grouped vesicles characterized by acantholytic cells and multinucleated giant cells (MGCs). Although viral factors have been studied as fusion proteins, little is known how the differentiation status of keratinocytes is involved in the formation of MGCs by HSV-1 infection.

Objective: As the human epidermis is composed of several layers of keratinocytes that undergo terminal differentiation, we aimed to elucidate whether the differentiation status of keratinocytes affects viral entry, propagation, cell-to-cell transmission of HSV-1, and MGC formation.

Methods: HaCaT cells and normal human epidermal keratinocytes were cultured in either low- or high-Ca²⁺ medium. After HSV-1 infection, cellular morphology, viral propagation, and expression of cytoskeletal and intercellular adhesion molecules were examined sequentially. Viral entry, replication, and expression of HSV receptors were analyzed. Cell-to-cell transmission and fusion after HSV-1 infection was evaluated using the Cell Tracker™ Red CMTPX dye system.

Results: Keratinocytes in high-Ca²⁺ medium formed MGCs, but those in low-Ca²⁺ medium formed single nuclear round cells in response to HSV-1 infection. HSV-1 entered the keratinocytes more effectively in low-Ca²⁺ than in high-Ca²⁺ medium, although transcripts of HSV receptors were comparable in both conditions. HSV-1 could replicate more efficiently in high-Ca²⁺ than in low-Ca²⁺ medium. A cell-to-cell fusion assay showed that HSV-1-infected and adjacent-uninfected keratinocytes were involved in MGCs in high-Ca²⁺ but not in low-Ca²⁺ medium.

Conclusion: Differentiated keratinocytes promote MGC formation by cell-to-cell fusion with resolution of cell membrane and cell-to-cell transmission of HSV-1 from infected keratinocytes to neighboring uninfected keratinocytes.

Keywords: Cell-to-cell fusion; Cell-to-cell transmission; Differentiation; Herpes simplex virus; Keratinocyte; Multinucleated giant cell.