Merkel cell polyomavirus exploits extracellular vesicles for skin infection and host immune evasion through activated Wnt signaling.

Research on small DNA tumor viruses has been limited by the lack of advanced virus culture systems that support robust viral infection. Thus, studies involving Merkel cell polyomavirus (MCPyV), a human oncogenic polyomavirus, primarily rely on model systems that might not fully reflect the native MCPyV skin infection. To decipher the mechanisms of natural human polyomavirus skin infection, we developed a 3D spheroid system for producing MCPyV virions derived from primary human dermal fibroblasts. The 3D spheroid microenvironment enhanced viral genome replication and genome maintenance, enabling the production and secretion of MCPyV virions that were associated with infectious extracellular vesicles (MCPyV-EVs) into the culture medium. EV-mediated MCPyV infection was moderately susceptible to antibody-mediated neutralization, implying the importance of EV-mediated MCPyV infection in host immune evasion. Proteomic analysis revealed a significant decrease in antiviral interferon-stimulated genes (ISGs) within MCPyV-EVs, indicating that MCPyV utilizes EVs as a means to enhance susceptibility of uninfected cells for viral transmission and infection. RNA-Seq gene expression analysis of primary human dermal fibroblasts with MCPyV infection suggests that the 3D microenvironment could replicate Wnt-mediated epithelial remodeling in the skin. Our results imply that Wnt-driven wound healing processes, accelerated by skin damage, UV radiation, and aging, regulate MCPyV viral replication and productive infection, which may promote the development of MCPyV-associated MCC.