Human Cytomegalovirus Infection of Primary Human Oral Keratinocytes Induces Intermediate Keratinocyte Differentiation and an Altered Innate Immune Response

Abstract

The oral mucosa is central for human cytomegalovirus (HCMV) acquisition. However, the initial infection events in the oral cavity, including how HCMV modulates oral cells to establish infection and spread, are supported by limited experimental data and therefore remain poorly understood. We investigated HCMV infection in cultures of primary human oral keratinocytes (HOKs) as a model to study HCMV infection in the oral mucosa using the TB40E-Gfp and MOLD strains of HCMV. Both viral strains successfully infected and replicated in HOKs as demonstrated by the appearance of cytopathic effects, full expression of viral transcripts, and the shedding of infectious virus. To investigate how HCMV modulates the transcriptome of primary HOKs, we performed single cell RNA sequencing (scRNA-seq) which revealed 11 subtypes of cells. Furthermore, infection resulted in significant changes in host gene expression. HCMV upregulated genes involved in immune responses, cell cycle regulation, cancer-related pathways, neuronal and synaptic functions, metabolism, stress responses, molecular chaperones, and vesicular trafficking which might be critical for viral protein expression and assembly. HCMV also increased the expression of genes related to the extracellular matrix, cell adhesion, microtubules, signal transduction, kinases, and transcriptional regulation. Conversely, HCMV downregulated genes associated with inflammation and immune responses, cell cycle control and apoptosis, cell adhesion and migration, as well as signaling pathways, growth factors, ion channels, transporters, and transcription factors when compared to uninfected cells. These findings suggest that HCMV modulates a wide range of host cellular pathways to create a favorable environment for its replication. HCMV also induced changes in HOK differentiation genes downregulating basal state genes and upregulating intermediate genes and select terminal differentiation genes, indicating HCMV might be driving HOK differentiation towards a dysregulated intermediate phenotype while inhibiting their terminal differentiation to maintain a cell state capable of sustaining viral replication. Using SciViewer, our recently developed scRNA-seq data visualization tool, we determined that as the infection progressed from low to high viral transcript accumulation, HCMV infection upregulated E2F targeted genes, some antiviral and immune responses as well as autophagy and cellular stress responses, while downregulating interferon-stimulated genes, immune and antiviral response genes, pyroptosis, inflammatory cell death, and membrane remodeling and viral entry genes. Overall, our study shows that primary human HOKs can be used as a model to study HCMV infection in the oral mucosa and that HCMV drives dysregulated intermediate oral keratinocyte differentiation and altered immune responses which likely facilitate viral replication in the oral mucosa and spread into the rest of the host.