Cytomegalovirus pentamer dependent endocytic cellular infection utilizes redundant entry receptors in the guinea pig model

Abstract The guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV (cCMV). GPCMV cell entry is dictated by specific viral gH/gL-based complexes: gH/gL/gO trimer (direct entry); pentamer complex, PC (endocytic entry). GPCMV gB as the fusogenic protein is also essential for all entry pathways. PDGFRA and NRP2 are receptors for direct and endocytic virus entry respectively based on strain 13 animal fibroblast ATCC cell line studies. All non-fibroblast guinea pig cell lines are derived from Dunkin-Hartley animals, the focus of cCMV studies. GPCMV infection of Dunkin-Hartley embryo fibroblasts (GEFh) and epithelial cells were compared. Knockout of PDGFRA on GEFh cells prevented GPCMV(PC-) direct entry but not endocytic GPCMV(PC+) infection, demonstrating both pathways of infection. Fibroblast generated virus poorly infected epithelial cells compared to epithelial virus stock, which exhibited full tropism to all cell types. Guinea pig epithelial cell lines are NRP2-positive and PDGFRA-negative requiring PC for GPCMV infection. Epithelial and GEFh cells, but not strain 13 fibroblasts, additionally expressed ThBD. In immunoprecipitation assays, PC and ThBD interacted unlike CD46 receptor candidate targeting gH/gL. Double-knockout of NRP2/ThBD in epithelial cells impaired infection unlike single knockouts. Individual ectopic species-specific receptor expression restored infection on double-knockout epithelial (NRP2/ThBD) and fibroblast (PDGFRA/NRP2) cell lines. Knockout of NRP2/ThBD receptors did not enhance GPCMV neutralization by gB antibodies on PDGFRA-negative cells demonstrating a limitation of a gB vaccine strategy. Overall, GPCMV and HCMV similarity for receptors and cell tropism maintains the translational importance of this model. 3. Impact Statement A CMV vaccine is a high priority as congenital cytomegalovirus (cCMV) is a leading cause of hearing loss and cognitive impairment in newborns. CMV species-specificity requires animal studies to utilize species-specific virus. The guinea pig is the only small animal model for cCMV and guinea pig cytomegalovirus (GPCMV) encodes functional HCMV homolog glycoprotein complexes for cell entry including a gH/gL-based PC for endocytic cell entry. The viral fusogenic gB glycoprotein is required for GPCMV infection of all cell types but antibody based gB vaccines fail to fully protect against endocytic infection. The PC is potentially an important vaccine antibody target, but PC-based cell entry is only partially understood and poorly characterized for GPCMV. Identifying GPCMV cell entry receptors is critical to the understanding of virus tropism and disease especially since the PC is necessary for cCMV. Correlation with HCMV improves translational impact of guinea pig based cCMV intervention strategies.