Selective knockout of key CMV receptors in fetal cells blocks direct and endocytic pathways of entry in the guinea pig

Cytomegalovirus is a leading cause of congenital disease and a vaccine is a high priority. Guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV (cCMV). GPCMV encodes functionally essential viral entry glycoprotein complexes similar to HCMV, which are neutralizing antibody targets. As with HCMV, GPCMV has two pathways of cell entry (direct and endocytic). Common to both pathways and essential for infection is the fusogenic viral glycoprotein gB. Additional gH/gL-based complexes are necessary for receptor interaction and cell entry: gH/gL/gO trimer (direct); pentamer complex, PC (endocytic). Direct cell entry requires host PDGFRA receptor and viral trimer. An endocytic PC receptor has not been identified for GPCMV or any animal CMV. GPCMV endocytic entry was blocked by acidic flux inhibition but not direct cell entry, requiring knockout of PDGFRA. We hypothesized that cellular knockout of GPCMV direct and endocytic receptors would completely block infection. Two PC receptor candidates, guinea pig NRP2 and CD147, present on all established guinea pig cell lines were evaluated. Results demonstrated that NRP2 interacted with PC unlike CD147 in immunoprecipitation assays. Double knockout of PDGFRA and NRP2 completely blocked GPCMV but had no impact on control HSV-1 infection. In contrast, CD147/PDGFRA double knockout had limited inhibition of GPCMV and no impact on HSV-1. Ectopic expression of cell receptors restored infection to normal levels on knockout cell lines. Overall, results demonstrate GPCMV conservation with HCMV for key receptors and cell entry pathways enhancing the translational importance of this model. ImportanceCongenital CMV is a leading cause of hearing loss and cognitive impairment in newborns and a vaccine is a high priority. Species-specificity of HCMV requires animal model studies to utilize species-specific virus. The guinea is the only small animal model for cCMV and GPCMV encodes functional HCMV homolog glycoprotein complexes for cell entry via direct or endocytic pathways. The gB glycoprotein is required for infection of all cell types but a gB vaccine fails to fully protect against cCMV. GPCMV encodes a functional PC required for infection of different cell types via endocytic pathway. The PC has emerged as 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 in this model. Correlation with HCMV improves translational impact of a GPCMV vaccine and antiviral cCMV intervention.