Guinea Pig Cytomegalovirus Entry and Post-Entry Determinants of Viral Tropism

Abstract

Congenital cytomegalovirus (cCMV) is a leading cause of developmental disabilities in newborns. The guinea pig, with guinea pig CMV (GPCMV) infection, is the only small animal model for cCMV. GPCMV encodes functional homologs to human CMV (HCMV) glycoprotein complexes, including gB, gH/gL/gO triplex, and gH-based pentamer complex (PC), enabling two pathways of cell entry: direct fusion (PC-independent) and endocytosis (PC-dependent). Guinea pig platelet-derived growth factor receptor alpha (PDGFRA) and guinea pig epidermal growth factor receptor (EGFR) were potential candidate cell receptors for GPCMV cell entry. PDGFRA was present only in fibroblast cells, which support PC-independent cell entry. A CRISPR/Cas9 strategy identified PDGFRA as the receptor for PC-independent fibroblast infection. PDGFRA was absent in renal epithelial, trophoblast, and amniotic sac epithelial cells, which require the PC for infection, unless PDGFRA was ectopically expressed. Immunoprecipitation and cell-cell fusion assays demonstrated direct interaction of gH/gL/gO trimer complex with PDGFRA, which was dependent upon gO but did not require gB. Subsequent studies found that gO N-terminal N-glycosylation was important for this interaction but not trimer formation. Evaluation of GPCMV viral particles demonstrated that gO is present on the virion, and gB is present in trimeric form. In HCMV and GPCMV, gB, gH/gL, and PC are important neutralizing antibody targets, but the immunogenicity of gO alone has not previously been defined. A defective recombinant adenovirus encoding gO (AdgO) was evaluated as a potential vaccine candidate, producing neutralizing antibodies which enhanced gB or gH/gL neutralizing activity on GPCMV infection. The functionality of GPCMV tegument protein GP83, the presumed homolog of HCMV pp65, was also evaluated to define a role in cells post-entry. A PC+, GP83 null deletion mutant GPCMV (GP83dPC+) was attenuated in animals and had normal growth kinetics on fibroblasts but was highly impaired for infection of epithelial and trophoblast cells. GP83dPC+ virus was highly sensitive to IFN-I, suggesting that GP83 had an innate immune evasion function. GP83 interacted with guinea pig IFI16 and cGAS, indicating a role in cGAS/STING pathway evasion. Ectopically expressed GP83 in trophoblasts rescued GP83dPC+ infection. Additionally, mutant virus growth was restored in epithelial cells by expression of Bovine Viral Diarrhea Virus (BVDV) NPRO protein targeting IRF3 as a part of the cGAS/STING pathway. Alternatively, GP83dPC+ virus growth was restored by ectopic expression of PDGFRA, which enabled entry via direct fusion pathway. Together, these results demonstrate that GP83 is a functional pp65 homolog and reveal a previously unappreciated relationship between the endocytic CMV entry pathway and post-entry innate immune activation or evasion.