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Glycoprotein B (gB) plays an important role in alphaherpesvirus cellular entry and acts in concert with gD and the gH/gL complex. To evaluate whether functional differences exist between gB1 and gB4, the corresponding genes were exchanged between the two viruses. The gB4-containing-EHV-1 (EHV-1_gB4) recombinant virus was analyzed for growth in culture, cell tropism, and cell entry revealing no significant differences when compared to parental virus. We also disrupted a potential integrin-binding motif, which did not affect the function of gB in culture. In contrast, a significant reduction of plaque sizes and growth kinetics of gB1-containing-EHV-4 (EHV-4_gB1) was evident when compared to parental EHV-4 and revertant viruses. The reduction in virus growth may be attributable to the loss of functional interaction between gB and the other envelope proteins involved in virus entry, including gD and gH/gL. Alternatively, gB4 might have an additional function, required for EHV-4 replication, which is not fulfilled by gB1. The significant attenuation of virus growth in the case of EHV-4_gB1 may be attributable to the loss of functional interaction between gB and other proteins involved in virus entry. One possible cause for the loss of function/interaction may be structural differences between gB1 and gB4. Our rationale was that this structural difference could be caused by the different locations of the furin cleavage site within the respective gBs.
To investigate the contribution of furin-mediated gB cleavage to EHV-1 and EHV-4 growth, the cleavage sites were mutated. While mitigating furin recognition motif did not affect in vitro growth of EHV-1, reconstitution of the mutant EHV-4 was not successful, which confirms previous results indicating different properties of gB4 when compared to gB1. Western blot and mass spectrometry analysis of mutated gB1 suggest that mutating the furin cleavage site indeed prevented gB cleavage and resulted in a partial misfolding. In addition, a novel signal peptide cleavage site was identified for gB1 between residues 98 and 99, which is different from that previously published. We conclude that furin cleavage is solely responsible for gB cleavage and involved in the protein folding. After addressing the structural and functional aspects of gB on a protein level, we looked at how this translated to the function of gB during a key step of EHV-1 pathogenesis, namely the viral transfer between infected PBMC to EC.
Infected peripheral blood mononuclear cells (PBMC) effectively transport equine herpesvirus type 1 (EHV-1), but not EHV-4, to endothelial cells (EC) lining the blood vessels of the pregnant uterus or central nervous system, a process that can result in abortion or myeloencephalopathy. We examined, using a dynamic in vitro model, the differences between EHV-1 and EHV-4 infection of PBMC and PBMC-EC interactions. Infection assays revealed that EHV-1 infected B-lymphocytes and monocytes more efficiently than EHV-4. In order to evaluate viral transfer between infected PBMC and EC, co-cultivation assays were performed. Only EHV-1 was transferred from PBMC to EC and viral glycoprotein B (gB) was shown to be mainly responsible for this form of cell-to-cell transfer. For addressing the more dynamic aspects of PBMC-EC interaction, infected PBMC were perfused through a flow channel containing EC in the presence of neutralizing antibodies. By simulating capillary blood flow and analyzing the behavior of infected PBMC through live fluorescence imaging and automated cell tracking, we observed that EHV-1 was able to maintain tethering and rolling of infected PBMC on EC more effectively than EHV-4. Deletion of US3 reduced the ability of infected PBMC to tether and roll compared to parental virus, which resulted in a significant reduction in virus transfer from PBMC to EC. Taken together, we conclude that systemic spread and EC infection of EHV-1, but not EHV-4, is caused by its ability to infect and/or reprogram mononuclear cells with respect to their tethering and rolling behavior on EC and consequent virus transfer.