The cytoplasmic tail of ovine herpesvirus 2 glycoprotein B affects cell surface expression and is required for membrane fusion

Authors: LYNCH, COLLEEN - Washington State University; HERNDON, MARIA - Washington State University; HULL, MCKENNA - Washington State University; MORÉ, DANIELA - Washington State University; Baker, Katherine; Cunha, Cristina; NICOLA, ANTHONY - Washington State University

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/9/2025
Publication Date: 7/16/2025
Citation: Lynch, C.M., Herndon, M.K., Hull, M.A., Moré, D.D., Baker, K.N., Cunha, C.W., Nicola, A.V. 2025. The cytoplasmic tail of ovine herpesvirus 2 glycoprotein B affects cell surface expression and is required for membrane fusion. Viruses. 17(7). Article 994. https://doi.org/10.3390/v17070994.
DOI: https://doi.org/10.3390/v17070994

Interpretive Summary: Ovine herpesvirus 2 (OvHV-2) is a prevalent cause of malignant catarrhal fever, a typically fatal disease in both wild and domestic ungulates. Currently, there is no vaccine or cure for the disease; therefore, strategies to minimize virus transmission and improve disease control are critical research priorities. The OvHV-2 entry mechanism is a key target for intervention. However, a culture system for propagating OvHV-2 is not available, so details of its entry process remain sparse. In this study, we identified the C-terminal cytoplasmic tail of OvHV-2 gB, the core herpesviral fusion protein, as a functional domain essential for fusion. We also discovered that a mutant retaining this domain, but with the remainder of the cytoplasmic tail removed, exhibits increased protein expression. This mutant proved to be a viable option for a robust cell-cell fusion assay, which can help to elucidate the entry mechanisms of this important pathogen. This study represents the first structure-function analysis of OvHV-2 gB, and the enhancement of laboratory techniques to investigate virus entry marks a significant step toward developing effective strategies to control OvHV-2-induced malignant catarrhal fever.

Technical Abstract: Ovine herpesvirus 2 (OvHV-2) is a cause of the fatal veterinary disease malignant catarrhal fever. Fusion is an essential step in host cell entry of enveloped viruses and is an important target for vaccine development. OvHV-2 cannot be propagated in vitro, so a robust virus-free cell-cell membrane fusion assay is necessary to elucidate its entry mechanism. OvHV-2 cell-cell fusion requires the three conserved herpesviral envelope glycoproteins: gB, gH, and gL. OvHV-2 fusion activity is detectable but low. We hypothesize that enhancing the cell surface expression of gB, the core herpesviral fusogen, will increase cell-cell fusion. The cytoplasmic tail of OvHV-2 gB contains two putative endocytosis motifs. We generated C-terminal truncation mutants of gB and determined their cell surface expression, subcellular distribution, and fusion activity. Two mutants, including one that lacked the entire cytoplasmic tail domain, failed to function in the cell-cell fusion assay, despite wild type levels of surface expression. This suggests that the OvHV-2 gB cytoplasmic tail is critical for fusion. A gB mutant truncated at amino acid 847 showed increased in surface expression relative to wild type and facilitated fusion at a much greater level than wild type. This suggests that the robust fusion activity of gB847 is the result of increased surface expression. gB847 may be used in place of wild type gB in an improved, more robust OvHV-2 fusion assay.