Mutational dissection of HCMV gB and gH cytoplasmic tails highlights conserved and divergent features of fusion regulation

In herpesviruses, the fusogenic activity of the conserved glycoproteins gB and gH is regulated by their cytoplasmic (or intraviral) tails. However, prior to this work, their regulatory mechanisms had only been investigated in <i>Alpha-</i> and <i>Gamma-</i> but not in <i>Betaherpesvirinae</i>. Here, we developed a plasmid-transfection-based split-luciferase assay as a quantitative platform for measuring cell-cell fusion mediated by glycoproteins from human cytomegalovirus (HCMV), a member of <i>Betaherpesvirinae</i>. Using this assay, we confirmed that glycoproteins gB, gH, gL, and gO, along with the PDGFRα receptor, are necessary and sufficient for efficient cell-cell fusion in two distinct HCMV strains, TR and AD169. To investigate the roles of the cytoplasmic tails of HCMV TR gB and gH in membrane fusion, we generated a series of truncation and point mutants analogous to those that have hyper- or hypo-fusogenic phenotypes in herpes simplex virus 1 (HSV-1). We found that, similarly to HSV-1, the C-terminal amphipathic helix in the HCMV gB cytoplasmic tail restricts fusion, whereas the entire HCMV gH cytoplasmic tail is required for fusion activation. However, the structure of the HCMV gB cytoplasmic tail and its interactions with the cytoplasmic tail of gH might be different from HSV-1. We hypothesize that while in HCMV-as in HSV-1-the cytoplasmic tails of HCMV gB and gH function as an inhibitory clamp and an activating wedge, their structures and interactions differ from HSV-1, implicating potential mechanistic differences in fusion regulation between herpesvirus subfamilies.IMPORTANCEHerpesviruses promote membrane fusion during infection using a complex multi-component membrane fusion machinery. Proper spatiotemporal deployment of such machinery is subject to precise regulatory inputs. One of these is the regulation of the fusogenic activity of the conserved herpesviral glycoproteins gB and gH by their cytoplasmic (or intraviral) domains. This regulatory mechanism has been investigated in <i>Alpha-</i> and <i>Gamma-</i> but not yet in <i>Betaherpesvirinae</i>. Here, we combined mutagenesis targeting the cytoplasmic tails of HCMV gB and gH with a plasmid-transfection-based split-luciferase assay for measuring cell-cell fusion, which we developed here. By testing a panel of truncation mutants, we showed that inhibitory and activating regulatory regions in gB and gH, respectively, are conserved in HCMV. However, none of the single point mutants of gB had expected phenotypes, suggesting that the cytoplasmic tails of HCMV gB and gH have distinct structures and interactions. This study introduces a robust and sensitive <i>in vitro</i> cell-cell fusion assay for probing HCMV fusion mechanism and shows that despite sequence conservation, the cytoplasmic domains of gB and gH may regulate fusion by distinct mechanisms. This knowledge may inform future efforts in rational vaccine design and antiviral development.