Human cytomegalovirus gH/gL/gO binding to PDGFRα provides a regulatory signal activating the fusion protein gB that can be blocked by neutralizing antibodies.

Unlabelled: Herpesviruses require membrane fusion for entry and spread, a process facilitated by the fusion glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL can be modified by the accessory protein gO, or the set of proteins UL128, UL130 and UL131. While the binding of the gH/gL/gO and gH/gL/UL128-131 complexes to cellular receptors including PDFGRα and NRP2 has been well-characterized structurally, the specific role of receptor engagements by the gH/gL/gO and gH/gL/UL128-131 in regulation of fusion has remained unclear. We describe a cell-cell fusion assay that can quantitatively measure fusion on a timescale of minutes and demonstrate that binding of gH/gL/gO to PDGFRα dramatically enhances gB-mediated cell-cell fusion. In contrast, gH/gL/pUL128-131-regulated fusion is significantly slower and gH/gL alone cannot promote gB fusion activity within this timescale. The genetic diversity of gO influenced the observed cell-cell fusion rates, correlating with previously reported effects on HCMV infectivity. Mutations in gL that had no effect on formation of gH/gL/gO or binding to PDGFRα dramatically reduced the cell-cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFRα receptor-binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFRα regulated cell-cell fusion, suggesting this mechanism as a therapeutic target.

Significance: Development of vaccines and therapeutics targeting the fusion apparatus of HCMV has been limited by the lack an cell-cell fusion assay that faithfully models the receptor-dependent fusion characteristic of HCMV entry. The cell-cell fusion assay described here demonstrated that the binding of gH/gL/gO to its receptor, PDGFRα serves to regulate the activity of the fusion protein gB. Moreover, this regulatory mechanism is specifically vulnerable to inhibition by neutralizing antibodies. The cell-cell fusion assay described here provides a new tool to characterize neutralizing mAbs as therapeutic agents.