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

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 PDGFRα 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 the formation of gH/gL/gO or binding to PDGFRa dramatically reduced the cell–cell fusion rate, suggesting that gL plays a critical role in linking the gH/gL/gO-PDGFRa receptor binding to activation of gB. Several neutralizing human monoclonal antibodies were found to potently block gH/gL/gO-PDGFRa-regulated cell–cell fusion, suggesting this mechanism as a therapeutic target. IMPORTANCE Development of vaccines and therapeutics targeting the fusion apparatus of human cytomegalovirus (HCMV) has been limited by the lack of an in vitro 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, and this is specifically vulnerable to inhibition by neutralizing antibodies. Moreover, the measurement of fusion kinetics allows for mutational studies of the fusion mechanism, assessing the influence of genetic diversity among the viral glycoproteins and studying the mechanism of neutralizing antibodies.