Functional dissection of HCMV gB's autonomous fusion activity provides insights into how polymorphisms in clinical...

Glycoprotein B (gB) serves as the viral fusogen facilitating entry and cell-to-cell spread of human cytomegalovirus (HCMV). Fusion is initiated when gH/gL-containing complexes bind to their receptors, leading to gB's transition from a metastable prefusion to a stable postfusion conformation. We recently demonstrated that substituting or truncating the carboxy-terminal domain of gB renders it intrinsically fusion-competent; however, the precise mechanisms of gB ectodomain reorganization-likely shared by both gH/gL-dependent and -independent fusion processes-remain unclear. In this study, a dual split protein (DSP)-based cell-cell fusion assay was used to assess the gH/gL-independent fusion activities across five major HCMV gB-genotypes (gB1-gB5). Our results show that gB of VR1814 (gB3) exhibits superior autonomous fusion activity compared to TB40/E (gB1) and AD169 (gB2), while gB from C194A (gB4) and UKNEQAS1 (gB5) were fusion-incompetent. Through domain-swapping and site-directed mutagenesis, we identified substitutions that enhance or abrogate fusion and mapped them onto the available pre- and post-fusion structures of gB. Our analysis uncovered a complex network of intra- and inter-domain interactions involving structural domains I, IV, and V of gB, which are crucial for fusion regulation. Functional characterization of gB polymorphisms from clinical HCMV isolates demonstrated opposing impacts, with some variants remaining inactive, while others conferred fusion competence. Collectively, our findings mechanistically elucidate how specific substitutions within the gB ectodomain of particular HCMV strains markedly alter gH/gL-independent fusogenicity. These insights have profound implications for developing diagnostic tools to detect functionally relevant gB polymorphisms and for optimizing vaccine design.IMPORTANCEHuman cytomegalovirus (HCMV) establishes lifelong, predominantly asymptomatic infections but remains a major cause of morbidity and mortality in immunocompromised individuals and newborns. This study elucidates the mechanisms driving the structural reorganization of the gB ectodomain, which likely regulate both gH/gL-independent and -dependent fusion activities across gB-genotypes gB1-gB5. Through a combinatorial in silico and functional approach, we determined that C-terminally truncated gB is fusion-incompetent when expressed alone. However, clinical isolates evolved diverse fusion-enhancing substitutions, which either destabilize the prefusion conformation or inhibit the formation of the postfusion conformation of gB, thus rendering the protein fusion-competent. The presence of these substitutions in isolates from diverse body compartments suggests a possible implication for HCMV pathogenicity and transmission. Assessing the gH/gL-independent cell-to-cell fusion activity of gB provides a valuable method for identifying functionally relevant polymorphisms, laying the foundation for further viral infection studies and guiding vaccine development.