Growth defect of domain III glycoprotein B mutants of human cytomegalovirus reverted by compensatory mutations co-localizing in post-fusion conformation

Cell entry is a crucial step for a virus to infect a host cell. Human cytomegalovirus (HCMV) utilizes the glycoprotein B (gB) to fuse the viral and host cell membrane upon receptor binding of gH/gL-containing complexes. Fusion is mediated by major conformational changes of gB from a metastable pre-fusion to a stable post-fusion whereby the central trimeric coiled-coils, formed by domain (D) III α helices, remain structurally nearly unchanged. To better understand the role of the stable core, we individually introduced three potentially helix-breaking and one disulfide bond-breaking mutation in the DIII α3 to alter the gB stability, and studied different aspects of the viral behavior upon long-term culturing. Two of the three helix-breaking mutations were lethal for the virus in either fibroblasts or epithelial cells and the third substitution led from mild to severe effects on viral replication and infection efficiency. gB_Y494P and gB_I495P suggest that the pre-fusion conformation was stabilized and the fusion process inhibited, gB_G493P on the other hand displayed a delayed replication increase and spread, more pronounced in epithelial cells, hinting at an impaired fusion. Interestingely, the disulfide bond-breaker mutation, gB_C507S, performed strikingly different in the two cell types – lethal in epithelial cells and an atypical phenotype in fibroblasts, respectively. Replication curve analyses paired with the infection efficiency and the spread morphology suggest a dysregulated fusion process which could be reverted by second-site mutations mapping predominantly to gB DV. This underlines the functional importance of a stable core for a well-regulated DV rearrangement during fusion. Importance Human cytomegalovirus (HCMV) can establish a lifelong infection. In most people, the infection follows an asymptomatic course, however it is a major cause of morbidity and mortality in immunocompromised patients or neonates. HCMV has a very broad cell tropism, ranging from fibroblasts to epi- and endothelial cells. It uses different entry pathways utilizing the core fusion machinery consisting of glycoprotein complexes gH/gL and gB. The fusion protein gB undergoes severe rearrangements from a metastable pre-fusion to a stable post-fusion. Here, we were able to characterize the viral behavior after the introduction of four single point mutations in gBs central core. These led to various cell type-specific atypical phenotypes and the emergence of compensatory mutations, demonstrating an important interaction between domains III and V. We provide a new basis for the delevopment of recombinant stable pre-fusion gB which can further serve as a tool for the drug and vaccine development.