@phdthesis{Vallbracht2018,
  author    = {Melina Vallbracht},
  title     = {Identification of essential, dispensable and modulatory domains in the core herpesvirus membrane fusion components gB and gH},
  journal    = {Identifizierung essentieller und nicht essentieller sowie regulatorischer Dom{\"a}nen in gB und gH, den Hauptkomponenten der herpesviralen Fusionsmaschinerie},
  url       = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-24350},
  pages     = {188},
  year      = {2018},
  abstract  = {Herpesviruses are a fascinating group of enveloped DNA viruses, which rely on membrane fusion for infectious entry and direct cell-to-cell spread. Compared with many other enveloped viruses, they utilize a remarkably complex fusion machinery. Three conserved virion proteins, the bona fide fusion protein gB, and the presumably gB activating gH/gL heterodimer constitute the conserved core fusion machinery and are believed to drive membrane fusion in a cascade-like fashion. Activation of this cascade in most alphaherpesviruses is proposed to be triggered by binding of gD to specific host cell receptors. The molecular details of this fusion process, however, remain largely elusive. Yet, a detailed mechanistic knowledge of this process would be greatly beneficial for the development of efficient countermeasures against a variety of diseases. In this thesis, the functional relevance of individual components of the essential gH/gL complex of the alphaherpesvirus PrV has been assessed by two different approaches: by reversion analysis (paper II) and site-directed mutagenesis (papers III-V). In contrast to other herpesviruses, gL-deleted PrV is able to perform limited cell-to-cell spread, providing the unique opportunity to passage the entry-deficient virus in cell culture to select for PrV revertants capable of infecting cells gL-independently. This approach already resulted in an infectious gL-negative PrV mutant (PrV-ΔgLPass), in which the function of gL was compensated by formation of a gDgH hybrid protein. Here, the requirements for gL-independent infectivity of a second independent revertant (PrV-ΔgLPassB4.1), were analyzed. Sequencing of the genes encoding for gB, gH and gD, revealed mutations in each of them. By means of a robust infection-free, transfection-based cell-cell fusion assay (paper I), we identified two amino acid substitutions in the gL-binding domain I of gHB4.1 (L70P, W103R) as sufficient to compensate for lack of gL. Two mutations in gB (G672R, ΔK883) were found to enhance fusogenicity, probably by lowering the energy, required for gB refolding from pre- to postfusion conformation. Coexpression of gHB4.1 and gBB4.1 led to an excess fusion, which was completely suppressed by gDB4.1 in the fusion assays. This was surprising since PrV gD is normally not required for in vitro fusion or direct viral cell-to-cell spread, clearly separating this process from fusion during entry, for which PrV gD is essential. The fusion inhibiting effect of gDB4.1 could be attributed to a single point mutation resulting in an amino acid substitution within the ectodomain (A106V). In conclusion, these results indicated that gL is not central to the fusion process, as its function can be compensated for. As found so far, gL-independent infectivity can be realized by compensatory mutations in gH (as in PrV-ΔgLPass) or in gH plus gB (as in PrV-ΔgLPassB4.1). Excessive fusion induced by gHB4.1 and gBB4.1 was counter-regulated by gDB4.1, indicating that the interplay between these proteins is precisely regulated and further implies that gL and gD, despite being not absolutely essential for the fusion process, have important regulatory functions on gH and/or gB. Both PrV-ΔgLPass mutants had acquired compensatory mutations in gH affecting the predicted gL-binding domain I in gH. By construction of an artificial gH32/98, which lacked the predicted gL-binding domain and was similar to the recently crystallized gH-core fragment present in the gDgH hybrid protein, we identified the N-terminal part of PrV gH as essential for gH function during fusion (paper III). gH32/98 was unable to promote fusion of wild-type gB in fusion assays and led to a total loss of function in the viral context. These results indicated that the gD moiety, present in gDgH, is critical for proper function of the gH-core fragment. We hypothesize that the gD moiety may adopt a stabilizing or modulating influence on the gH structure, which is normally executed by gL and important for interaction of gH with wild-type gB. Remarkably, substitution of wild-type gB by gBB4.1 rescued function of gH32/98 in the cellular and viral contexts. These findings suggest that gBB4.1 has been selected for interaction with “gL-less” gH. In conclusion, these results demonstrated that gL and the gL-binding domain are not strictly required for membrane fusion during virus entry and spread but that compensatory mutations must be present in gB to restore a fully functional fusion machinery. These results strongly support the notion of a functional gH-gB interaction as a prerequisite for membrane fusion. In addition to the N-terminal domain, we identified the transmembrane domain of PrV gH as an essential component of the fusion machinery, while the cytoplasmic domain was demonstrated to play a modulatory but nonessential role (paper IV). Whereas truncation or substitution of the PrV gH TMD by a gpi-anchor or the analogous sequence from PrV gD rendered gH non-functional, the HSV-1 gH TMD was found to functionally substitute for the PrV gH TMD in cell-cell fusion and complementation assays. Since residues in the TMD which are conserved between HSV and PrV gH but absent in PrV gD, are placed on one face of an α-helical wheel plot, we hypothesize that the gH TMD has an intrinsic property to interact with membrane components such as lipids or other molecules as a requirement for promoting membrane fusion. In a final study focusing on the function of gH, we identified the N-glycosylation sites utilized by PrV gH, and determined their individual role in viral infection (paper V). PrV gH was found to be modified by N-glycans at five potential glycosylation sites. N-glycans at PrV specific N77 and the highly conserved site N627 were found to be critical for efficient membrane fusion in the fusion assays, and during viral entry and cell-to-cell spread. N627 was further shown to be crucial for proper gH transport and maturation. In contrast, inactivation of N604, conserved in the Varicellovirus genus, enhanced in vitro fusion activity and viral cell-to-cell spread. These findings demonstrated a role of the N-glycans in proper localization and function of PrV gH.},
  language  = {en}
}