Mutagenesis of Human Cytomegalovirus Glycoprotein L Disproportionately Disrupts gH/gL/gO over gH/gL/pUL128-131.
TL;DR: In this paper, the authors characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL/gO-gB-dependent cell-cell fusion but were still able to form gH, gL/pUL128-131 and induce receptor interference.
Abstract: Cell-free and cell-to-cell spread of herpesviruses involves a core fusion apparatus comprised of the fusion protein glycoprotein B (gB) and the regulatory factor gH/gL. The human cytomegalovirus (HCMV) gH/gL/gO and gH/gL/pUL128-131 facilitate spread in different cell types. The gO and pUL128-131 components bind distinct receptors, but how the gH/gL portions of the complexes functionally compare is not understood. We previously characterized a panel of gL mutants by transient expression and showed that many were impaired for gH/gL-gB-dependent cell-cell fusion but were still able to form gH/gL/pUL128-131 and induce receptor interference. Here, the gL mutants were engineered into the HCMV BAC clones TB40/e-BAC4 (TB), TR, and Merlin (ME), which differ in their utilization of the two complexes for entry and spread. Several of the gL mutations disproportionately impacted gH/gL/gO-dependent entry and spread over gH/gL/pUL128-131 processes. The effects of some mutants could be explained by impaired gH/gL/gO assembly, but other mutants impacted gH/gL/gO function. Soluble gH/gL/gO containing the L201 mutant failed to block HCMV infection despite unimpaired binding to PDGFRα, indicating the existence of other important gH/gL/gO receptors. Another mutant (L139) enhanced the gH/gL/gO-dependent cell-free spread of TR, suggesting a "hyperactive" gH/gL/gO. Recently published crystallography and cryo-electron microscopy studies suggest structural conservation of the gH/gL underlying gH/gL/gO and gH/gL/pUL128-131. However, our data suggest important differences in the gH/gL of the two complexes and support a model in which gH/gL/gO can provide an activation signal for gB. IMPORTANCE The endemic betaherpesvirus HCMV circulates in human populations as a complex mixture of genetically distinct variants, establishes lifelong persistent infections, and causes significant disease in neonates and immunocompromised adults. This study capitalizes on our recent characterizations of three genetically distinct HCMV BAC clones to discern the functions of the envelope glycoprotein complexes gH/gL/gO and gH/gL/pUL128-13, which are promising vaccine targets that share the herpesvirus core fusion apparatus component, gH/gL. Mutations in the shared gL subunit disproportionally affected gH/gL/gO, demonstrating mechanistic differences between the two complexes, and may provide a basis for more refined evaluations of neutralizing antibodies.
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TL;DR: The difficulties of determining the cellular provenance of these structures in light of viral redistribution of cellular marker proteins are discussed and potential paths to illuminate their genesis are provided.
Abstract: Human cytomegalovirus (HCMV) is a ubiquitous herpesvirus and the leading cause of congenital disabilities as well as a significant cause of disease in immunocompromised patients. The envelopment and egress of HCMV particles is an essential step of the viral life cycle as it determines viral spread and potentially tropism. Here we review the current literature on HCMV envelopment and egress with a particular focus on the role of virus‐containing multivesicular body‐like vesicles for virus egress and spread. We discuss the difficulties of determining the cellular provenance of these structures in light of viral redistribution of cellular marker proteins and provide potential paths to illuminate their genesis. Finally, we discuss how divergent egress pathways could result in virions of different tropisms.
2 citations
TL;DR: This paper studied the structure and interactions of the HCMV trimer, formed by AD169 strain gH and gL and TR strain gO proteins, with the human platelet derived growth factor receptor alpha (PDGFRα).
Abstract: Human cytomegalovirus (HCMV) is a herpesvirus that produces disease in transplant patients and newborn children. Entry of HCMV into cells relies on gH/gL trimer (gHgLgO) and pentamer (gHgLUL128-131) complexes that bind cellular receptors. Here, we studied the structure and interactions of the HCMV trimer, formed by AD169 strain gH and gL and TR strain gO proteins, with the human platelet-derived growth factor receptor alpha (PDGFRα). Three trimer surfaces make extensive contacts with three PDGFRα N-terminal domains, causing PDGFRα to wrap around gO in a structure similar to a human hand, explaining the high-affinity interaction. gO is among the least conserved HCMV proteins, with 8 distinct genotypes. We observed high conservation of residues mediating gO-gL interactions but more extensive gO variability in the PDGFRα interface. Comparisons between our trimer structure and a previously determined structure composed of different subunit genotypes indicate that gO variability is accommodated by adjustments in the gO-PDGFRα interface. We identified two loops within gO that were disordered and apparently glycosylated, which could be deleted without disrupting PDGFRα binding. We also identified four gO residues that contact PDGFRα, which when mutated produced markedly reduced receptor binding. These residues fall within conserved contact sites of gO with PDGFRα and may represent key targets for anti-trimer neutralizing antibodies and HCMV vaccines. Finally, we observe that gO mutations distant from the gL interaction site impact trimer expression, suggesting that the intrinsic folding or stability of gO can impact the efficiency of trimer assembly. IMPORTANCE HCMV is a herpesvirus that infects a large percentage of the adult population and causes significant levels of disease in immunocompromised individuals and birth defects in the developing fetus. The virus encodes a complex protein machinery that coordinates infection of different cell types in the body, including a trimer formed of gH, gL, and gO subunits. Here, we studied the interactions of the HCMV trimer with its receptor on cells, the platelet derived growth factor receptor α (PDGFRα), to better understand how HCMV coordinates virus entry into cells. Our results add to our understanding of HCMV strain-specific differences and identify sites on the trimer that represent potential targets for therapeutic antibodies or vaccine development.
2 citations
TL;DR: In this article , the pentamer complex/trimer complex (PC/TC) ratio varies according to the producer cell culture type used for the virus preparation to be employed in the neutralizing antibody (NAb) assay, and is lower in fibroblasts and higher in epithelial and especially endothelial cells.
Abstract: In sequential sera from pregnant women with HCMV primary infection (PI), the serum neutralizing activity is higher against virions produced in epithelial and endothelial cells than in fibroblasts. Immunoblotting shows that the pentamer complex/trimer complex (PC/TC) ratio varies according to the producer cell culture type used for the virus preparation to be employed in the neutralizing antibody (NAb) assay, and is lower in fibroblasts and higher in epithelial, and especially endothelial cells. The blocking activity of TC- and PC-specific inhibitors varies according to the PC/TC ratio of virus preparations. The rapid reversion of the virus phenotype following its back passage to the original cell culture (fibroblasts) potentially argues in favor of a producer cell effect on virus phenotype. However, the role of genetic factors cannot be overlooked. In addition to the producer cell type, the PC/TC ratio may differ in single HCMV strains. In conclusion, the NAb activity not only varies with different HCMV strains, but is a dynamic parameter changing according to virus strain, type of target and producer cells, and number of cell culture passages. These findings may have some important implications for the development of both therapeutic antibodies and subunit vaccines.
1 citations
TL;DR: In this paper , the role of the stable core of the glycoprotein B (gB) was investigated in the context of HCMV, where the authors individually introduced three potentially helix-breaking and one disulfide bond-breaking mutations in the DIII α3 to alter the gB stability and studied different aspects of the viral behavior upon long-term culturing.
Abstract: 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.
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TL;DR: Two unusual extensions are presented: Multiscale, which adds the ability to visualize large‐scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales.
Abstract: The design, implementation, and capabilities of an extensible visualization system, UCSF Chimera, are discussed. Chimera is segmented into a core that provides basic services and visualization, and extensions that provide most higher level functionality. This architecture ensures that the extension mechanism satisfies the demands of outside developers who wish to incorporate new features. Two unusual extensions are presented: Multiscale, which adds the ability to visualize large-scale molecular assemblies such as viral coats, and Collaboratory, which allows researchers to share a Chimera session interactively despite being at separate locales. Other extensions include Multalign Viewer, for showing multiple sequence alignments and associated structures; ViewDock, for screening docked ligand orientations; Movie, for replaying molecular dynamics trajectories; and Volume Viewer, for display and analysis of volumetric data. A discussion of the usage of Chimera in real-world situations is given, along with anticipated future directions. Chimera includes full user documentation, is free to academic and nonprofit users, and is available for Microsoft Windows, Linux, Apple Mac OS X, SGI IRIX, and HP Tru64 Unix from http://www.cgl.ucsf.edu/chimera/.
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533 citations
TL;DR: Six strains of human cytomegalovirus have been sequenced, and a total of 252 ORFs with the potential to encode proteins have been identified that are conserved in all four clinical isolates of the virus.
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527 citations