Hepatitis B virus envelope proteins can serve as therapeutic targets embedded in the host cell plasma membrane.

Hepatitis B virus (HBV) infection is a major health threat causing 880,000 deaths each year. Available therapies control viral replication but do not cure HBV, leaving patients at risk to develop hepatocellular carcinoma. Here, we show that HBV envelope proteins (HBs)-besides their integration into endosomal membranes-become embedded in the plasma membrane where they can be targeted by redirected T-cells. HBs was detected on the surface of HBV-infected cells, in livers of mice replicating HBV and in HBV-induced hepatocellular carcinoma. Staining with HBs-specific recombinant antibody MoMab recognising a conformational epitope indicated that membrane-associated HBs remains correctly folded in HBV-replicating cells in cell culture and in livers of HBV-transgenic mice in vivo. MoMab coated onto superparamagnetic iron oxide nanoparticles allowed to detect membrane-associated HBs after HBV infection by electron microscopy in distinct stretches of the hepatocyte plasma membrane. Last but not least, we demonstrate that HBs located on the cell surface allow therapeutic targeting of HBV-positive cells by T-cells either engrafted with a chimeric antigen receptor or redirected by bispecific, T-cell engager antibodies. TAKE AWAYS: HBs become translocated to the plasma membrane. Novel, recombinant antibody confirmed proper conformation of HBs on the membrane. HBs provide an interesting target by T-cell-based, potentially curative therapies.

Novel hereditary angioedema linked with a heparan sulfate 3-O-sulfotransferase 6 gene mutation.

BACKGROUND: Hereditary angioedema (HAE) is a potentially fatal disorder resulting in recurrent attacks of severe swelling. It may be associated with a genetic deficiency of functional C1 inhibitor or with normal C1 inhibitor (HAEnCI). In families with HAEnCI, HAE-linked mutations in the F12, PLG, KNG1, ANGPT1, or MYOF genes have been identified. In many families with HAEnCI the genetic cause of the disease is currently unknown. OBJECTIVE: The aim of this study was to identify a novel disease-linked mutation for HAEnCI. METHODS: The study methods comprised whole exome sequencing, Sanger sequencing analysis, pedigree analysis, bioinformatic analysis of the mutation, and biochemical analysis of parameters of the kallikrein-kinin (contact) system. RESULTS: By performing whole exome sequencing on a multigenerational family with HAEnCI we were able to identify the heparan sulfate (HS)-glucosamine 3-O-sulfotransferase 6 (HS3ST6) mutation c.430A>T (p.Thr144Ser) in all 3 affected family members who were sequenced. This gene encodes HS-glucosamine 3-O-sulfotransferase 6 (3-OST-6), which is involved in the last step of HS biosynthesis. The p.Thr144Ser mutation is likely to affect the interaction between 2 ß-sheets stabilizing the active center of the 3-OST-6 protein. CONCLUSIONS: We conclude that mutant 3-OST-6 fails to transfer sulfo groups to the 3-OH position of HS, resulting in incomplete HS biosynthesis. This likely affects cell surface interactions of key players in angioedema formation and is a novel mechanism for disease development.

Inhibition of EBV-mediated membrane fusion by anti-gHgL antibodies.

Herpesvirus entry into cells requires the coordinated action of multiple virus envelope glycoproteins, including gH, gL, and gB. For EBV, the gp42 protein assembles into complexes with gHgL heterodimers and binds HLA class II to activate gB-mediated membrane fusion with B cells. EBV tropism is dictated by gp42 levels in the virion, as it inhibits entry into epithelial cells while promoting entry into B cells. The gHgL and gB proteins are targets of neutralizing antibodies and potential candidates for subunit vaccine development, but our understanding of their neutralizing epitopes and the mechanisms of inhibition remain relatively unexplored. Here we studied the structures and mechanisms of two anti-gHgL antibodies, CL40 and CL59, that block membrane fusion with both B cells and epithelial cells. We determined the structures of the CL40 and CL59 complexes with gHgL using X-ray crystallography and EM to identify their epitope locations. CL59 binds to the C-terminal domain IV of gH, while CL40 binds to a site occupied by the gp42 receptor binding domain. CL40 binding to gHgL/gp42 complexes is not blocked by gp42 and does not interfere with gp42 binding to HLA class II, indicating that its ability to block membrane fusion with B cells represents a defect in gB activation. These data indicate that anti-gHgL neutralizing antibodies can block gHgL-mediated activation of gB through different surface epitopes and mechanisms.

Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

Comparative Mutagenesis of Pseudorabies Virus and Epstein-Barr Virus gH Identifies a Structural Determinant within Domain III of gH Required for Surface Expression and Entry Function.

UNLABELLED: Herpesviruses infect cells using the conserved core fusion machinery composed of glycoprotein B (gB) and gH/gL. The gH/gL complex plays an essential but still poorly characterized role in membrane fusion and cell tropism. Our previous studies demonstrated that the conserved disulfide bond (DB) C278/C335 in domain II (D-II) of Epstein-Barr virus (EBV) gH has an epithelial cell-specific function, whereas the interface of D-II/D-III is involved in formation of the B cell entry complex by binding to gp42. To extend these studies, we compared gH of the alphaherpesvirus pseudorabies virus (PrV) with gH of the gammaherpesvirus EBV to identify functionally equivalent regions critical for gH function during entry. We identified several conserved amino acids surrounding the conserved DB that connects three central helices of D-III of PrV and EBV gH. The present study verified that the conserved DB and several contacting amino acids in D-III modulate cell surface expression and thereby contribute to gH function. In line with this finding, we found that DB C404/C439 and T401 are important for cell-to-cell spread and efficient entry of PrV. This parallel comparison between PrV and EBV gH function brings new insights into how gH structure impacts fusion function during herpesvirus entry. IMPORTANCE: The alphaherpesvirus PrV is known for its neuroinvasion, whereas the gammaherpesvirus EBV is associated with cancer of epithelial and B cell origin. Despite low amino acid conservation, PrV gH and EBV gH show strikingly similar structures. Interestingly, both PrV gH and EBV gH contain a structural motif composed of a DB and supporting amino acids which is highly conserved within the Herpesviridae. Our study verified that PrV gH uses a minimal motif with the DB as the core, whereas the DB of EBV gH forms extensive connections through hydrogen bonds to surrounding amino acids, ensuring the cell surface expression of gH/gL. Our study verifies that the comparative analysis of distantly related herpesviruses, such as PrV and EBV, allows the identification of common gH functions. In addition, we provide an understanding of how functional domains can evolve over time, resulting in subtle differences in domain structure and function.

Assembly and architecture of the EBV B cell entry triggering complex.

Epstein-Barr Virus (EBV) is an enveloped double-stranded DNA virus of the gammaherpesvirinae sub-family that predominantly infects humans through epithelial cells and B cells. Three EBV glycoproteins, gH, gL and gp42, form a complex that targets EBV infection of B cells. Human leukocyte antigen (HLA) class II molecules expressed on B cells serve as the receptor for gp42, triggering membrane fusion and virus entry. The mechanistic role of gHgL in herpesvirus entry has been largely unresolved, but it is thought to regulate the activation of the virally-encoded gB protein, which acts as the primary fusogen. Here we study the assembly and function of the reconstituted B cell entry complex comprised of gHgL, gp42 and HLA class II. The structure from negative-stain electron microscopy provides a detailed snapshot of an intermediate state in EBV entry and highlights the potential for the triggering complex to bring the two membrane bilayers into proximity. Furthermore, gHgL interacts with a previously identified, functionally important hydrophobic pocket on gp42, defining the overall architecture of the complex and playing a critical role in membrane fusion activation. We propose a macroscopic model of the initiating events in EBV B cell fusion centered on the formation of the triggering complex in the context of both viral and host membranes. This model suggests how the triggering complex may bridge the two membrane bilayers, orienting critical regions of the N- and C- terminal ends of gHgL to promote the activation of gB and efficient membrane fusion.

The conserved disulfide bond within domain II of Epstein-Barr virus gH has divergent roles in membrane fusion with epithelial cells and B cells.

UNLABELLED: Epstein-Barr virus (EBV) infects target cells via fusion with cellular membranes. For entry into epithelial cells, EBV requires the herpesvirus conserved core fusion machinery, composed of glycoprotein B (gB) and gH/gL. In contrast, for B cell fusion it requires gB and gH/gL with gp42 serving as a cell tropism switch. The available crystal structures for gH/gL allow the targeted analysis of structural determinants of gH to identify functional regions critical for membrane fusion. Domain II of EBV gH contains two disulfide bonds (DBs). The first is unique for EBV and closely related gammaherpesviruses. The second is conserved across the beta- and gammaherpesviruses and is positioned to stabilize a putative syntaxin-like bundle motif. To analyze the role of these DBs in membrane fusion, gH was mutated by amino acid substitution of the DB cysteines. Mutation of the EBV-specific DB resulted in diminished gH/gL cell surface expression that correlated with diminished B cell and epithelial cell fusion. In contrast, mutation of the conserved DB resulted in wild-type-like B cell fusion, whereas epithelial cell fusion was greatly reduced. The gH mutants bound well to gp42 but had diminished binding to epithelial cells. Tyrosine 336, located adjacent to cysteine 335 of the conserved DB, also was found to be important for DB stabilization and gH/gL function. We conclude that the conserved DB has a cell type-specific function, since it is important for the binding of gH to epithelial cells initiating epithelial cell fusion but not for fusion with B cells and gp42 binding. IMPORTANCE: EBV predominantly infects epithelial and B cells in humans, which can result in EBV-associated cancers, such as Burkitt and Hodgkin lymphoma, as well as nasopharyngeal carcinoma. EBV is also associated with a variety of lymphoproliferative disorders, typically of B cell origin, observed in immunosuppressed individuals, such as posttransplant or HIV/AIDS patients. The gH/gL complex plays an essential but still poorly characterized role as an important determinant for EBV cell tropism. In the current studies, we found that mutants in the DB C278/C335 and the neighboring tyrosine 336 have cell type-specific functional deficits with selective decreases in epithelial cell, but not B cell, binding and fusion. The present study brings new insights into the gH function as a determinant for epithelial cell tropism during herpesvirus-induced membrane fusion and highlights a specific gH motif required for epithelial cell fusion.

Mutant plasminogen in hereditary angioedema is bypassing FXII/kallikrein to generate bradykinin.

Hereditary angioedema (HAE) is characterized by recurrent localized edema in various organs, which can be potentially fatal. There are different types of hereditary angioedema, which include genetic deficiency of C1 inhibitor (C1-INH) and hereditary angioedema with normal C1-INH (HAEnCI). In HAEnCI patients mutations have been identified in the F12, PLG, KNG1, ANGPT1, MYOF, and HS3ST6 genes. The release of bradykinin from kininogen via the kallikrein-kinin system (KKS) has been shown to be the main mediator in HAE-FXII, but for HAE-PLG there are only first indications how the PLG mutations can result in bradykinin release. Here we identified in a multi-generation HAE-PLG family an additional F12 mutation, resulting in the loss of one F12 allele. There were no differences in the clinical presentation between HAE-PLG patients with and without the additional F12 mutation, thus we concluded that the kallikrein-kinin system is bypassed in HAE-PLG. Structural modeling and in vitro assays using purified proteins confirmed the PLG mutation c.988A>G; p.K330E to be a gain of function mutation resulting in an increased bradykinin release by direct cleavage of high molecular weight kininogen (HMWK). Thus, we can provide clinical and experimental evidence that mutant plasminogen in HAE-PLG is bypassing FXII/kallikrein to generate bradykinin.

Cockayne syndrome without UV-sensitivity in Vietnamese siblings with novel ERCC8 variants.

Cockayne syndrome (CS) is a rare progeroid disorder characterized by growth failure, microcephaly, photosensitivity, and premature aging, mainly arising from biallelic ERCC8 (CS-A) or ERCC6 (CS-B) variants. In this study we describe siblings suffering from classical Cockayne syndrome but without photosensitivity, which delayed a clinical diagnosis for 16 years. By whole-exome sequencing we identified the two novel compound heterozygous ERCC8 variants c.370_371del (p.L124Efs*15) and c.484G>C (p.G162R). The causality of the ERCC8 variants, of which one results in a frameshift and the other affects the WD3 domain, was tested and confirmed by a rescue experiment investigating DNA repair in H2O2 treated patient fibroblasts. Structural modeling of the p.G162R variant indicates effects on protein-protein interaction. This case shows the importance to test for ERCC6 and ERCC8 variants even if patients do not present with a complete CS phenotype.

Random transposon-mediated mutagenesis of the essential large tegument protein pUL36 of pseudorabies virus.

Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.

Intracellular localization of the pseudorabies virus large tegument protein pUL36.

Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated "inner" tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.

Gammaherpesvirus entry and fusion: A tale how two human pathogenic viruses enter their host cells.

The prototypical human γ-herpesviruses Epstein-Barr virus (EBV) and Kaposi Sarcoma-associated herpesvirus (KSHV) are involved in the development of malignancies. Like all herpesviruses, they share the establishment of latency, the typical architecture, and the conserved fusion machinery to initiate infection. The fusion machinery reflects virus-specific adaptations due to the requirements of the respective herpesvirus. For example, EBV evolved a tropism switch involving either the B- or epithelial cell-tropism complexes to activate fusion driven by gB. Most of the EBV entry proteins and their cellular receptors have been crystallized providing molecular details of the initial steps of infection. For KSHV, a variety of entry and binding receptors has also been reported but the mechanism how receptor binding activates gB-driven fusion is not as well understood as that for EBV. However, the downstream signaling pathways that promote the early steps of KSHV entry are well described. This review summarizes the current knowledge of the key players involved in EBV and KSHV entry and the cell-type specific mechanisms that allow infection of a wide variety of cell types.

Structural and Mechanistic Insights into the Tropism of Epstein-Barr Virus.

Epstein-Barr virus (EBV) is the prototypical γ-herpesvirus and an obligate human pathogen that infects mainly epithelial cells and B cells, which can result in malignancies. EBV infects these target cells by fusing with the viral and cellular lipid bilayer membranes using multiple viral factors and host receptor(s) thus exhibiting a unique complexity in its entry machinery. To enter epithelial cells, EBV requires minimally the conserved core fusion machinery comprised of the glycoproteins gH/gL acting as the receptor-binding complex and gB as the fusogen. EBV can enter B cells using gp42, which binds tightly to gH/gL and interacts with host HLA class II, activating fusion. Previously, we published the individual crystal structures of EBV entry factors, such as gH/gL and gp42, the EBV/host receptor complex, gp42/HLA-DR1, and the fusion protein EBV gB in a postfusion conformation, which allowed us to identify structural determinants and regions critical for receptor-binding and membrane fusion. Recently, we reported different low resolution models of the EBV B cell entry triggering complex (gHgL/gp42/HLA class II) in "open" and "closed" states based on negative-stain single particle electron microscopy, which provide further mechanistic insights. This review summarizes the current knowledge of these key players in EBV entry and how their structures impact receptor-binding and the triggering of gB-mediated fusion.

Structural basis for Epstein-Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins.

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein-Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, here we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator of EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. These observations clarify key determinants of EBV host cell tropism.