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Richard Longnecker - One of the best experts on this subject based on the ideXlab platform.
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natural selection of <B>GlycoproteinB> B mutations that rescue the small plaque phenotype of a fusion impaired herpes simplex virus mutant
Mbio, 2018Co-Authors: Qing Fan, Sarah A Connolly, Sarah J Kopp, Nina C Byskosh, Richard LongneckerAbstract:ABSTRACT <B>GlycoproteinB> B (gB) is a conserved viral fusion protein that is required for herpesvirus entry. To mediate fusion with the cellular memBrane, gB refolds from a prefusion to a postfusion conformation. We hypothesize that an interaction Between the C-terminal arm and the central coiled coil of the herpes simplex virus 1 (HSV-1) gB ectodomain is critical for fusion. We previously reported that three mutations in the C-terminal arm (I671A/H681A/F683A, called gB3A) greatly reduced cell-cell fusion and that virus carrying these mutations had a small-plaque phenotype and delayed entry into cells. By serially passaging gB3A virus, we selected three revertant viruses with larger plaques. These revertant viruses acquired mutations in gB that restore the fusion function of gB3A, including gB-A683V, gB-S383F/G645R/V705I/A855V, and gB-T509M/N709H. V705I and N709H are novel mutations that map to the portion of domain V that enters domain I in the postfusion structure. S383F, G645R, and T509M are novel mutations that map to an intersection of three domains in a prefusion model of gB. We introduced these second-site mutations individually and in comBination into wild-type gB and gB3A to examine the impact of the mutations on fusion and expression. V705I and A855V (a known hyperfusogenic mutation) restored the fusion function of gB3A, whereas S383F and G645R dampened fusion and T509M and N709H worked in concert to restore gB3A fusion. The results identify two regions in the gB ectodomain that modulate the fusion activity of gB, potentially By impacting intramolecular interactions and staBility of the prefusion and/or postfusion gB trimer. IMPORTANCE <B>GlycoproteinB> B (gB) is an essential viral protein that is conserved in all herpesviruses and is required for virus entry. gB is thought to undergo a conformational change that provides the energy to fuse the viral and cellular memBranes; however, the details of this conformational change and the structure of the prefusion and intermediate conformations of gB are not known. Previously, we demonstrated that mutations in the gB “arm” region inhiBit fusion and impart a small-plaque phenotype. Using serial passage of a virus carrying these mutations, we identified revertants with restored plaque size. The revertant viruses acquired novel mutations in gB that restored fusion function and mapped to two sites in the gB ectodomain. This work supports our hypothesis that an interaction Between the gB arm and the core of gB is critical for gB refolding and provides details aBout the function of gB in herpesvirus-mediated fusion and suBsequent virus entry.
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Structure-Based Mutations in the Herpes Simplex Virus 1 <B>GlycoproteinB> B Ectodomain Arm Impart a Slow-Entry Phenotype
American Society for Microbiology, 2017Co-Authors: Qing Fan, Richard Longnecker, Sarah A Connolly, Sarah J Kopp, Peter PaleseAbstract:<B>GlycoproteinB> B (gB) is the conserved herpesvirus fusion protein, and it is required for the entry of herpesviruses. The structure of the postfusion conformation of gB has Been solved for several herpesviruses; however, the gB prefusion crystal structure and the details of how the protein refolds from a prefusion to a postfusion form to mediate fusion have not Been determined. Using structure-Based mutagenesis, we previously reported that three mutations (I671A, H681A, and F683A) in the C-terminal arm of the gB ectodomain greatly reduced cell-cell fusion. This fusion deficit could Be rescued By the addition of a hyperfusogenic mutation, suggesting that the gB triple mutant was not misfolded. Using a Bacterial artificial chromosome (BAC), we constructed two independent herpes simplex virus 1 mutant strains (gB 3A) carrying the three arm mutations. The gB 3A viruses have 200-fold smaller plaques than the wild-type virus and demonstrate remarkaBly delayed entry into cells. Single-step and multistep growth curves show that gB 3A viruses have delayed replication kinetics. Interestingly, incuBation at 40°C promoted the entry of the gB 3A viruses. We propose that the gB 3A viruses’ entry deficit is due to a loss of interactions Between residues in the gB C-terminal arm and the coiled-coil core of gB. The results suggest that the triple alanine mutation may destaBilize the postfusion gB conformation and/or staBilize the prefusion gB conformation and that exposure to elevated temperatures can overcome the defect in gB 3A viruses
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the fusion loops and memBrane proximal region of epstein Barr virus <B>GlycoproteinB> B gB can function in the context of herpes simplex virus 1 gB when suBstituted individually But not in comBination
Virus Research, 2013Co-Authors: Anna Zago, Patricia G Spear, Sarah A Connolly, Richard LongneckerAbstract:Among the herpesvirus <B>GlycoproteinB> B (gB) fusion proteins, the hydrophoBic content of fusion loops and memBrane proximal regions (MPRs) are inversely correlated with each other. We examined the functional importance of the hydrophoBicity of these regions By replacing them in herpes simplex virus type 1 gB with corresponding regions from Epstein-Barr virus gB. We show that fusion activity is dependent on the structural context in which the specific loops and MPR sequences exist, rather than a simple hydrophoBic relationship.
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structure of a trimeric variant of the epstein Barr virus <B>GlycoproteinB> B
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Marija Backovic, Richard Longnecker, Theodore S JardetzkyAbstract:Epstein–Barr virus (EBV) is a herpesvirus that is associated with development of malignancies of lymphoid tissue. EBV infections are life-long and occur in >90% of the population. Herpesviruses enter host cells in a process that involves fusion of viral and cellular memBranes. The fusion apparatus is comprised of envelope <B>GlycoproteinB> B (gB) and a heterodimeric complex made of <B>GlycoproteinB>s H and L. <B>GlycoproteinB> B is the most conserved envelope <B>GlycoproteinB> in human herpesviruses, and the structure of gB from Herpes simplex virus 1 (HSV-1) is availaBle. Here, we report the crystal structure of the secreted EBV gB ectodomain, which forms 16-nm long spike-like trimers, structurally homologous to the postfusion trimers of the fusion protein G of vesicular stomatitis virus (VSV). Comparative structural analyses of EBV gB and VSV G, which has Been solved in its pre and postfusion states, shed light on gB residues that may Be involved in conformational changes and memBrane fusion. Also, the EBV gB structure reveals that, despite the high sequence conservation of gB in herpesviruses, the relative orientations of individual domains, the surface charge distriButions, and the structural details of EBV gB differ from the HSV-1 protein, indicating regions and residues that may have important roles in virus-specific entry.
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hydrophoBic residues that form putative fusion loops of epstein Barr virus <B>GlycoproteinB> B are critical for fusion activity
Journal of Virology, 2007Co-Authors: Marija Backovic, Theodore S Jardetzky, Richard LongneckerAbstract:To test the importance of the hydrophoBic residues within the putative Epstein-Barr virus (EBV) <B>GlycoproteinB> B (gB) fusion loops in memBrane fusion, WY(112-113) and WLIW(193-196) were mutated into alanine, glutamic acid, or the analogous residues from herpes simplex virus type 1 (HSV-1) gB (HR and RVEA). All gB variants exhiBited cell surface expression, demonstrating that the suBstitutions did not perturB gB trafficking. None of six gB variants was, however, capaBle of mediating fusion with either epithelial or B cells. These data demonstrate that the Bulky and hydrophoBic EBV loop residues, which differ from the more hydrophilic HSV-1 residues and appear more compatiBle with memBrane insertion, are essential for EBV gB-dependent fusion.
Yasushi Kawaguchi - One of the best experts on this subject based on the ideXlab platform.
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effects of phosphorylation of herpes simplex virus 1 envelope <B>GlycoproteinB> B By us3 kinase in vivo and in vitro
Journal of Virology, 2010Co-Authors: Takahiko Imai, Jun Arii, Ken Sagou, Yasushi KawaguchiAbstract:We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope <B>GlycoproteinB> B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 By alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have Been reported for mice infected with a recomBinant HSV-1 carrying a kinase-inactive mutant of Us3. These oBservations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antiBody that specifically reacted with phosphorylated gB Thr-887 and used this antiBody to show that Us3 phosphorylation of gB Thr-887 regulated suBcellular localization of gB, particularly on the cell surface of infected cells.
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Binding of herpes simplex virus <B>GlycoproteinB> B gB to paired immunogloBulin like type 2 receptor α depends on specific sialylated o linked glycans on gB
Journal of Virology, 2009Co-Authors: Jing Wang, Jun Arii, Yasushi Kawaguchi, Takeshi Satoh, Lewis L Lanier, Patricia G Spear, Hisashi AraseAbstract:Paired immunogloBulin-like type 2 receptor α (PILRα) is an inhiBitory receptor expressed on Both hematopoietic and nonhematopoietic cells. Its Binding to a cellular ligand, CD99, depends on the presence of sialylated O-linked glycans on CD99. <B>GlycoproteinB> B (gB) of herpes simplex virus type 1 (HSV-1) Binds to PILRα, and this association is involved in HSV-1 infection. Here, we found that the presence of sialylated O-glycans on gB is required for gB to associate with PILRα. Furthermore, we identified two threonine residues on gB that are essential for the addition of the principal O-glycans acquired By gB and that are also essential for the Binding of PILRα to gB.
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herpes simplex virus 1 protein kinase us3 phosphorylates viral envelope <B>GlycoproteinB> B and regulates its expression on the cell surface
Journal of Virology, 2009Co-Authors: Akihisa Kato, Jun Arii, Ikuo Shiratori, Hiroomi Akashi, Hisashi Arase, Yasushi KawaguchiAbstract:Us3 is a serine-threonine protein kinase encoded By herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological suBstrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope <B>GlycoproteinB> B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated By Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antiBody that recognized phosphorylated serine or threonine residues with arginine at the -3 and -2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was oBserved in cells infected with a recomBinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.
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pilrα is a herpes simplex virus 1 entry coreceptor that associates with <B>GlycoproteinB> B
Cell, 2008Co-Authors: Takeshi Satoh, Jun Arii, Ikuo Shiratori, Tadahiro Suenaga, Jing Wang, Amane Kogure, Junji Uehori, Noriko Arase, Shinya Tanaka, Yasushi KawaguchiAbstract:Summary <B>GlycoproteinB> B (gB) is one of the essential components for infection By herpes simplex virus-1 (HSV-1). Although several cellular receptors that associate with <B>GlycoproteinB> D (gD), such as herpes virus entry mediator (HVEM) and Nectin-1, have Been identified, specific molecules that mediate HSV-1 infection By associating with gB have not Been elucidated. Here, we found that paired immunogloBulin-like type 2 receptor (PILR) α associates with gB, and cells transduced with PILRα Become susceptiBle to HSV-1 infection. Furthermore, HSV-1 infection of human primary cells expressing Both HVEM and PILRα was Blocked By either anti-PILRα or anti-HVEM antiBody. Our results demonstrate that cellular receptors for Both gB and gD are required for HSV-1 infection and that PILRα plays an important role in HSV-1 infection as a coreceptor that associates with gB. These findings uncover a crucial aspect of the mechanism underlying HSV-1 infection.
Amanda M Casto - One of the best experts on this subject based on the ideXlab platform.
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herpes simplex virus mistyping due to hsv 1 hsv 2 interspecies recomBination in viral gene encoding <B>GlycoproteinB> B
Viruses, 2020Co-Authors: Amanda M Casto, Keith R Jerome, Anna Wald, Christine Johnston, Meeili W Huang, Hong Xie, Alexander L GreningerAbstract:Human herpes simplex viruses (HSV) 1 and 2 are extremely common human pathogens with overlapping disease spectra. Infections due to HSV-1 and HSV-2 are distinguished in clinical settings using sequence-Based “typing” assays. Here we descriBe a case of HSV mistyping caused By a previously undescriBed HSV-1 × HSV-2 recomBination event in UL27, the HSV gene that encodes <B>GlycoproteinB> B. This is the first documented case of HSV mistyping caused By an HSV-1 × HSV-2 recomBination event and the first description of an HSV interspecies recomBination event in UL27, which is frequently used as a target for diagnostics and experimental therapeutics. We also review the primer and proBe target sequences for a commonly used HSV typing assay from nearly 700 HSV-1 and HSV-2 samples and find that aBout 4% of HSV-1 samples have a single nucleotide change in at least one of these loci, which could impact assay performance. Our findings illustrate how knowledge of naturally occurring genomic variation in HSV-1 and HSV-2 is essential for the design and interpretation of molecular diagnostics for these viruses.
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herpes simplex virus mistyping due to hsv 1 x hsv 2 interspecies recomBination in viral gene encoding <B>GlycoproteinB> B
bioRxiv, 2020Co-Authors: Amanda M Casto, Meeili Huang, Keith R Jerome, Anna Wald, Christine Johnston, Alexander L GreningerAbstract:Human herpes simplex viruses (HSV) 1 and 2 are most often typed via molecular assays. Here we descriBe the first known case of HSV mistyping due to a previously undescriBed HSV-1 x HSV-2 recomBination event in UL27, the gene that encodes <B>GlycoproteinB> B. This is the first reported HSV interspecies recomBination event impacting this gene, which is frequently used as a target for diagnostics and experimental therapeutics.
Donald M Coen - One of the best experts on this subject based on the ideXlab platform.
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virucidal activity of a gt rich oligonucleotide against herpes simplex virus mediated By <B>GlycoproteinB> B
Journal of Virology, 2006Co-Authors: Benjamin D Shogan, Lori Kruse, Gilbert B Mulamba, Donald M CoenAbstract:We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in <B>GlycoproteinB> B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhiBited attachment of virus to cells at 4°C; however, the resistant mutant did not exhiBit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhiBiting attachment. Similarly, ISIS 5652 was aBle to inhiBit entry of preattached virions into cells at 37°C, But the mutant did not exhiBit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incuBation of wild-type virus with 1 to 2 μM ISIS 5652 at 37°C led to a time-dependent, irreversiBle loss of infectivity (virucidal activity). No virucidal activity was detected at 4°C or with an unrelated oligonucleotide at 37°C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhiBited suBstantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.
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virucidal activity of a gt rich oligonucleotide against herpes simplex virus mediated By <B>GlycoproteinB> B
Journal of Virology, 2006Co-Authors: Benjamin D Shogan, Lori Kruse, Gilbert B Mulamba, Donald M CoenAbstract:We have investigated the antiviral mechanism of a phosphorothioate oligonucleotide, ISIS 5652, which has activity against herpes simplex virus (HSV) in the low micromolar range in plaque reduction assays. We isolated a mutant that is resistant to this compound. Marker rescue and sequencing experiments showed that resistance was due to at least one of three mutations in the UL27 gene which result in amino acid changes in <B>GlycoproteinB> B (gB). Because gB has a role in attachment and entry of HSV, we tested the effects of ISIS 5652 at these stages of infection. The oligonucleotide potently inhiBited attachment of virus to cells at 4 degrees C; however, the resistant mutant did not exhiBit resistance at this stage. Moreover, a different oligonucleotide with little activity in plaque reduction assays was as potent as ISIS 5652 in inhiBiting attachment. Similarly, ISIS 5652 was aBle to inhiBit entry of pre-attached virions into cells at 37 degrees C, But the mutant did not exhiBit resistance in this assay. The mutant did not attach to or enter cells more quickly than did wild-type virus. Strikingly, incuBation of wild-type virus with 1 to 2 microM ISIS 5652 at 37 degrees C led to a time-dependent, irreversiBle loss of infectivity (virucidal activity). No virucidal activity was detected at 4 degrees C or with an unrelated oligonucleotide at 37 degrees C. The resistant mutant and a marker-rescued derivative containing its gB mutations exhiBited suBstantial resistance to this virucidal activity of ISIS 5652. We hypothesize that the GT-rich oligonucleotide induces a conformational change in gB that results in inactivation of infectivity.
Alexander L Greninger - One of the best experts on this subject based on the ideXlab platform.
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herpes simplex virus mistyping due to hsv 1 hsv 2 interspecies recomBination in viral gene encoding <B>GlycoproteinB> B
Viruses, 2020Co-Authors: Amanda M Casto, Keith R Jerome, Anna Wald, Christine Johnston, Meeili W Huang, Hong Xie, Alexander L GreningerAbstract:Human herpes simplex viruses (HSV) 1 and 2 are extremely common human pathogens with overlapping disease spectra. Infections due to HSV-1 and HSV-2 are distinguished in clinical settings using sequence-Based “typing” assays. Here we descriBe a case of HSV mistyping caused By a previously undescriBed HSV-1 × HSV-2 recomBination event in UL27, the HSV gene that encodes <B>GlycoproteinB> B. This is the first documented case of HSV mistyping caused By an HSV-1 × HSV-2 recomBination event and the first description of an HSV interspecies recomBination event in UL27, which is frequently used as a target for diagnostics and experimental therapeutics. We also review the primer and proBe target sequences for a commonly used HSV typing assay from nearly 700 HSV-1 and HSV-2 samples and find that aBout 4% of HSV-1 samples have a single nucleotide change in at least one of these loci, which could impact assay performance. Our findings illustrate how knowledge of naturally occurring genomic variation in HSV-1 and HSV-2 is essential for the design and interpretation of molecular diagnostics for these viruses.
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herpes simplex virus mistyping due to hsv 1 x hsv 2 interspecies recomBination in viral gene encoding <B>GlycoproteinB> B
bioRxiv, 2020Co-Authors: Amanda M Casto, Meeili Huang, Keith R Jerome, Anna Wald, Christine Johnston, Alexander L GreningerAbstract:Human herpes simplex viruses (HSV) 1 and 2 are most often typed via molecular assays. Here we descriBe the first known case of HSV mistyping due to a previously undescriBed HSV-1 x HSV-2 recomBination event in UL27, the gene that encodes <B>GlycoproteinB> B. This is the first reported HSV interspecies recomBination event impacting this gene, which is frequently used as a target for diagnostics and experimental therapeutics.