The Experts below are selected from a list of 34716 Experts worldwide ranked by ideXlab platform
Ralph A. Tripp - One of the best experts on this subject based on the ideXlab platform.
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Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.
Journal of virology, 2017Co-Authors: Seyhan Boyoglu-barnum, S O Todd, S J Jadhao, A G Oomens, Tatiana Chirkova, Lia M. Haynes, J Meng, Thomas R. Barnum, Ralph A. Tripp, M. L. MooreAbstract:Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C Chemokine motif ( 182 CWAIC 186 ) in the G protein, RSV binds to the corresponding Chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log 10 -fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine. IMPORTANCE RSV binds to the corresponding Chemokine receptor, CX3CR1, through a CX3C Chemokine motif ( 182 CWAIC 186 ) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab′) 2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.
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Nanoparticle Vaccines Encompassing the Respiratory Syncytial Virus (RSV) G Protein CX3C Chemokine Motif Induce Robust Immunity Protecting from Challenge and Disease
PloS one, 2013Co-Authors: Patricia A. Jorquera, Lia M. Haynes, Larry J. Anderson, Youngjoo Choi, Katie E. Oakley, Thomas Powell, James Gorham Boyd, Naveen Palath, Ralph A. TrippAbstract:Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C Chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN-γ secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8+ T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2- specific T cell responses, and a reduction in RSV disease pathogenesis.
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Antibodies to the central conserved region of respiratory syncytial virus (RSV) G protein block RSV G protein CX3C-CX3CR1 binding and cross-neutralize RSV A and B strains.
Viral immunology, 2012Co-Authors: Youngjoo Choi, Les P. Jones, Patricia A. Jorquera, Caleb S. Mason, Jackelyn Crabtree, Ralph A. TrippAbstract:Respiratory syncytial virus (RSV) is a primary cause of severe lower respiratory tract disease in infants, young children, and the elderly worldwide, and despite decades of effort, there remains no safe and effective vaccine. RSV modifies the host immune response during infection by CX3C Chemokine mimicry adversely affecting pulmonary leukocyte chemotaxis and CX3CR1+ RSV-specific T-cell responses. In this study we investigated whether immunization of mice with RSV G protein polypeptides from strain A2 could induce antibodies that block G protein–CX3CR1 interactions of both RSV A and B strains. The results show that mice immunized with RSV A2 G polypeptides generate antibodies that block binding of RSV A2 and B1 native G proteins to CX3CR1, and that these antibodies effectively cross-neutralize both A and B strains of RSV. These findings suggest that vaccines that induce RSV G protein–CX3CR1 blocking antibodies may provide a disease intervention strategy in the efforts to develop safe and efficacious RSV vaccines.
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Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice
Journal of virology, 2009Co-Authors: Wenliang Zhang, Lia M. Haynes, Jennifer L Harcourt, Larry J. Anderson, Les P. Jones, Youngjoo Choi, Ralph A. TrippAbstract:Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C Chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.
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Therapeutic Monoclonal Antibody Treatment Targeting Respiratory Syncytial Virus (RSV) G Protein Mediates Viral Clearance and Reduces the Pathogenesis of RSV Infection in BALB/c Mice
The Journal of infectious diseases, 2009Co-Authors: Lia M. Haynes, Ralph A. Tripp, Jennifer L Harcourt, Congrong Miao, Hayat Caidi, Gertrud U. Radu, Larry J. AndersonAbstract:Because the G protein of respiratory syncytial virus (RSV) has a CX3C Chemokine motif that has been associated with the ability of RSV G protein to modulate the virus-induced host immune response, we examined whether therapeutic treatment with an anti-RSV G monoclonal antibody (mAb), 131-2G, that blocks the CX3C-associated activity of RSV G protein might decrease the pulmonary inflammation associated with infection in BALB/c mice. The results show that treatment with mAb 131-2G on day 3 after RSV infection reduces both inflammation and RSV titer in the lungs. Later administration of anti-RSV G mAb (day 5 after RSV infection) effectively reduced the viral titer but had a minimal effect on pulmonary inflammation. This study suggests that an anti-RSV G mAb might be an effective antiviral, either alone or in combination with anti-RSV F protein neutralizing antibodies, for decreasing the virus-induced host response to infection and improve treatment outcome.
Larry J. Anderson - One of the best experts on this subject based on the ideXlab platform.
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Mutation of Respiratory Syncytial Virus G Protein’s CX3C Motif Attenuates Infection in Cotton Rats and Primary Human Airway Epithelial Cells
Vaccines, 2019Co-Authors: Tatiana Chirkova, Marina S. Boukhvalova, He Ying Sun, Edward E. Walsh, Christopher S. Anderson, Thomas J. Mariani, Larry J. AndersonAbstract:Despite being a high priority for vaccine development, no vaccine is yet available for respiratory syncytial virus (RSV). A live virus vaccine is the primary type of vaccine being developed for young children. In this report, we describe our studies of infected cotton rats and primary human airway epithelial cells (pHAECs) using an RSV r19F with a mutation in the CX3C Chemokine motif in the RSV G protein (CX4C). Through this CX3C motif, RSV binds to the corresponding Chemokine receptor, CX3CR1, and this binding contributes to RSV infection of pHAECs and virus induced host responses that contribute to disease. In both the cotton rat and pHAECs, the CX4C mutation decreased virus replication and disease and/or host responses to infection. Thus, this mutation, or other mutations that block binding to CX3CR1, has the potential to improve a live attenuated RSV vaccine by attenuating both infection and disease pathogenesis.
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Nanoparticle Vaccines Encompassing the Respiratory Syncytial Virus (RSV) G Protein CX3C Chemokine Motif Induce Robust Immunity Protecting from Challenge and Disease
PloS one, 2013Co-Authors: Patricia A. Jorquera, Lia M. Haynes, Larry J. Anderson, Youngjoo Choi, Katie E. Oakley, Thomas Powell, James Gorham Boyd, Naveen Palath, Ralph A. TrippAbstract:Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C Chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN-γ secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8+ T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2- specific T cell responses, and a reduction in RSV disease pathogenesis.
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Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice
Journal of virology, 2009Co-Authors: Wenliang Zhang, Lia M. Haynes, Jennifer L Harcourt, Larry J. Anderson, Les P. Jones, Youngjoo Choi, Ralph A. TrippAbstract:Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C Chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.
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Therapeutic Monoclonal Antibody Treatment Targeting Respiratory Syncytial Virus (RSV) G Protein Mediates Viral Clearance and Reduces the Pathogenesis of RSV Infection in BALB/c Mice
The Journal of infectious diseases, 2009Co-Authors: Lia M. Haynes, Ralph A. Tripp, Jennifer L Harcourt, Congrong Miao, Hayat Caidi, Gertrud U. Radu, Larry J. AndersonAbstract:Because the G protein of respiratory syncytial virus (RSV) has a CX3C Chemokine motif that has been associated with the ability of RSV G protein to modulate the virus-induced host immune response, we examined whether therapeutic treatment with an anti-RSV G monoclonal antibody (mAb), 131-2G, that blocks the CX3C-associated activity of RSV G protein might decrease the pulmonary inflammation associated with infection in BALB/c mice. The results show that treatment with mAb 131-2G on day 3 after RSV infection reduces both inflammation and RSV titer in the lungs. Later administration of anti-RSV G mAb (day 5 after RSV infection) effectively reduced the viral titer but had a minimal effect on pulmonary inflammation. This study suggests that an anti-RSV G mAb might be an effective antiviral, either alone or in combination with anti-RSV F protein neutralizing antibodies, for decreasing the virus-induced host response to infection and improve treatment outcome.
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Respiratory Syncytial Virus G Protein and G Protein CX3C Motif Adversely Affect CX3CR1+ T Cell Responses
Journal of immunology (Baltimore Md. : 1950), 2006Co-Authors: Jennifer L Harcourt, Larry J. Anderson, Les P. Jones, Rene Alvarez, Christine Henderson, Ralph A. TrippAbstract:Interactions between fractalkine (CX3CL1) and its receptor, CX3CR1, mediate leukocyte adhesion, activation, and trafficking. The respiratory syncytial virus (RSV) G protein has a CX3C Chemokine motif that can bind CX3CR1 and modify CXCL1-mediated responses. In this study, we show that expression of the RSV G protein or the G protein CX3C motif during infection is associated with reduced CX3CR1 + T cell trafficking to the lung, reduced frequencies of RSV-specific, MHC class I-restricted IFN-γ-expressing cells, and lower numbers of IL-4- and CX3CL1-expressing cells. In addition, we show that CX3CR1 + cells constitute a major component of the cytotoxic response to RSV infection. These results suggest that G protein and the G protein CX3C motif reduce the antiviral T cell response to RSV infection.
Lia M. Haynes - One of the best experts on this subject based on the ideXlab platform.
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Mutating the CX3C Motif in the G Protein Should Make a Live Respiratory Syncytial Virus Vaccine Safer and More Effective.
Journal of virology, 2017Co-Authors: Seyhan Boyoglu-barnum, S O Todd, S J Jadhao, A G Oomens, Tatiana Chirkova, Lia M. Haynes, J Meng, Thomas R. Barnum, Ralph A. Tripp, M. L. MooreAbstract:Respiratory syncytial virus (RSV) belongs to the family Paramyxoviridae and is the single most important cause of serious lower respiratory tract infections in young children, yet no highly effective treatment or vaccine is available. Through a CX3C Chemokine motif ( 182 CWAIC 186 ) in the G protein, RSV binds to the corresponding Chemokine receptor, CX3CR1. Since RSV binding to CX3CR1 contributes to disease pathogenesis, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), which is known to block binding to CX3CR1, might decrease disease. We studied the effect of the CX4C mutation in two strains of RSV (A2 and r19F) in a mouse challenge model. We included RSV r19F because it induces mucus production and airway resistance, two manifestations of RSV infection in humans, in mice. Compared to wild-type (wt) virus, mice infected with CX4C had a 0.7 to 1.2 log 10 -fold lower virus titer in the lung at 5 days postinfection (p.i.) and had markedly reduced weight loss, pulmonary inflammatory cell infiltration, mucus production, and airway resistance after challenge. This decrease in disease was not dependent on decrease in virus replication but did correspond to a decrease in pulmonary Th2 and inflammatory cytokines. Mice infected with CX4C viruses also had higher antibody titers and a Th1-biased T cell memory response at 75 days p.i. These results suggest that the CX4C mutation in the G protein could improve the safety and efficacy of a live attenuated RSV vaccine. IMPORTANCE RSV binds to the corresponding Chemokine receptor, CX3CR1, through a CX3C Chemokine motif ( 182 CWAIC 186 ) in the G protein. RSV binding to CX3CR1 contributes to disease pathogenesis; therefore, we investigated whether a mutation in the CX3C motif by insertion of an alanine, A 186 , within the CX3C motif, mutating it to CX4C ( 182 CWAIAC 187 ), known to block binding to CX3CR1, might decrease disease. The effect of this mutation and treatment with the F(ab′) 2 form of the anti-RSV G 131-2G monoclonal antibody (MAb) show that mutating the CX3C motif to CX4C blocks much of the disease and immune modulation associated with the G protein and should improve the safety and efficacy of a live attenuated RSV vaccine.
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Nanoparticle Vaccines Encompassing the Respiratory Syncytial Virus (RSV) G Protein CX3C Chemokine Motif Induce Robust Immunity Protecting from Challenge and Disease
PloS one, 2013Co-Authors: Patricia A. Jorquera, Lia M. Haynes, Larry J. Anderson, Youngjoo Choi, Katie E. Oakley, Thomas Powell, James Gorham Boyd, Naveen Palath, Ralph A. TrippAbstract:Nanoparticle vaccines were produced using layer-by-layer fabrication and incorporating respiratory syncytial virus (RSV) G protein polypeptides comprising the CX3C Chemokine motif. BALB/c mice immunized with G protein nanoparticle vaccines produced a neutralizing antibody response that inhibited RSV replication in the lungs following RSV challenge. ELISPOT analysis showed that G nanoparticle vaccinated mice had increased levels of RSV G protein-specific IL-4 and IFN-γ secreting cells compared to controls following RSV challenge. Remarkably, RSV challenge of G protein nanoparticle vaccinated mice resulted in increased RSV M2-specific IL-4 and IFN-γ secreting T cells, and increased M2-specific H-2Kd-tetramer positive CD8+ T cells in the lungs compared to controls. Cell type analysis showed vaccination was not associated with increased pulmonary eosinophilia following RSV challenge. These results demonstrate that vaccination of mice with the RSV G protein nanoparticle vaccines induces a potent neutralizing antibody response, increased G protein- and M2- specific T cell responses, and a reduction in RSV disease pathogenesis.
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Effect of Chemokine receptor CX3CR1 deficiency in a murine model of respiratory syncytial virus infection.
Comparative medicine, 2012Co-Authors: Crystal H Johnson, Jennifer L Harcourt, Congrong Miao, Elisabeth G. Blanchard, Hayat Caidi, Gertrud U. Radu, Lia M. HaynesAbstract:Respiratory syncytial virus (RSV) is the most common cause of serious lower respiratory illness in infants and young children worldwide, making it a high priority for development of strategies for prevention and treatment. RSV can cause repeat infections throughout life, with serious complications in elderly and immunocompromised patients. Previous studies indicate that the RSV G protein binds through a CX3C Chemokine motif to the host Chemokine receptor, CX3CR1, and modulates the inflammatory immune response. In the current study, we examined the contribution of CX3CR1 to the immune response to RSV infection in mice. CX3CR1-deficient mice showed an impaired innate immune response to RSV infection, characterized by substantially decreased NK1.1+ natural killer, CD11b+, and RB6-8C5+ polymorphonuclear cell trafficking to the lung and reduced IFNγ production compared with those in wildtype control mice. Leukocytes from CX3CR1-deficient mice were poorly chemotactic toward RSV G protein and CX3CL1. These results substantiate the importance of the RSV G CX3C–CX3CR1 interaction in the innate immune response to RSV infection.
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Vaccination To Induce Antibodies Blocking the CX3C-CX3CR1 Interaction of Respiratory Syncytial Virus G Protein Reduces Pulmonary Inflammation and Virus Replication in Mice
Journal of virology, 2009Co-Authors: Wenliang Zhang, Lia M. Haynes, Jennifer L Harcourt, Larry J. Anderson, Les P. Jones, Youngjoo Choi, Ralph A. TrippAbstract:Respiratory syncytial virus (RSV) infection causes substantial morbidity and some deaths in the young and elderly worldwide. There is no safe and effective vaccine available, although it is possible to reduce the hospitalization rate for high-risk children by anti-RSV antibody prophylaxis. RSV has been shown to modify the immune response to infection, a feature linked in part to RSV G protein CX3C Chemokine mimicry. This study determined if vaccination with G protein polypeptides or peptides spanning the central conserved region of the G protein could induce antibodies that blocked G protein CX3C-CX3CR1 interaction and disease pathogenesis mediated by RSV infection. The results show that mice vaccinated with G protein peptides or polypeptides containing the CX3C motif generate antibodies that inhibit G protein CX3C-CX3CR1 binding and chemotaxis, reduce lung virus titers, and prevent body weight loss and pulmonary inflammation. The results suggest that RSV vaccines that induce antibodies that block G protein CX3C-CX3CR1 interaction may offer a new, safe, and efficacious RSV vaccine strategy.
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Therapeutic Monoclonal Antibody Treatment Targeting Respiratory Syncytial Virus (RSV) G Protein Mediates Viral Clearance and Reduces the Pathogenesis of RSV Infection in BALB/c Mice
The Journal of infectious diseases, 2009Co-Authors: Lia M. Haynes, Ralph A. Tripp, Jennifer L Harcourt, Congrong Miao, Hayat Caidi, Gertrud U. Radu, Larry J. AndersonAbstract:Because the G protein of respiratory syncytial virus (RSV) has a CX3C Chemokine motif that has been associated with the ability of RSV G protein to modulate the virus-induced host immune response, we examined whether therapeutic treatment with an anti-RSV G monoclonal antibody (mAb), 131-2G, that blocks the CX3C-associated activity of RSV G protein might decrease the pulmonary inflammation associated with infection in BALB/c mice. The results show that treatment with mAb 131-2G on day 3 after RSV infection reduces both inflammation and RSV titer in the lungs. Later administration of anti-RSV G mAb (day 5 after RSV infection) effectively reduced the viral titer but had a minimal effect on pulmonary inflammation. This study suggests that an anti-RSV G mAb might be an effective antiviral, either alone or in combination with anti-RSV F protein neutralizing antibodies, for decreasing the virus-induced host response to infection and improve treatment outcome.
Thue W. Schwartz - One of the best experts on this subject based on the ideXlab platform.
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CC and CX3C Chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor
The Journal of biological chemistry, 2004Co-Authors: Paola Casarosa, Thomas N Kledal, Thue W. Schwartz, Maria Waldhoer, Patricia J. Liwang, Henry F. Vischer, Henk Timmerman, Martine J. Smit, Rob LeursAbstract:Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human Chemokine receptor CCR1 and binds several Chemokines of the CC family as well as the CX3C Chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a Chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between Chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C Chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of Chemokine dimerization and interaction with glycosaminoglycans (GAGs) on Chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing Chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.
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Molecular Determinants of Receptor Binding and Signaling by the CX3C Chemokine Fractalkine
The Journal of biological chemistry, 2001Co-Authors: Laura S. Mizoue, Kevin B Bacon, Thomas N Kledal, Thue W. Schwartz, Susan K. Sullivan, David S. King, Tracy M. HandelAbstract:Fractalkine/CX3CL1 is a membrane-tethered Chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's Chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to Chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC Chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.
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The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling.
Molecular biology of the cell, 2001Co-Authors: Alberto Fraile-ramos, Thomas N Kledal, Thue W. Schwartz, Annegret Pelchen-matthews, Katherine Bowers, Mark MarshAbstract:Genes encoding Chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) Chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX3C-Chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host Chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.
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Selective recognition of the membrane‐bound CX3C Chemokine, fractalkine, by the human cytomegalovirus‐encoded broad‐spectrum receptor US28
FEBS letters, 1998Co-Authors: Thomas N Kledal, Mette M. Rosenkilde, Thue W. SchwartzAbstract:The 7TM receptor, US28, encoded by human cytomegalovirus binds a broad spectrum of endogenous CC Chemokines with sub-nanomolar affinity as determined in homologous competition binding assays. We here find that US28 also recognizes the membrane-associated CX3C Chemokine, fractalkine, with sub-nanomolar affinity (IC50=0.42+/-0.09 nM). Importantly, although fractalkine could compete with high affinity against the binding of CC Chemokines, the secreted CC Chemokines were only able to compete for binding against radioactive fractalkine with very low affinity. It is concluded that US28, which is known to enhance cell-cell fusion processes through interaction with an as yet unidentified, human cell-specific factor, has been optimized by cytomegalovirus to selectively recognize the membrane-associated fractalkine. It is suggested that US28 expressed on the surface of infected cells and possibly on the envelope of the virion is involved in transfer of the virus from cell to cell.
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selective recognition of the membrane bound CX3C Chemokine fractalkine by the human cytomegalovirus encoded broad spectrum receptor us28
FEBS Letters, 1998Co-Authors: Thomas N Kledal, Mette M. Rosenkilde, Thue W. SchwartzAbstract:The 7TM receptor, US28, encoded by human cytomegalovirus binds a broad spectrum of endogenous CC Chemokines with sub-nanomolar affinity as determined in homologous competition binding assays. We here find that US28 also recognizes the membrane-associated CX3C Chemokine, fractalkine, with sub-nanomolar affinity (IC50=0.42+/-0.09 nM). Importantly, although fractalkine could compete with high affinity against the binding of CC Chemokines, the secreted CC Chemokines were only able to compete for binding against radioactive fractalkine with very low affinity. It is concluded that US28, which is known to enhance cell-cell fusion processes through interaction with an as yet unidentified, human cell-specific factor, has been optimized by cytomegalovirus to selectively recognize the membrane-associated fractalkine. It is suggested that US28 expressed on the surface of infected cells and possibly on the envelope of the virion is involved in transfer of the virus from cell to cell.
Thomas N Kledal - One of the best experts on this subject based on the ideXlab platform.
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human cytomegalovirus Chemokine receptor us28 induces migration of cells on a CX3Cl1 presenting surface
Journal of General Virology, 2013Co-Authors: Gertrud Malene Hjorto, Katrine Kiilerichpedersen, D Selmeczi, Thomas N Kledal, Niels Bent LarsenAbstract:Human cytomegalovirus (HCMV)-encoded G protein-coupled-receptor US28 is believed to participate in virus dissemination through modulation of cell migration and immune evasion. US28 binds different CC Chemokines and the CX3C Chemokine CX3CL1. Membrane-anchored CX3CL1 is expressed by immune-activated endothelial cells, causing redirection of CX3CR1-expressing leukocytes in the blood to sites of infection. Here, we used stable transfected cell lines to examine how US28 expression affects cell migration on immobilized full-length CX3CL1, to model how HCMV-infected leukocytes interact with inflamed endothelium. We observed that US28-expressing cells migrated more than CX3CR1-expressing cells when adhering to immobilized CX3CL1. US28-induced migration was G protein-signalling dependent and was blocked by the phospholipase Cβ inhibitor U73122 and the intracellular calcium chelator BAPTA-AM. In addition, migration was inhibited in a dose-dependent manner by competition from CCL2 and CCL5, whereas CCL3 had little effect. Instead of migrating, CX3CR1-expressing cells performed ‘dancing-on-the-spot’ movements, demonstrating that anchored CX3CL1 acts as a strong tether for these cells. At low receptor expression levels, however, no significant difference in migration potential was observed when comparing the migration of CX3CR1- and US28-expressing cells. Thus, these data showed that, in contrast to CX3CR1, which promotes efficient cell capture upon binding to anchored CX3CL1, US28 acts to increase the migration of cells upon binding to the same ligand. Overall, this indicates that infected cells probably move more than uninfected cells in inflamed tissues with high CX3CL1 expression, with soluble Chemokines affecting the final migration.
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CC and CX3C Chemokines differentially interact with the N terminus of the human cytomegalovirus-encoded US28 receptor
The Journal of biological chemistry, 2004Co-Authors: Paola Casarosa, Thomas N Kledal, Thue W. Schwartz, Maria Waldhoer, Patricia J. Liwang, Henry F. Vischer, Henk Timmerman, Martine J. Smit, Rob LeursAbstract:Human cytomegalovirus (HCMV) is the causative agent of life-threatening systemic diseases in immunocompromised patients as well as a risk factor for vascular pathologies, like atherosclerosis, in immunocompetent individuals. HCMV encodes a G-protein-coupled receptor (GPCR), referred to as US28, that displays homology to the human Chemokine receptor CCR1 and binds several Chemokines of the CC family as well as the CX3C Chemokine fractalkine with high affinity. Most importantly, following HCMV infection, US28 activates several intracellular pathways, either constitutively or in a Chemokine-dependent manner. In this study, our goal was to understand the molecular interactions between Chemokines and the HCMV-encoded US28 receptor. To achieve this goal, a double approach has been used, consisting in the analysis of both receptor and ligand mutants. This approach has led us to identify several amino acids located in the N terminus of US28 that differentially contribute to the high affinity binding of CC versus CX3C Chemokines. Additionally, our results highlight the importance of secondary modifications occurring at US28, such as sulfation, for ligand recognition. Finally, the effects of Chemokine dimerization and interaction with glycosaminoglycans (GAGs) on Chemokine binding and activation of US28 were investigated as well using CCL4 as model ligand. In line with the two-state model describing Chemokine/receptor interaction, we show that an aromatic residue in the N-loop region of CCL4 promotes tight binding to US28, whereas receptor activation depends on the presence of the N terminus of CCL4, as shown previously for CCR5.
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Molecular Determinants of Receptor Binding and Signaling by the CX3C Chemokine Fractalkine
The Journal of biological chemistry, 2001Co-Authors: Laura S. Mizoue, Kevin B Bacon, Thomas N Kledal, Thue W. Schwartz, Susan K. Sullivan, David S. King, Tracy M. HandelAbstract:Fractalkine/CX3CL1 is a membrane-tethered Chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's Chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to Chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC Chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.
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The human cytomegalovirus US28 protein is located in endocytic vesicles and undergoes constitutive endocytosis and recycling.
Molecular biology of the cell, 2001Co-Authors: Alberto Fraile-ramos, Thomas N Kledal, Thue W. Schwartz, Annegret Pelchen-matthews, Katherine Bowers, Mark MarshAbstract:Genes encoding Chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) Chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX3C-Chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host Chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.
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Selective recognition of the membrane‐bound CX3C Chemokine, fractalkine, by the human cytomegalovirus‐encoded broad‐spectrum receptor US28
FEBS letters, 1998Co-Authors: Thomas N Kledal, Mette M. Rosenkilde, Thue W. SchwartzAbstract:The 7TM receptor, US28, encoded by human cytomegalovirus binds a broad spectrum of endogenous CC Chemokines with sub-nanomolar affinity as determined in homologous competition binding assays. We here find that US28 also recognizes the membrane-associated CX3C Chemokine, fractalkine, with sub-nanomolar affinity (IC50=0.42+/-0.09 nM). Importantly, although fractalkine could compete with high affinity against the binding of CC Chemokines, the secreted CC Chemokines were only able to compete for binding against radioactive fractalkine with very low affinity. It is concluded that US28, which is known to enhance cell-cell fusion processes through interaction with an as yet unidentified, human cell-specific factor, has been optimized by cytomegalovirus to selectively recognize the membrane-associated fractalkine. It is suggested that US28 expressed on the surface of infected cells and possibly on the envelope of the virion is involved in transfer of the virus from cell to cell.
