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Pavithra Aravamudhan - One of the best experts on this subject based on the ideXlab platform.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
bioRxiv, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:ABSTRACT Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA-in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
Kelly Urbanek - One of the best experts on this subject based on the ideXlab platform.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
bioRxiv, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:ABSTRACT Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA-in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
Danica M Sutherland - One of the best experts on this subject based on the ideXlab platform.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
bioRxiv, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:ABSTRACT Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA-in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
Jonathan J Knowlton - One of the best experts on this subject based on the ideXlab platform.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
Journal of Virology, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:Engagement of cell surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein σ1 binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements for different cell types are not entirely known. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, CMP N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains that differ in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for reovirus infection. Following CRISPR-Cas9 disruption of either gene, cell surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Furthermore, infectivity of T3SA+ was diminished to levels similar to those of T3SA- in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection.IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface and mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
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cytidine monophosphate n acetylneuraminic acid synthetase and solute carrier family 35 member a1 are required for reovirus binding and infection
bioRxiv, 2020Co-Authors: Kelly Urbanek, Danica M Sutherland, Robert C Orchard, Craig B Wilen, Jonathan J Knowlton, Pavithra AravamudhanAbstract:ABSTRACT Engagement of cell-surface receptors by viruses is a critical determinant of viral tropism and disease. The reovirus attachment protein, σ1, binds sialylated glycans and proteinaceous receptors to mediate infection, but the specific requirements on different cell types are unknown. To identify host factors required for reovirus-induced cell death, we conducted a CRISPR-knockout screen targeting over 20,000 genes in murine microglial BV2 cells. Candidate genes identified as required for reovirus to cause cell death were highly enriched for sialic acid synthesis and transport. Two of the top candidates identified, cytidine monophosphate N-acetylneuraminic acid synthetase (Cmas) and solute carrier family 35 member A1 (Slc35a1), promote sialic acid expression on the cell surface. Two reovirus strains differing in the capacity to bind sialic acid, T3SA+ and T3SA-, were used to evaluate Cmas and Slc35a1 as potential host genes required for infection. Following CRISPR-Cas9 disruption of either gene, cell-surface expression of sialic acid was diminished. These results correlated with decreased binding of strain T3SA+, which is capable of engaging sialic acid. Disruption of either gene did not alter the low-level binding of T3SA-, which does not engage sialic acid. Infectivity of T3SA+ was diminished to levels of T3SA-in cells lacking Cmas and Slc35a1 by CRISPR ablation. However, exogenous expression of Cmas and Slc35a1 into the respective null cells restored sialic acid expression and T3SA+ binding and infectivity. These results demonstrate that Cmas and Slc35a1, which mediate cell-surface expression of sialic acid, are required in murine microglial cells for efficient reovirus binding and infection. IMPORTANCE Attachment factors and receptors are important determinants of dissemination and tropism during reovirus-induced disease. In a CRISPR cell-survival screen, we discovered two genes, Cmas and Slc35a1, which encode proteins required for sialic acid expression on the cell surface, that mediate reovirus infection of microglial cells. This work elucidates host genes that render microglial cells susceptible to reovirus infection and expands current understanding of the receptors on microglial cells that are engaged by reovirus. Such knowledge may lead to new strategies to selectively target microglial cells for oncolytic applications.
Dirk Lefeber - One of the best experts on this subject based on the ideXlab platform.
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high resolution mass spectrometry glycoprofiling of intact transferrin for diagnosis and subtype identification in the congenital disorders of glycosylation
Translational Research, 2015Co-Authors: Monique Van Scherpenzeel, Ron A. Wevers, Eva Morava, Gerry Steenbergen, Dirk LefeberAbstract:Diagnostic screening of the congenital disorders of glycosylation (CDG) generally involves isoelectric focusing of plasma transferrin, a robust method easily integrated in medical laboratories. Structural information is needed as the next step, as required for the challenging classification of Golgi glycosylation defects (CDG-II). Here, we present the use of high-resolution nano liquid chromatography-chip (C8)-quadrupole time of flight mass spectrometry (nanoLC-chip [C8]-QTOF MS) for protein-specific glycoprofiling of intact transferrin, which allows screening and direct diagnosis of a number of CDG-II defects. Transferrin was immunopurified from 10 μL of plasma and analyzed by nanoLC-chip-QTOF MS. Charge distribution raw data were deconvoluted by Mass Hunter software to reconstructed mass spectra. Plasma samples were processed from controls (n = 56), patients with known defects (n = 30), and patients with secondary (n = 6) or unsolved (n = 3) cause of abnormal glycosylation. This fast and robust method, established for CDG diagnostics, requires only 2 hours analysis time, including sample preparation and analysis. For CDG-I patients, the characteristic loss of complete N-glycans could be detected with high sensitivity. Known CDG-II defects (phosphoglucomutase 1 [PGM1-CDG], mannosyl (α-1,6-)-glycoprotein β-1,2-N-acetylglucosaminyltransferase [MGAT2-CDG], β-1,4-galactosyltransferase 1 [B4GALT1-CDG], CMP-sialic acid transporter [SLC35A1-CDG], UDP-galactose transporter [SLC35A2-CDG] and mannosyl-oligosaccharide 1,2-alpha-mannosidase [MAN1B1-CDG]) resulted in characteristic diagnostic profiles. Moreover, in the group of Golgi trafficking defects and unsolved CDG-II patients, distinct profiles were observed, which facilitate identification of the specific CDG subtype. The established QTOF method affords high sensitivity and resolution for the detection of complete glycan loss and structural assignment of truncated glycans in a single assay. The speed and robustness allow its clinical diagnostic application as a first step in the diagnostic procedure for CDG defects.
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plasma n glycan profiling by mass spectrometry for congenital disorders of glycosylation type ii
Clinical Chemistry, 2011Co-Authors: Mailys Guillard, Christian Korner, Ron A. Wevers, Eva Morava, Floris L Van Delft, Rosie Hague, Maciej Adamowicz, Dirk LefeberAbstract:BACKGROUND: Determination of the genetic defect in patients with a congenital disorder of glycosylation (CDG) is challenging because of the wide clinical presentation, the large number of gene products involved, and the occurrence of secondary causes of underglycosylation. Transferrin isoelectric focusing has been the method of choice for CDG screening; however, improved methods are required for the molecular diagnosis of patients with CDG type II. METHODS: Plasma samples with a typical transferrin isofocusing profile were analyzed. N-glycans were released from these samples by PNGase F [peptide- N 4-(acetyl-β-glucosaminyl)-asparagine amidase] digestion, permethylated and purified, and measured on a MALDI linear ion trap mass spectrometer. A set of 38 glycans was used for quantitative comparison and to establish reference intervals for such glycan features as the number of antennae, the level of truncation, and fucosylation. Plasma N-glycans from control individuals, patients with known CDG type II defects, and patients with a secondary cause of underglycosylation were analyzed. RESULTS: CDGs due to mannosyl (α-1,6-)-glycoprotein β-1,2- N -acetylglucosaminyltransferase (MGAT2), β-1,4-galactosyltransferase 1 (B4GALT1), and SLC35C1 (a GDP-fucose transporter) defects could be diagnosed directly from the N-glycan profile. CDGs due to defects in proteins involved in Golgi trafficking, such as subunit 7 of the conserved oligomeric Golgi complex (COG7) and subunit V0 a2 of the lysosomal H+-transporting ATPase (ATP6V0A2) caused a loss of triantennary N-glycans and an increase of truncated structures. Secondary causes with liver involvement were characterized by increased fucosylation, whereas the presence of plasma sialidase produced isolated undersialylation. CONCLUSIONS: MALDI ion trap analysis of plasma N-glycans documents features that discriminate between primary and secondary causes of underglycosylation and should be applied as the first step in the diagnostic track of all patients with an unsolved CDG type II.