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The human polyoma JC virus agnoprotein acts as a viroporin.

Suzuki T, Orba Y, Okada Y, Sunden Y, Kimura T, Tanaka S, Nagashima K, Hall WW, Sawa H - PLoS Pathog. (2010)

Bottom Line: Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV.Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins.Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.

ABSTRACT
Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca(2+) homeostasis leading to membrane dysfunction and enhancement of virus release.

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The N-terminus of agnoprotein is necessary for targeting to the ER.(A) Hydrophobicity plot of JCV agnoprotein. The plot was drawn using the Lyte-Doolittle method of calculating hydrophilicity over a window length of seven [54]. (B) The N-terminal region of agnoprotein is characterized by the presence of positively charged residues. Schematic representation of GST-EGFP fusion constructs of wild type (WT) agnoprotein and its various mutants. The green boxes indicate the basic amino acid clusters, which could be important for determining the orientation to the membrane. A gray box indicates a hydrophobic amino acid stretch. (C) Immunoblot analyses of GST-EGFP–fused agnoprotein and its mutants prepared from the ER-nuclear fraction of transfected HEK293 cells prepared by sucrose density centrifugation as described in the Materials and Methods. WT and N46 mutant were detected in the ER-rich fractions, whereas the C6 mutant and GST-EGFP were not.
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ppat-1000801-g004: The N-terminus of agnoprotein is necessary for targeting to the ER.(A) Hydrophobicity plot of JCV agnoprotein. The plot was drawn using the Lyte-Doolittle method of calculating hydrophilicity over a window length of seven [54]. (B) The N-terminal region of agnoprotein is characterized by the presence of positively charged residues. Schematic representation of GST-EGFP fusion constructs of wild type (WT) agnoprotein and its various mutants. The green boxes indicate the basic amino acid clusters, which could be important for determining the orientation to the membrane. A gray box indicates a hydrophobic amino acid stretch. (C) Immunoblot analyses of GST-EGFP–fused agnoprotein and its mutants prepared from the ER-nuclear fraction of transfected HEK293 cells prepared by sucrose density centrifugation as described in the Materials and Methods. WT and N46 mutant were detected in the ER-rich fractions, whereas the C6 mutant and GST-EGFP were not.

Mentions: We next sought to identify the motif(s) of agnoprotein which are necessary for targeting the ER. A Kyte and Doolittle hydrophobicity plot of agnoprotein revealed that the middle region of agnoprotein contains the only hydrophobic region. This is comprised of 18 amino acids and would serve as a potential transmembrane segment (Figure 4A). To determine the domain of agnoprotein important for its ER distribution, we generated a series of mutants as fusion proteins with the GST-EGFP tag (∼50 kDa) (Figure 4B). Immunoblot analyses of GST-EGFP–fused agnoprotein and its mutants were prepared from the ER-nuclear fraction of transfected HEK293 cells by sucrose density centrifugation. WT and N46 mutant were detected in the ER-rich fractions, but the C6 mutant and GST-EGFP were not (Figure 4C). This result suggested that the N-terminus of agnoprotein was necessary for targeting to the ER.


The human polyoma JC virus agnoprotein acts as a viroporin.

Suzuki T, Orba Y, Okada Y, Sunden Y, Kimura T, Tanaka S, Nagashima K, Hall WW, Sawa H - PLoS Pathog. (2010)

The N-terminus of agnoprotein is necessary for targeting to the ER.(A) Hydrophobicity plot of JCV agnoprotein. The plot was drawn using the Lyte-Doolittle method of calculating hydrophilicity over a window length of seven [54]. (B) The N-terminal region of agnoprotein is characterized by the presence of positively charged residues. Schematic representation of GST-EGFP fusion constructs of wild type (WT) agnoprotein and its various mutants. The green boxes indicate the basic amino acid clusters, which could be important for determining the orientation to the membrane. A gray box indicates a hydrophobic amino acid stretch. (C) Immunoblot analyses of GST-EGFP–fused agnoprotein and its mutants prepared from the ER-nuclear fraction of transfected HEK293 cells prepared by sucrose density centrifugation as described in the Materials and Methods. WT and N46 mutant were detected in the ER-rich fractions, whereas the C6 mutant and GST-EGFP were not.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2837404&req=5

ppat-1000801-g004: The N-terminus of agnoprotein is necessary for targeting to the ER.(A) Hydrophobicity plot of JCV agnoprotein. The plot was drawn using the Lyte-Doolittle method of calculating hydrophilicity over a window length of seven [54]. (B) The N-terminal region of agnoprotein is characterized by the presence of positively charged residues. Schematic representation of GST-EGFP fusion constructs of wild type (WT) agnoprotein and its various mutants. The green boxes indicate the basic amino acid clusters, which could be important for determining the orientation to the membrane. A gray box indicates a hydrophobic amino acid stretch. (C) Immunoblot analyses of GST-EGFP–fused agnoprotein and its mutants prepared from the ER-nuclear fraction of transfected HEK293 cells prepared by sucrose density centrifugation as described in the Materials and Methods. WT and N46 mutant were detected in the ER-rich fractions, whereas the C6 mutant and GST-EGFP were not.
Mentions: We next sought to identify the motif(s) of agnoprotein which are necessary for targeting the ER. A Kyte and Doolittle hydrophobicity plot of agnoprotein revealed that the middle region of agnoprotein contains the only hydrophobic region. This is comprised of 18 amino acids and would serve as a potential transmembrane segment (Figure 4A). To determine the domain of agnoprotein important for its ER distribution, we generated a series of mutants as fusion proteins with the GST-EGFP tag (∼50 kDa) (Figure 4B). Immunoblot analyses of GST-EGFP–fused agnoprotein and its mutants were prepared from the ER-nuclear fraction of transfected HEK293 cells by sucrose density centrifugation. WT and N46 mutant were detected in the ER-rich fractions, but the C6 mutant and GST-EGFP were not (Figure 4C). This result suggested that the N-terminus of agnoprotein was necessary for targeting to the ER.

Bottom Line: Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV.Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins.Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Pathobiology, Hokkaido University Research Center for Zoonosis Control, Sapporo, Japan.

ABSTRACT
Virus infections can result in a range of cellular injuries and commonly this involves both the plasma and intracellular membranes, resulting in enhanced permeability. Viroporins are a group of proteins that interact with plasma membranes modifying permeability and can promote the release of viral particles. While these proteins are not essential for virus replication, their activity certainly promotes virus growth. Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease resulting from lytic infection of oligodendrocytes by the polyomavirus JC virus (JCV). The genome of JCV encodes six major proteins including a small auxiliary protein known as agnoprotein. Studies on other polyomavirus agnoproteins have suggested that the protein may contribute to viral propagation at various stages in the replication cycle, including transcription, translation, processing of late viral proteins, assembly of virions, and viral propagation. Previous studies from our and other laboratories have indicated that JCV agnoprotein plays an important, although as yet incompletely understood role in the propagation of JCV. Here, we demonstrate that agnoprotein possesses properties commonly associated with viroporins. Our findings demonstrate that: (i) A deletion mutant of agnoprotein is defective in virion release and viral propagation; (ii) Agnoprotein localizes to the ER early in infection, but is also found at the plasma membrane late in infection; (iii) Agnoprotein is an integral membrane protein and forms homo-oligomers; (iv) Agnoprotein enhances permeability of cells to the translation inhibitor hygromycin B; (v) Agnoprotein induces the influx of extracellular Ca(2+); (vi) The basic residues at amino acid positions 8 and 9 of agnoprotein key are determinants of the viroporin activity. The viroporin-like properties of agnoprotein result in increased membrane permeability and alterations in intracellular Ca(2+) homeostasis leading to membrane dysfunction and enhancement of virus release.

Show MeSH
Related in: MedlinePlus