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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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“RK” residues of Agnoprotein are necessary for viroporin activity.(A) HygB permeability assays with HeLa cells transfected with control vector (Mock), pCFPNLS-Agno (Agno), pCFPNLS-N46 (N46), or pCFPNLS-RK8AA (RK8AA). (B) Viral growth was monitored by indirect immunofluorescence of VP1 (*p<0.001). (C) Whole cell lysates (WCL) and culture supernatants (SUP) from SVG-A cells transfected with wtJCV or RK8AAJCV at the indicated days post-transfection were analyzed by immunoblotting with an anti-VP1 antibody. Culture supernatants were concentrated to 25 times by centrifugation. (D) Electron microscopic analysis of wtJCV- or RK8AAJCV–transfected SVG-A cells. NT: not transfected. The boxed areas in the upper panel are shown at higher magnification in the lower panel. Viral particles of about 40-nm in diameter were in the nuclei (N) of cells transfected with either wtJCV or RK8AAJCV. Scale bars in upper and lower panels, 500 and 100 nm, respectively. C: cytoplasm. E: extracellular space.
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ppat-1000801-g008: “RK” residues of Agnoprotein are necessary for viroporin activity.(A) HygB permeability assays with HeLa cells transfected with control vector (Mock), pCFPNLS-Agno (Agno), pCFPNLS-N46 (N46), or pCFPNLS-RK8AA (RK8AA). (B) Viral growth was monitored by indirect immunofluorescence of VP1 (*p<0.001). (C) Whole cell lysates (WCL) and culture supernatants (SUP) from SVG-A cells transfected with wtJCV or RK8AAJCV at the indicated days post-transfection were analyzed by immunoblotting with an anti-VP1 antibody. Culture supernatants were concentrated to 25 times by centrifugation. (D) Electron microscopic analysis of wtJCV- or RK8AAJCV–transfected SVG-A cells. NT: not transfected. The boxed areas in the upper panel are shown at higher magnification in the lower panel. Viral particles of about 40-nm in diameter were in the nuclei (N) of cells transfected with either wtJCV or RK8AAJCV. Scale bars in upper and lower panels, 500 and 100 nm, respectively. C: cytoplasm. E: extracellular space.

Mentions: To further investigate the viroporin activity, we performed a HygB permeability assay with agnoprotein mutants (N46 mutant and RK8AA mutant), which localize at the ER similar to WT protein (Figure 5). In N46 mutant- or WT Agno-transfected HeLa cells, the amount of radio-labeled CFPNLS was markedly decreased in the presence of HygB compared to its absence (Figure 8A). In contrast, the levels of CFPNLS were unaltered in mock or RK8AA- transfected cells (Figure 8A), suggesting that WT Agno and N46, but not RK8AA, enhances membrane permeabilization. To further demonstrate the importance of this viroporin activity, we generated viral genomes containing the RK8AA mutation of agnoprotein (RK8AAJCV) and performed JCV growth assays. Viral growth was measured by monitoring the percentage of VP1 positive cells (Figure 8B). At 3 and 5 days post-transfection, the percentage of VP1 positive cells were similar in wtJCV- and RK8AAJCV-transfected cells (Figure 8B). However, by 9 days the percentage of VP1 positive cells of RK8AAJCV-transfected cells had significantly decreased compared to that at day 5, whereas that continued to increase in wtJCV-transfected cells (Figure 8B), suggesting that RK8AA mutation of agnoprotein was associated with impaired viral propagation. The amount of VP1 in the culture supernatant of RK8AAJCV-transfected cells was, as expected, substantially decreased (SUP in Figure 8C and S6B). The intracellular localization and expression level of agnoprotein of RK8AAJCV-transfected cells was similar to those of wtJCV-transfected cells (Figures S5A and B). These results suggested that RK8AAJCV has a defect in the release of progeny virus. Transmission electron microscopy was carried out to evaluate whether the RK8AA mutant of agnoprotein influences the virion assembly. This revealed the presence of virus particles of 40-nm in diameter in the nuclei of RK8AAJCV-transfected cells similar to that observed in wtJCV-transfected cells at 5 days post-transfection (Figure 8D, left and middle columns). In contrast, particles were not observed in cells without transfection (Figure 8D, right columns). The virions extracted from the cells transfected with RK8AAJCV were able to infect to the SVG-A cells similar to that of wtJCV and thus were not defective (Figure S5C). These findings show that the inability of the RK8AAJCV to propagate viral infection is due to a defect(s) in virion release. Taken together, Arg-8 and Lys-9 in the NH2-terminus of agnoprotein are necessary for viroporin activities, which permeabilize the cell membrane and release virions from the JCV infected cells.


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)

“RK” residues of Agnoprotein are necessary for viroporin activity.(A) HygB permeability assays with HeLa cells transfected with control vector (Mock), pCFPNLS-Agno (Agno), pCFPNLS-N46 (N46), or pCFPNLS-RK8AA (RK8AA). (B) Viral growth was monitored by indirect immunofluorescence of VP1 (*p<0.001). (C) Whole cell lysates (WCL) and culture supernatants (SUP) from SVG-A cells transfected with wtJCV or RK8AAJCV at the indicated days post-transfection were analyzed by immunoblotting with an anti-VP1 antibody. Culture supernatants were concentrated to 25 times by centrifugation. (D) Electron microscopic analysis of wtJCV- or RK8AAJCV–transfected SVG-A cells. NT: not transfected. The boxed areas in the upper panel are shown at higher magnification in the lower panel. Viral particles of about 40-nm in diameter were in the nuclei (N) of cells transfected with either wtJCV or RK8AAJCV. Scale bars in upper and lower panels, 500 and 100 nm, respectively. C: cytoplasm. E: extracellular space.
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Related In: Results  -  Collection

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

ppat-1000801-g008: “RK” residues of Agnoprotein are necessary for viroporin activity.(A) HygB permeability assays with HeLa cells transfected with control vector (Mock), pCFPNLS-Agno (Agno), pCFPNLS-N46 (N46), or pCFPNLS-RK8AA (RK8AA). (B) Viral growth was monitored by indirect immunofluorescence of VP1 (*p<0.001). (C) Whole cell lysates (WCL) and culture supernatants (SUP) from SVG-A cells transfected with wtJCV or RK8AAJCV at the indicated days post-transfection were analyzed by immunoblotting with an anti-VP1 antibody. Culture supernatants were concentrated to 25 times by centrifugation. (D) Electron microscopic analysis of wtJCV- or RK8AAJCV–transfected SVG-A cells. NT: not transfected. The boxed areas in the upper panel are shown at higher magnification in the lower panel. Viral particles of about 40-nm in diameter were in the nuclei (N) of cells transfected with either wtJCV or RK8AAJCV. Scale bars in upper and lower panels, 500 and 100 nm, respectively. C: cytoplasm. E: extracellular space.
Mentions: To further investigate the viroporin activity, we performed a HygB permeability assay with agnoprotein mutants (N46 mutant and RK8AA mutant), which localize at the ER similar to WT protein (Figure 5). In N46 mutant- or WT Agno-transfected HeLa cells, the amount of radio-labeled CFPNLS was markedly decreased in the presence of HygB compared to its absence (Figure 8A). In contrast, the levels of CFPNLS were unaltered in mock or RK8AA- transfected cells (Figure 8A), suggesting that WT Agno and N46, but not RK8AA, enhances membrane permeabilization. To further demonstrate the importance of this viroporin activity, we generated viral genomes containing the RK8AA mutation of agnoprotein (RK8AAJCV) and performed JCV growth assays. Viral growth was measured by monitoring the percentage of VP1 positive cells (Figure 8B). At 3 and 5 days post-transfection, the percentage of VP1 positive cells were similar in wtJCV- and RK8AAJCV-transfected cells (Figure 8B). However, by 9 days the percentage of VP1 positive cells of RK8AAJCV-transfected cells had significantly decreased compared to that at day 5, whereas that continued to increase in wtJCV-transfected cells (Figure 8B), suggesting that RK8AA mutation of agnoprotein was associated with impaired viral propagation. The amount of VP1 in the culture supernatant of RK8AAJCV-transfected cells was, as expected, substantially decreased (SUP in Figure 8C and S6B). The intracellular localization and expression level of agnoprotein of RK8AAJCV-transfected cells was similar to those of wtJCV-transfected cells (Figures S5A and B). These results suggested that RK8AAJCV has a defect in the release of progeny virus. Transmission electron microscopy was carried out to evaluate whether the RK8AA mutant of agnoprotein influences the virion assembly. This revealed the presence of virus particles of 40-nm in diameter in the nuclei of RK8AAJCV-transfected cells similar to that observed in wtJCV-transfected cells at 5 days post-transfection (Figure 8D, left and middle columns). In contrast, particles were not observed in cells without transfection (Figure 8D, right columns). The virions extracted from the cells transfected with RK8AAJCV were able to infect to the SVG-A cells similar to that of wtJCV and thus were not defective (Figure S5C). These findings show that the inability of the RK8AAJCV to propagate viral infection is due to a defect(s) in virion release. Taken together, Arg-8 and Lys-9 in the NH2-terminus of agnoprotein are necessary for viroporin activities, which permeabilize the cell membrane and release virions from the JCV infected cells.

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