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Infectious bursal disease virus VP5 polypeptide: a phosphoinositide-binding protein required for efficient cell-to-cell virus dissemination.

Méndez F, de Garay T, Rodríguez D, Rodríguez JF - PLoS ONE (2015)

Bottom Line: We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting.Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding.Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología-CSIC, Cantoblanco, 28049, Madrid, Spain.

ABSTRACT
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a major avian pathogen responsible for an immunosuppressive disease affecting juvenile chickens. The IBDV genome is formed by two dsRNA segments. The largest one harbors two partially overlapping open reading frames encoding a non-structural polypeptide, known as VP5, and a large polyprotein, respectively. VP5 is non-essential for virus replication. However, it plays a major role in IBDV pathogenesis. VP5 accumulates at the plasma membrane (PM) of IBDV-infected cells. We have analyzed the mechanism underlying the VP5 PM targeting. Updated topological prediction algorithm servers fail to identify a transmembrane domain within the VP5 sequence. However, the VP5 polycationic C-terminal region, harboring three closely spaced patches formed by two or three consecutive basic amino acid residues (lysine or arginine), might account for its PM tropism. We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting. Lipid overlay assays performed with an affinity-purified Flag-tagged VP5 (FVP5) protein version show that this polypeptide binds several phosphoinositides (PIP), exhibiting a clear preference for monophosphate species. Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding. Data gathered with IBDV mutants expressing C-terminal deleted VP5 polypeptides generated by reverse genetics demonstrate that the VP5-PIP binding domain is required both for its PM targeting in infected cells, and for efficient virus dissemination. Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism.

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The positively charged VP5 C-terminal domain is required for binding to PIPs.A. Affinity purified VP5 polypeptides. The wild type (FVP5) and C-terminal mutant (FVP5Δ15CT and FVP5CTM1) proteins used for this analysis were expressed in E. coli and purified by affinity chromatography using anti-Flag agarose. Purified protein samples were analyzed by SDS-PAGE followed by Coomasie blue staining. B. Protein lipid overlay assay. Membranes spotted with 100 pmol of various lipids were incubated with 5μg/ml of the different proteins. Bound proteins were detected using an anti-Flag mouse mAb, followed by incubation with goat-anti mouse Ig coupled to peroxidase and developed with ECL. The right panel indicates the positions of spots corresponding to the different lipids. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI(3)P, PI(3) phosphate; PI(4)P, PI(4) phosphate; PI(5)P, PI(5) phosphate; PI(3,4)P2, PI(3,4) bisphosphate; PI(3,5)P2, PI(3,5) bisphosphate; PI(4,5)P2, PI(4,5) bisphosphate; PI(3,4,5)P3, PI(3,4,5) trisphosphate; PS, phosphatidylserine; S1P, sphingosine-1-phosphate. C. FVP5 PIP binding. Membranes spotted with a concentration gradient of all eight phosphoinositides were incubated with FVP5. Overlay assays were performed as described above. PIP amounts (pmol) are indicated.
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pone.0123470.g005: The positively charged VP5 C-terminal domain is required for binding to PIPs.A. Affinity purified VP5 polypeptides. The wild type (FVP5) and C-terminal mutant (FVP5Δ15CT and FVP5CTM1) proteins used for this analysis were expressed in E. coli and purified by affinity chromatography using anti-Flag agarose. Purified protein samples were analyzed by SDS-PAGE followed by Coomasie blue staining. B. Protein lipid overlay assay. Membranes spotted with 100 pmol of various lipids were incubated with 5μg/ml of the different proteins. Bound proteins were detected using an anti-Flag mouse mAb, followed by incubation with goat-anti mouse Ig coupled to peroxidase and developed with ECL. The right panel indicates the positions of spots corresponding to the different lipids. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI(3)P, PI(3) phosphate; PI(4)P, PI(4) phosphate; PI(5)P, PI(5) phosphate; PI(3,4)P2, PI(3,4) bisphosphate; PI(3,5)P2, PI(3,5) bisphosphate; PI(4,5)P2, PI(4,5) bisphosphate; PI(3,4,5)P3, PI(3,4,5) trisphosphate; PS, phosphatidylserine; S1P, sphingosine-1-phosphate. C. FVP5 PIP binding. Membranes spotted with a concentration gradient of all eight phosphoinositides were incubated with FVP5. Overlay assays were performed as described above. PIP amounts (pmol) are indicated.

Mentions: In view of results described above, it was important to assess the capacity of the VP5 polypeptide to interact with membrane lipids. For this, affinity-purified FVP5 protein (Fig 5A) was incubated with lipid arrays containing biologically relevant lipids, including phosphatidylinositol (PI) and its seven phosphorylated derivatives (phosphoinositides, PIP), lysophosphatidic acid (LPA), lysophosphocholine (LPC), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), phosphatidic acid (PA) and phosphatidylserine (PS), found at the cytosolic face of membranous cell compartments [27]. As controls for these experiments, assays were also performed with affinity-purified FVP5Δ15CT and FVP5CTM1 mutant proteins (Fig 5A).


Infectious bursal disease virus VP5 polypeptide: a phosphoinositide-binding protein required for efficient cell-to-cell virus dissemination.

Méndez F, de Garay T, Rodríguez D, Rodríguez JF - PLoS ONE (2015)

The positively charged VP5 C-terminal domain is required for binding to PIPs.A. Affinity purified VP5 polypeptides. The wild type (FVP5) and C-terminal mutant (FVP5Δ15CT and FVP5CTM1) proteins used for this analysis were expressed in E. coli and purified by affinity chromatography using anti-Flag agarose. Purified protein samples were analyzed by SDS-PAGE followed by Coomasie blue staining. B. Protein lipid overlay assay. Membranes spotted with 100 pmol of various lipids were incubated with 5μg/ml of the different proteins. Bound proteins were detected using an anti-Flag mouse mAb, followed by incubation with goat-anti mouse Ig coupled to peroxidase and developed with ECL. The right panel indicates the positions of spots corresponding to the different lipids. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI(3)P, PI(3) phosphate; PI(4)P, PI(4) phosphate; PI(5)P, PI(5) phosphate; PI(3,4)P2, PI(3,4) bisphosphate; PI(3,5)P2, PI(3,5) bisphosphate; PI(4,5)P2, PI(4,5) bisphosphate; PI(3,4,5)P3, PI(3,4,5) trisphosphate; PS, phosphatidylserine; S1P, sphingosine-1-phosphate. C. FVP5 PIP binding. Membranes spotted with a concentration gradient of all eight phosphoinositides were incubated with FVP5. Overlay assays were performed as described above. PIP amounts (pmol) are indicated.
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pone.0123470.g005: The positively charged VP5 C-terminal domain is required for binding to PIPs.A. Affinity purified VP5 polypeptides. The wild type (FVP5) and C-terminal mutant (FVP5Δ15CT and FVP5CTM1) proteins used for this analysis were expressed in E. coli and purified by affinity chromatography using anti-Flag agarose. Purified protein samples were analyzed by SDS-PAGE followed by Coomasie blue staining. B. Protein lipid overlay assay. Membranes spotted with 100 pmol of various lipids were incubated with 5μg/ml of the different proteins. Bound proteins were detected using an anti-Flag mouse mAb, followed by incubation with goat-anti mouse Ig coupled to peroxidase and developed with ECL. The right panel indicates the positions of spots corresponding to the different lipids. PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI(3)P, PI(3) phosphate; PI(4)P, PI(4) phosphate; PI(5)P, PI(5) phosphate; PI(3,4)P2, PI(3,4) bisphosphate; PI(3,5)P2, PI(3,5) bisphosphate; PI(4,5)P2, PI(4,5) bisphosphate; PI(3,4,5)P3, PI(3,4,5) trisphosphate; PS, phosphatidylserine; S1P, sphingosine-1-phosphate. C. FVP5 PIP binding. Membranes spotted with a concentration gradient of all eight phosphoinositides were incubated with FVP5. Overlay assays were performed as described above. PIP amounts (pmol) are indicated.
Mentions: In view of results described above, it was important to assess the capacity of the VP5 polypeptide to interact with membrane lipids. For this, affinity-purified FVP5 protein (Fig 5A) was incubated with lipid arrays containing biologically relevant lipids, including phosphatidylinositol (PI) and its seven phosphorylated derivatives (phosphoinositides, PIP), lysophosphatidic acid (LPA), lysophosphocholine (LPC), phosphatidylethanolamine (PE), phosphatidylcholine (PC), sphingosine 1-phosphate (S1P), phosphatidic acid (PA) and phosphatidylserine (PS), found at the cytosolic face of membranous cell compartments [27]. As controls for these experiments, assays were also performed with affinity-purified FVP5Δ15CT and FVP5CTM1 mutant proteins (Fig 5A).

Bottom Line: We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting.Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding.Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular and Cellular Biology, Centro Nacional de Biotecnología-CSIC, Cantoblanco, 28049, Madrid, Spain.

ABSTRACT
Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, is a major avian pathogen responsible for an immunosuppressive disease affecting juvenile chickens. The IBDV genome is formed by two dsRNA segments. The largest one harbors two partially overlapping open reading frames encoding a non-structural polypeptide, known as VP5, and a large polyprotein, respectively. VP5 is non-essential for virus replication. However, it plays a major role in IBDV pathogenesis. VP5 accumulates at the plasma membrane (PM) of IBDV-infected cells. We have analyzed the mechanism underlying the VP5 PM targeting. Updated topological prediction algorithm servers fail to identify a transmembrane domain within the VP5 sequence. However, the VP5 polycationic C-terminal region, harboring three closely spaced patches formed by two or three consecutive basic amino acid residues (lysine or arginine), might account for its PM tropism. We have found that mutations, either C-terminal VP5 deletions or replacement of basic amino acids by alanine residues, that reduce the electropositive charge of the VP5 C-terminus abolish PM targeting. Lipid overlay assays performed with an affinity-purified Flag-tagged VP5 (FVP5) protein version show that this polypeptide binds several phosphoinositides (PIP), exhibiting a clear preference for monophosphate species. Experiments performed with FVP5 mutant proteins lacking the polycationic domain demonstrate that this region is essential for PIP binding. Data gathered with IBDV mutants expressing C-terminal deleted VP5 polypeptides generated by reverse genetics demonstrate that the VP5-PIP binding domain is required both for its PM targeting in infected cells, and for efficient virus dissemination. Data presented here lead us to hypothesize that IBDV might use a non-lytic VP5-dependent cell-to-cell spreading mechanism.

Show MeSH
Related in: MedlinePlus