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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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VP5 C-terminal ablation affects the IBDV plaque size phenotype.A. Plaque assays. QM7 cell monolayers were infected with the VP5 knockout mutant (KO) virus, the wild type (WT) virus and mutant IBDV viruses lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) VP5 C-terminal residues, respectively. After infection, monolayers were covered with semisolid medium. After three days, the medium was removed and virus-induced plaques were detected by immunostaining using serum specifically recognizing the VP2 IBDV capsid polypeptide. B. Statistical analysis. The statistical analysis was carried on images from immnunostained monolayers using the GraphPad Prism software 4.0. A Kruskal-Wallis test was performed followed by Dunn’s multiple comparison tests between lysis plaque areas detected in monolayers infected with the different mutant viruses and those generated by the wild type virus. Presented data correspond to three independent experiments totalizing 750 plaques for each virus. Significant differences (p<0.001) between KO, Δ10CT, Δ14CT and WT viruses are indicated with asterisks (***). C. Extracellular virus yields. QM7 cells were infected with the VP5 knockout mutant (white columns), wild type (black) and mutants viruses VP5Δ3CT (light grey), VP5Δ10CT (medium grey), and Δ14CT (dark grey), at an MOI of 3 PFU/cell. At 8, 16 and 24 h p.i. cultures were harvested and subjected to centrifugation at 15,000xg for 15 min to eliminate cells and large debris. The corresponding supernatants were used to determine extracellular virus titres. Presented data correspond to three independent experiments.
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pone.0123470.g007: VP5 C-terminal ablation affects the IBDV plaque size phenotype.A. Plaque assays. QM7 cell monolayers were infected with the VP5 knockout mutant (KO) virus, the wild type (WT) virus and mutant IBDV viruses lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) VP5 C-terminal residues, respectively. After infection, monolayers were covered with semisolid medium. After three days, the medium was removed and virus-induced plaques were detected by immunostaining using serum specifically recognizing the VP2 IBDV capsid polypeptide. B. Statistical analysis. The statistical analysis was carried on images from immnunostained monolayers using the GraphPad Prism software 4.0. A Kruskal-Wallis test was performed followed by Dunn’s multiple comparison tests between lysis plaque areas detected in monolayers infected with the different mutant viruses and those generated by the wild type virus. Presented data correspond to three independent experiments totalizing 750 plaques for each virus. Significant differences (p<0.001) between KO, Δ10CT, Δ14CT and WT viruses are indicated with asterisks (***). C. Extracellular virus yields. QM7 cells were infected with the VP5 knockout mutant (white columns), wild type (black) and mutants viruses VP5Δ3CT (light grey), VP5Δ10CT (medium grey), and Δ14CT (dark grey), at an MOI of 3 PFU/cell. At 8, 16 and 24 h p.i. cultures were harvested and subjected to centrifugation at 15,000xg for 15 min to eliminate cells and large debris. The corresponding supernatants were used to determine extracellular virus titres. Presented data correspond to three independent experiments.

Mentions: As shown in Fig 7A, lysis plaques formed by the VP5 knockout mutant (IBDV_VP5 KO) are remarkably smaller (over 14xfold) than those observed in cells infected with the wild type virus, thus showing that the blockade of VP5 expression caused by the elimination of its translation initiation codon results in a dramatic reduction of the cell-to-cell spreading capacity of the virus. Noteworthy, the three VP5 C-terminal truncation mutants also exhibited conspicuous reductions, ranging from 1.7–3.1xfold, on the average plaque diameter. Statistical analysis showed that plaque size reductions observed with the VP5Δ10CT and VP5Δ14CT mutants are statistically significant (p<0.001) (Fig 7B).


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)

VP5 C-terminal ablation affects the IBDV plaque size phenotype.A. Plaque assays. QM7 cell monolayers were infected with the VP5 knockout mutant (KO) virus, the wild type (WT) virus and mutant IBDV viruses lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) VP5 C-terminal residues, respectively. After infection, monolayers were covered with semisolid medium. After three days, the medium was removed and virus-induced plaques were detected by immunostaining using serum specifically recognizing the VP2 IBDV capsid polypeptide. B. Statistical analysis. The statistical analysis was carried on images from immnunostained monolayers using the GraphPad Prism software 4.0. A Kruskal-Wallis test was performed followed by Dunn’s multiple comparison tests between lysis plaque areas detected in monolayers infected with the different mutant viruses and those generated by the wild type virus. Presented data correspond to three independent experiments totalizing 750 plaques for each virus. Significant differences (p<0.001) between KO, Δ10CT, Δ14CT and WT viruses are indicated with asterisks (***). C. Extracellular virus yields. QM7 cells were infected with the VP5 knockout mutant (white columns), wild type (black) and mutants viruses VP5Δ3CT (light grey), VP5Δ10CT (medium grey), and Δ14CT (dark grey), at an MOI of 3 PFU/cell. At 8, 16 and 24 h p.i. cultures were harvested and subjected to centrifugation at 15,000xg for 15 min to eliminate cells and large debris. The corresponding supernatants were used to determine extracellular virus titres. Presented data correspond to three independent experiments.
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pone.0123470.g007: VP5 C-terminal ablation affects the IBDV plaque size phenotype.A. Plaque assays. QM7 cell monolayers were infected with the VP5 knockout mutant (KO) virus, the wild type (WT) virus and mutant IBDV viruses lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) VP5 C-terminal residues, respectively. After infection, monolayers were covered with semisolid medium. After three days, the medium was removed and virus-induced plaques were detected by immunostaining using serum specifically recognizing the VP2 IBDV capsid polypeptide. B. Statistical analysis. The statistical analysis was carried on images from immnunostained monolayers using the GraphPad Prism software 4.0. A Kruskal-Wallis test was performed followed by Dunn’s multiple comparison tests between lysis plaque areas detected in monolayers infected with the different mutant viruses and those generated by the wild type virus. Presented data correspond to three independent experiments totalizing 750 plaques for each virus. Significant differences (p<0.001) between KO, Δ10CT, Δ14CT and WT viruses are indicated with asterisks (***). C. Extracellular virus yields. QM7 cells were infected with the VP5 knockout mutant (white columns), wild type (black) and mutants viruses VP5Δ3CT (light grey), VP5Δ10CT (medium grey), and Δ14CT (dark grey), at an MOI of 3 PFU/cell. At 8, 16 and 24 h p.i. cultures were harvested and subjected to centrifugation at 15,000xg for 15 min to eliminate cells and large debris. The corresponding supernatants were used to determine extracellular virus titres. Presented data correspond to three independent experiments.
Mentions: As shown in Fig 7A, lysis plaques formed by the VP5 knockout mutant (IBDV_VP5 KO) are remarkably smaller (over 14xfold) than those observed in cells infected with the wild type virus, thus showing that the blockade of VP5 expression caused by the elimination of its translation initiation codon results in a dramatic reduction of the cell-to-cell spreading capacity of the virus. Noteworthy, the three VP5 C-terminal truncation mutants also exhibited conspicuous reductions, ranging from 1.7–3.1xfold, on the average plaque diameter. Statistical analysis showed that plaque size reductions observed with the VP5Δ10CT and VP5Δ14CT mutants are statistically significant (p<0.001) (Fig 7B).

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