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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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Contribution of the electropositive C-terminal charge to VP5 PM targeting.A. VP5 alanine replacement mutants. Diagram depicting Flag-tagged VP5 proteins expressed by the different recombinant VACVs used in this analysis. The M1 protein lacks the three cationic clusters whilst M2, M3, and M4 lack the 132KR133, the 136KRR138 or the 142RK143 clusters, respectively. A Flag-tagged version of the wild type protein (WT) was used as control. The black box indicates the position of the Flag tag. Positively charged amino acids at the VP5 C-terminus are highlighted in blue, and the replacing alanine residues in black. B. Protein expression analysis. Samples from cells infected with recombinant VACV expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using anti-Flag mAbs. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different recombinant VACVs were processed for IF using a mouse anti-Flag mAb followed by incubation with goat anti-mouse coupled to Alexa-488 (Green). Nuclei were stained with DAPI (Blue). Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the GFP-specific immunofluorescence observed in cells infected with the different recombinant VACVs. D. Statistical analysis. Cells showing more than 70% of the total Flag-specific signal at the plasma membrane or the cell cytoplasm were scored as having a predominant plasma membrane (P) or cytoplasmic (C) distribution. The remaining cell fraction was considered as exhibiting a mixed (Mx) plasma membrane/cytoplasmic distribution. Analyses were performed with data collected from three independent experiments using a total of over three hundred single cell images.
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pone.0123470.g004: Contribution of the electropositive C-terminal charge to VP5 PM targeting.A. VP5 alanine replacement mutants. Diagram depicting Flag-tagged VP5 proteins expressed by the different recombinant VACVs used in this analysis. The M1 protein lacks the three cationic clusters whilst M2, M3, and M4 lack the 132KR133, the 136KRR138 or the 142RK143 clusters, respectively. A Flag-tagged version of the wild type protein (WT) was used as control. The black box indicates the position of the Flag tag. Positively charged amino acids at the VP5 C-terminus are highlighted in blue, and the replacing alanine residues in black. B. Protein expression analysis. Samples from cells infected with recombinant VACV expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using anti-Flag mAbs. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different recombinant VACVs were processed for IF using a mouse anti-Flag mAb followed by incubation with goat anti-mouse coupled to Alexa-488 (Green). Nuclei were stained with DAPI (Blue). Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the GFP-specific immunofluorescence observed in cells infected with the different recombinant VACVs. D. Statistical analysis. Cells showing more than 70% of the total Flag-specific signal at the plasma membrane or the cell cytoplasm were scored as having a predominant plasma membrane (P) or cytoplasmic (C) distribution. The remaining cell fraction was considered as exhibiting a mixed (Mx) plasma membrane/cytoplasmic distribution. Analyses were performed with data collected from three independent experiments using a total of over three hundred single cell images.

Mentions: Previous results suggested that the interaction of VP5 with the plasmalemma involves the establishment of an electrostatic interaction between the VP5 polybasic C-terminal tail and anionic PM lipids. Provided that was the case, the reduction of the net charge of the VP5 C-terminal tail should significantly affect PM targeting. To test this hypothesis, four Flag-tagged VP5 mutant versions, in which cationic clusters were selectively replaced by non-polar alanine (A) residues, were engineered. A diagram depicting the configuration of the C-terminal tails of the different mutants is shown in Fig 4A. The FVP5CTM1 recombinant gene lacks the three cationic clusters. The FVP5CTM2, FVP5CTM3, and FVP5CTM4 polypeptides lack the 132KR133, the 136KRR138 or the 142RK143 cluster, respectively. The described constructs were used to generate inducible recombinant VACVs. The resulting viruses were employed to infect HeLa cells. The electrophoretic mobility and the subcellular distribution of the different FVP5 mutant versions were analyzed by Western blot and CLSM analysis. VT7/FVP5, expressing the full length Flag-tagged VP5 protein was used as a control for these experiments.


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)

Contribution of the electropositive C-terminal charge to VP5 PM targeting.A. VP5 alanine replacement mutants. Diagram depicting Flag-tagged VP5 proteins expressed by the different recombinant VACVs used in this analysis. The M1 protein lacks the three cationic clusters whilst M2, M3, and M4 lack the 132KR133, the 136KRR138 or the 142RK143 clusters, respectively. A Flag-tagged version of the wild type protein (WT) was used as control. The black box indicates the position of the Flag tag. Positively charged amino acids at the VP5 C-terminus are highlighted in blue, and the replacing alanine residues in black. B. Protein expression analysis. Samples from cells infected with recombinant VACV expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using anti-Flag mAbs. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different recombinant VACVs were processed for IF using a mouse anti-Flag mAb followed by incubation with goat anti-mouse coupled to Alexa-488 (Green). Nuclei were stained with DAPI (Blue). Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the GFP-specific immunofluorescence observed in cells infected with the different recombinant VACVs. D. Statistical analysis. Cells showing more than 70% of the total Flag-specific signal at the plasma membrane or the cell cytoplasm were scored as having a predominant plasma membrane (P) or cytoplasmic (C) distribution. The remaining cell fraction was considered as exhibiting a mixed (Mx) plasma membrane/cytoplasmic distribution. Analyses were performed with data collected from three independent experiments using a total of over three hundred single cell images.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4401730&req=5

pone.0123470.g004: Contribution of the electropositive C-terminal charge to VP5 PM targeting.A. VP5 alanine replacement mutants. Diagram depicting Flag-tagged VP5 proteins expressed by the different recombinant VACVs used in this analysis. The M1 protein lacks the three cationic clusters whilst M2, M3, and M4 lack the 132KR133, the 136KRR138 or the 142RK143 clusters, respectively. A Flag-tagged version of the wild type protein (WT) was used as control. The black box indicates the position of the Flag tag. Positively charged amino acids at the VP5 C-terminus are highlighted in blue, and the replacing alanine residues in black. B. Protein expression analysis. Samples from cells infected with recombinant VACV expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using anti-Flag mAbs. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different recombinant VACVs were processed for IF using a mouse anti-Flag mAb followed by incubation with goat anti-mouse coupled to Alexa-488 (Green). Nuclei were stained with DAPI (Blue). Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the GFP-specific immunofluorescence observed in cells infected with the different recombinant VACVs. D. Statistical analysis. Cells showing more than 70% of the total Flag-specific signal at the plasma membrane or the cell cytoplasm were scored as having a predominant plasma membrane (P) or cytoplasmic (C) distribution. The remaining cell fraction was considered as exhibiting a mixed (Mx) plasma membrane/cytoplasmic distribution. Analyses were performed with data collected from three independent experiments using a total of over three hundred single cell images.
Mentions: Previous results suggested that the interaction of VP5 with the plasmalemma involves the establishment of an electrostatic interaction between the VP5 polybasic C-terminal tail and anionic PM lipids. Provided that was the case, the reduction of the net charge of the VP5 C-terminal tail should significantly affect PM targeting. To test this hypothesis, four Flag-tagged VP5 mutant versions, in which cationic clusters were selectively replaced by non-polar alanine (A) residues, were engineered. A diagram depicting the configuration of the C-terminal tails of the different mutants is shown in Fig 4A. The FVP5CTM1 recombinant gene lacks the three cationic clusters. The FVP5CTM2, FVP5CTM3, and FVP5CTM4 polypeptides lack the 132KR133, the 136KRR138 or the 142RK143 cluster, respectively. The described constructs were used to generate inducible recombinant VACVs. The resulting viruses were employed to infect HeLa cells. The electrophoretic mobility and the subcellular distribution of the different FVP5 mutant versions were analyzed by Western blot and CLSM analysis. VT7/FVP5, expressing the full length Flag-tagged VP5 protein was used as a control for these experiments.

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