Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of C-terminal deletions on VP5 PM targeting.A. C-terminal deleted VP5 polypeptides. Diagram depicting the wild type Flag-tagged VP5 protein (FVP5WT) and versions of the protein lacking either 5 (Δ5CT), 10 (Δ10CT) or 15 (Δ15CT) C-terminal residues expressed by recombinant VACVs used in this analysis. The black box indicates the position of the Flag tag. Positively-charged amino acid residues at the C-terminal are highlighted in blue. B. Protein expression analysis. Samples from cells infected with recombinant VACVs expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using an anti-Flag mAb. 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

License
getmorefigures.php?uid=PMC4401730&req=5

pone.0123470.g003: Effect of C-terminal deletions on VP5 PM targeting.A. C-terminal deleted VP5 polypeptides. Diagram depicting the wild type Flag-tagged VP5 protein (FVP5WT) and versions of the protein lacking either 5 (Δ5CT), 10 (Δ10CT) or 15 (Δ15CT) C-terminal residues expressed by recombinant VACVs used in this analysis. The black box indicates the position of the Flag tag. Positively-charged amino acid residues at the C-terminal are highlighted in blue. B. Protein expression analysis. Samples from cells infected with recombinant VACVs expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using an anti-Flag mAb. 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: Data described above suggested the involvement of the VP5 C-terminal tail in PM targeting. To further analyze this hypothesis, three recombinant VP5 genes encoding polypeptides lacking 5 (FVP5Δ5CT), 10 (FVP5Δ10CT) and 15 (FVP5Δ15CT) C-terminal VP5 residues, respectively, were constructed. To facilitate their detection, a FLAG tag was fused to the N-termini of the recombinant polypeptides. These constructs were used to generate recombinant VACVs. The resulting recombinant VACVs were termed VT7/FVP5Δ5CT, VT7/FVP5Δ10CT and VT7/FVP5Δ15CT, respectively. A fourth recombinant VACV, VT7/FVP5, expressing the full length Flag-tagged VP5 protein was also generated and used as a control for subsequent experiments. A cartoon depicting the structure of the described constructs is shown in Fig 3A.


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)

Effect of C-terminal deletions on VP5 PM targeting.A. C-terminal deleted VP5 polypeptides. Diagram depicting the wild type Flag-tagged VP5 protein (FVP5WT) and versions of the protein lacking either 5 (Δ5CT), 10 (Δ10CT) or 15 (Δ15CT) C-terminal residues expressed by recombinant VACVs used in this analysis. The black box indicates the position of the Flag tag. Positively-charged amino acid residues at the C-terminal are highlighted in blue. B. Protein expression analysis. Samples from cells infected with recombinant VACVs expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using an anti-Flag mAb. 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

License
Show All Figures
getmorefigures.php?uid=PMC4401730&req=5

pone.0123470.g003: Effect of C-terminal deletions on VP5 PM targeting.A. C-terminal deleted VP5 polypeptides. Diagram depicting the wild type Flag-tagged VP5 protein (FVP5WT) and versions of the protein lacking either 5 (Δ5CT), 10 (Δ10CT) or 15 (Δ15CT) C-terminal residues expressed by recombinant VACVs used in this analysis. The black box indicates the position of the Flag tag. Positively-charged amino acid residues at the C-terminal are highlighted in blue. B. Protein expression analysis. Samples from cells infected with recombinant VACVs expressing the different proteins were subjected to SDS-PAGE and Western blot analysis using an anti-Flag mAb. 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: Data described above suggested the involvement of the VP5 C-terminal tail in PM targeting. To further analyze this hypothesis, three recombinant VP5 genes encoding polypeptides lacking 5 (FVP5Δ5CT), 10 (FVP5Δ10CT) and 15 (FVP5Δ15CT) C-terminal VP5 residues, respectively, were constructed. To facilitate their detection, a FLAG tag was fused to the N-termini of the recombinant polypeptides. These constructs were used to generate recombinant VACVs. The resulting recombinant VACVs were termed VT7/FVP5Δ5CT, VT7/FVP5Δ10CT and VT7/FVP5Δ15CT, respectively. A fourth recombinant VACV, VT7/FVP5, expressing the full length Flag-tagged VP5 protein was also generated and used as a control for subsequent experiments. A cartoon depicting the structure of the described constructs is shown in Fig 3A.

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