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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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GFP PM targeting mediated by the 24 VP5 C-terminal residues.HeLa cells were transfected with plasmids encoding GFP (GFP), GFP fused to the VP5 ORF (VP5/GFP) or GFP fused to the 24 VP5 C-terminal residues (GFP/CT122-145). At 12 h post-transfection cultures were fixed and processed for IF using a mouse anti-GFP mAb followed by incubation with goat anti-mouse coupled to Alexa-488. Nuclei were stained with DAPI. Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Presented 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 transfected with the different plasmids.
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pone.0123470.g002: GFP PM targeting mediated by the 24 VP5 C-terminal residues.HeLa cells were transfected with plasmids encoding GFP (GFP), GFP fused to the VP5 ORF (VP5/GFP) or GFP fused to the 24 VP5 C-terminal residues (GFP/CT122-145). At 12 h post-transfection cultures were fixed and processed for IF using a mouse anti-GFP mAb followed by incubation with goat anti-mouse coupled to Alexa-488. Nuclei were stained with DAPI. Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Presented 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 transfected with the different plasmids.

Mentions: Plasmids harboring the different constructs were transfected to preconfluent HeLa cell monolayers. At 12 h post-transfection cultures were fixed, processed for IF using a mouse anti-GFP mAb, and visualized by CLSM. As shown in Fig 2, the GFP protein showed a diffuse cytoplasmic/nuclear signal. As expected, the GFP-VP5 polypeptide accumulated at the PM. Noteworthy, the GFP/CT122-145 IF signal was specifically found to accumulate at both the PM and nuclei of transfected cells, thus indicating that this region is sufficient for an efficient PM targeting. PM targeting motifs resemble nuclear localization sequences [25]. Furthermore, it has been shown that the polybasic C-terminal tails from some small GTPases, e.g. Rit and Rin, drive GFP fusion proteins to both PM and nuclei [26]. Accordingly, the presence of the GFP/CT122-145 fusion protein at cell nuclei was not an unexpected finding.


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)

GFP PM targeting mediated by the 24 VP5 C-terminal residues.HeLa cells were transfected with plasmids encoding GFP (GFP), GFP fused to the VP5 ORF (VP5/GFP) or GFP fused to the 24 VP5 C-terminal residues (GFP/CT122-145). At 12 h post-transfection cultures were fixed and processed for IF using a mouse anti-GFP mAb followed by incubation with goat anti-mouse coupled to Alexa-488. Nuclei were stained with DAPI. Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Presented 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 transfected with the different plasmids.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123470.g002: GFP PM targeting mediated by the 24 VP5 C-terminal residues.HeLa cells were transfected with plasmids encoding GFP (GFP), GFP fused to the VP5 ORF (VP5/GFP) or GFP fused to the 24 VP5 C-terminal residues (GFP/CT122-145). At 12 h post-transfection cultures were fixed and processed for IF using a mouse anti-GFP mAb followed by incubation with goat anti-mouse coupled to Alexa-488. Nuclei were stained with DAPI. Cells were visualized by CLSM. Fluorescence signals were recorded separately by using appropriate filters. Presented 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 transfected with the different plasmids.
Mentions: Plasmids harboring the different constructs were transfected to preconfluent HeLa cell monolayers. At 12 h post-transfection cultures were fixed, processed for IF using a mouse anti-GFP mAb, and visualized by CLSM. As shown in Fig 2, the GFP protein showed a diffuse cytoplasmic/nuclear signal. As expected, the GFP-VP5 polypeptide accumulated at the PM. Noteworthy, the GFP/CT122-145 IF signal was specifically found to accumulate at both the PM and nuclei of transfected cells, thus indicating that this region is sufficient for an efficient PM targeting. PM targeting motifs resemble nuclear localization sequences [25]. Furthermore, it has been shown that the polybasic C-terminal tails from some small GTPases, e.g. Rit and Rin, drive GFP fusion proteins to both PM and nuclei [26]. Accordingly, the presence of the GFP/CT122-145 fusion protein at cell nuclei was not an unexpected finding.

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