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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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C-terminal ablation abolishes PM VP5 targeting in IBDV-infected cells.A. C-terminal VP5 ablation IBDV mutants. Diagram depicting the VP5 proteins expressed by IBDV mutants lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) residues used in this analysis. The wild type virus (WT) and a VP5 knockout mutant (KO) were used as controls. Positively charged amino acids at the C-terminal are highlighted in blue. B. VP5 expression. Samples from cells infected with different viruses were subjected to SDS-PAGE and Western blot analysis using anti-VP5 serum. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different viruses were processed for IF using rabbit anti-VP5 serum followed by incubation with goat anti-rabbit coupled to Alexa-488. Nuclei were stained with DAPI. 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. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the VP5-specific immunofluorescence observed in cells infected with the different viruses. D. Statistical analysis. Cells showing more than 70% of the total VP5-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.g006: C-terminal ablation abolishes PM VP5 targeting in IBDV-infected cells.A. C-terminal VP5 ablation IBDV mutants. Diagram depicting the VP5 proteins expressed by IBDV mutants lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) residues used in this analysis. The wild type virus (WT) and a VP5 knockout mutant (KO) were used as controls. Positively charged amino acids at the C-terminal are highlighted in blue. B. VP5 expression. Samples from cells infected with different viruses were subjected to SDS-PAGE and Western blot analysis using anti-VP5 serum. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different viruses were processed for IF using rabbit anti-VP5 serum followed by incubation with goat anti-rabbit coupled to Alexa-488. Nuclei were stained with DAPI. 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. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the VP5-specific immunofluorescence observed in cells infected with the different viruses. D. Statistical analysis. Cells showing more than 70% of the total VP5-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: Experiments described above were carried out using recombinant VP5 genes expressed in a heterologous context. Indeed, data obtained from recombinant expression systems should be treated with some caution. It was therefore important to investigate the effect of mutations affecting the PIP-binding domain within the context of the IBDV infection. To perform this analysis, three IBDV mutants, namely IBDV_VP5Δ3CT, IBDV_VP5Δ10CT, and IBDV_VP5Δ14CT, expressing truncated versions of the VP5 polypeptides lacking 3, 10, and 14 VP5 C-terminal residues, respectively, were generated by reverse genetics (Fig 6A). The segment A of these three viruses contain a single nucleotide substitution A489→U, A501→U or A522→U, respectively, that convert the “AAA” codons coding for VP5 K132 and K136, or the “AAG” codon coding for K142, into “UAA” or “UAG” translation termination codons. Noteworthy, these substitutions result in the introduction of synonymous mutations within the overlapping ORF, thus not altering the amino acid sequence of the virus polyprotein. Two additional viruses, i.e. wild type IBDV (IBDV_WT), expressing an unmodified VP5 version of the IBDV Soroa strain (WT), and IBDV VP5 knockout (IBDV_VP5 KO), were also generated by reverse genetics and used as controls for subsequent experiments. IBDV_VP5 KO contains a single nucleotide substitution, i.e. segment A: G98→A, converting the VP5 “AUG” translation initiation codon into an “AUA” triplet, thus abolishing VP5 expression [8]. A diagram depicting the configuration of the C-terminal tails of the different mutants is shown in Fig 6A.


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)

C-terminal ablation abolishes PM VP5 targeting in IBDV-infected cells.A. C-terminal VP5 ablation IBDV mutants. Diagram depicting the VP5 proteins expressed by IBDV mutants lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) residues used in this analysis. The wild type virus (WT) and a VP5 knockout mutant (KO) were used as controls. Positively charged amino acids at the C-terminal are highlighted in blue. B. VP5 expression. Samples from cells infected with different viruses were subjected to SDS-PAGE and Western blot analysis using anti-VP5 serum. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different viruses were processed for IF using rabbit anti-VP5 serum followed by incubation with goat anti-rabbit coupled to Alexa-488. Nuclei were stained with DAPI. 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. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the VP5-specific immunofluorescence observed in cells infected with the different viruses. D. Statistical analysis. Cells showing more than 70% of the total VP5-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.g006: C-terminal ablation abolishes PM VP5 targeting in IBDV-infected cells.A. C-terminal VP5 ablation IBDV mutants. Diagram depicting the VP5 proteins expressed by IBDV mutants lacking 3 (Δ3CT), 10 (Δ10CT), and 14 (Δ14CT) residues used in this analysis. The wild type virus (WT) and a VP5 knockout mutant (KO) were used as controls. Positively charged amino acids at the C-terminal are highlighted in blue. B. VP5 expression. Samples from cells infected with different viruses were subjected to SDS-PAGE and Western blot analysis using anti-VP5 serum. Subcellular VP5 distribution. C. Single cell images. Coverslips containing cells infected with the different viruses were processed for IF using rabbit anti-VP5 serum followed by incubation with goat anti-rabbit coupled to Alexa-488. Nuclei were stained with DAPI. 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. Images correspond to confocal sections showing the overlay of the two fluorescence signals, and are characteristic examples of the VP5-specific immunofluorescence observed in cells infected with the different viruses. D. Statistical analysis. Cells showing more than 70% of the total VP5-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: Experiments described above were carried out using recombinant VP5 genes expressed in a heterologous context. Indeed, data obtained from recombinant expression systems should be treated with some caution. It was therefore important to investigate the effect of mutations affecting the PIP-binding domain within the context of the IBDV infection. To perform this analysis, three IBDV mutants, namely IBDV_VP5Δ3CT, IBDV_VP5Δ10CT, and IBDV_VP5Δ14CT, expressing truncated versions of the VP5 polypeptides lacking 3, 10, and 14 VP5 C-terminal residues, respectively, were generated by reverse genetics (Fig 6A). The segment A of these three viruses contain a single nucleotide substitution A489→U, A501→U or A522→U, respectively, that convert the “AAA” codons coding for VP5 K132 and K136, or the “AAG” codon coding for K142, into “UAA” or “UAG” translation termination codons. Noteworthy, these substitutions result in the introduction of synonymous mutations within the overlapping ORF, thus not altering the amino acid sequence of the virus polyprotein. Two additional viruses, i.e. wild type IBDV (IBDV_WT), expressing an unmodified VP5 version of the IBDV Soroa strain (WT), and IBDV VP5 knockout (IBDV_VP5 KO), were also generated by reverse genetics and used as controls for subsequent experiments. IBDV_VP5 KO contains a single nucleotide substitution, i.e. segment A: G98→A, converting the VP5 “AUG” translation initiation codon into an “AUA” triplet, thus abolishing VP5 expression [8]. A diagram depicting the configuration of the C-terminal tails of the different mutants is shown in Fig 6A.

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