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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 amino acid sequence.A. Deduced VP5 sequence corresponding to the IBDV Soroa strain. Positively charged amino acid residues at the C-terminal are highlighted in blue. Residues corresponding to the putative transmembrane domain are underlined. B. Multiple sequence alignment of the 23 VP5 C-terminal residues from classical virulent serotype I Soroa, CEF94 (accession number: AF194428), and Cu1 (D00867); very virulent serotype I D6948 (AF240686) and UPM97/61 (AF247006); and avirulent serotype II 23/82 (AF362773) and OH (IBU30818) strains. Conserved electropositive residues are highlighted in blue.
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pone.0123470.g001: VP5 amino acid sequence.A. Deduced VP5 sequence corresponding to the IBDV Soroa strain. Positively charged amino acid residues at the C-terminal are highlighted in blue. Residues corresponding to the putative transmembrane domain are underlined. B. Multiple sequence alignment of the 23 VP5 C-terminal residues from classical virulent serotype I Soroa, CEF94 (accession number: AF194428), and Cu1 (D00867); very virulent serotype I D6948 (AF240686) and UPM97/61 (AF247006); and avirulent serotype II 23/82 (AF362773) and OH (IBU30818) strains. Conserved electropositive residues are highlighted in blue.

Mentions: Inspection of the VP5 sequence revealed the presence of a polycationic C-terminal region spanning amino acid residues 132–143 containing three closely spaced clusters formed by two or three consecutive basic amino acid residues, namely 132KR133, 136KRR138 and 142RK143 (Fig 1A). A comparison of VP5 sequences from classical and very virulent serotype I and avirulent serotype II IBDV strains indicates that this region is fairly well conserved (Fig 1B). A wide variety of cellular proteins employ surface exposed positively charged domains to interact with anionic lipids anchored to the inner PM leaflet [24]. To explore whether the VP5 C-terminal tail might be involved in PM tropism, a sequence coding for the 24 VP5 C-terminal residues was fused in-frame to the 3′ end of the GFP ORF. This construct was named GFP/CT122-145. Two additional plasmids were used as control for subsequent experiments, i.e. pGFP/VP5 expressing the EGFP gene fused to the VP5 ORF lacking the initiation ATG codon (GFP/VP5), and pEGFPC1 expressing EGFP gene (GFP).


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 amino acid sequence.A. Deduced VP5 sequence corresponding to the IBDV Soroa strain. Positively charged amino acid residues at the C-terminal are highlighted in blue. Residues corresponding to the putative transmembrane domain are underlined. B. Multiple sequence alignment of the 23 VP5 C-terminal residues from classical virulent serotype I Soroa, CEF94 (accession number: AF194428), and Cu1 (D00867); very virulent serotype I D6948 (AF240686) and UPM97/61 (AF247006); and avirulent serotype II 23/82 (AF362773) and OH (IBU30818) strains. Conserved electropositive residues are highlighted in blue.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0123470.g001: VP5 amino acid sequence.A. Deduced VP5 sequence corresponding to the IBDV Soroa strain. Positively charged amino acid residues at the C-terminal are highlighted in blue. Residues corresponding to the putative transmembrane domain are underlined. B. Multiple sequence alignment of the 23 VP5 C-terminal residues from classical virulent serotype I Soroa, CEF94 (accession number: AF194428), and Cu1 (D00867); very virulent serotype I D6948 (AF240686) and UPM97/61 (AF247006); and avirulent serotype II 23/82 (AF362773) and OH (IBU30818) strains. Conserved electropositive residues are highlighted in blue.
Mentions: Inspection of the VP5 sequence revealed the presence of a polycationic C-terminal region spanning amino acid residues 132–143 containing three closely spaced clusters formed by two or three consecutive basic amino acid residues, namely 132KR133, 136KRR138 and 142RK143 (Fig 1A). A comparison of VP5 sequences from classical and very virulent serotype I and avirulent serotype II IBDV strains indicates that this region is fairly well conserved (Fig 1B). A wide variety of cellular proteins employ surface exposed positively charged domains to interact with anionic lipids anchored to the inner PM leaflet [24]. To explore whether the VP5 C-terminal tail might be involved in PM tropism, a sequence coding for the 24 VP5 C-terminal residues was fused in-frame to the 3′ end of the GFP ORF. This construct was named GFP/CT122-145. Two additional plasmids were used as control for subsequent experiments, i.e. pGFP/VP5 expressing the EGFP gene fused to the VP5 ORF lacking the initiation ATG codon (GFP/VP5), and pEGFPC1 expressing EGFP gene (GFP).

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