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Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction.

ABSTRACT: Proper assembly of viruses must occur through specific interactions between capsid proteins. Many double-stranded DNA viruses and bacteriophages require internal scaffolding proteins to assemble their coat proteins into icosahedral capsids. The 303 amino acid bacteriophage P22 scaffolding protein is mostly helical, and its C-terminal helix-turn-helix (HTH) domain binds to the coat protein during virion assembly, directing the formation of an intermediate structure called the procapsid. The interaction between coat and scaffolding protein HTH domain is electrostatic, but the amino acids that form the protein-protein interface have yet to be described. In the present study, we used alanine scanning mutagenesis of charged surface residues of the C-terminal HTH domain of scaffolding protein. We have determined that P22 scaffolding protein residues R293 and K296 are crucial for binding to coat protein and that the neighboring charges are not essential but do modulate the affinity between the two proteins.

SUBMITTER: Cortines JR

PROVIDER: S-EPMC3208733 | biostudies-literature | 2011 Dec

REPOSITORIES: biostudies-literature

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Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction.

Cortines Juliana R JR Weigele Peter R PR Gilcrease Eddie B EB Casjens Sherwood R SR Teschke Carolyn M CM

Virology 20111004 1

Proper assembly of viruses must occur through specific interactions between capsid proteins. Many double-stranded DNA viruses and bacteriophages require internal scaffolding proteins to assemble their coat proteins into icosahedral capsids. The 303 amino acid bacteriophage P22 scaffolding protein is mostly helical, and its C-terminal helix-turn-helix (HTH) domain binds to the coat protein during virion assembly, directing the formation of an intermediate structure called the procapsid. The inter ...[more]

PMID: 21974803

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Project description:Icosahedral virus assembly requires a series of concerted and highly specific protein-protein interactions to produce a proper capsid. In bacteriophage P22, only coat protein (gp5) and scaffolding protein (gp8) are needed to assemble a procapsid-like particle, both in vivo and in vitro. In scaffolding protein's coat binding domain, residue R293 is required for procapsid assembly, while residue K296 is important but not essential. Here, we investigate the interaction of scaffolding protein with acidic residues in the N-arm of coat protein, since this interaction has been shown to be electrostatic. Through site-directed mutagenesis of genes 5 and 8, we show that changing coat protein N-arm residue 14 from aspartic acid to alanine causes a lethal phenotype. Coat protein residue D14 is shown by cross-linking to interact with scaffolding protein residue R293 and, thus, is intimately involved in proper procapsid assembly. To a lesser extent, coat protein N-arm residue E18 is also implicated in the interaction with scaffolding protein and is involved in capsid size determination, since a cysteine mutation at this site generated petite capsids. The final acidic residue in the N-arm that was tested, E15, is shown to only weakly interact with scaffolding protein's coat binding domain. This work supports growing evidence that surface charge density may be the driving force of virus capsid protein interactions.Bacteriophage P22 infects Salmonella enterica serovar Typhimurium and is a model for icosahedral viral capsid assembly. In this system, coat protein interacts with an internal scaffolding protein, triggering the assembly of an intermediate called a procapsid. Previously, we determined that there is a single amino acid in scaffolding protein required for P22 procapsid assembly, although others modulate affinity. Here, we identify partners in coat protein. We show experimentally that relatively weak interactions between coat and scaffolding proteins are capable of driving correctly shaped and sized procapsids and that the lack of these proper protein-protein interfaces leads to aberrant structures. The present work represents an important contribution supporting the hypothesis that virus capsid assembly is governed by seemingly simple interactions. The highly specific nature of the subunit interfaces suggests that these could be good targets for antivirals.

Dataset Information

Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction.

Publications

Decoding bacteriophage P22 assembly: identification of two charged residues in scaffolding protein responsible for coat protein interaction.

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