Highly Specific Salt Bridges Govern Bacteriophage P22 Icosahedral Capsid Assembly: Identification of the Site in Coat Protein Responsible for Interaction with Scaffolding Protein

Abstract

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. IMPORTANCEBacteriophage 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.

Nature׳s favorite building block: Deciphering folding and capsid assembly of proteins with the HK97-fold

“…Specifically, a basic residue (R293) of scaffolding protein is essential for coat protein binding and procapsid assembly (Cortines et al, 2011). Likewise, a single acidic amino acid in coat protein (D14) is indispensable for interaction with scaffolding protein (Cortines et al, 2014). Changing the essential coat protein D14 leads to a lethal phenotype for phage production and results in assembly of aberrant structures, similar to the spiral polymers seen in infections without scaffolding protein present (Earnshaw and King, 1978).…”

ϕX174 Procapsid Assembly: Effects of an Inhibitory External Scaffolding Protein and Resistant Coat Proteins In Vitro

“…The microvirus internal scaffolding protein shares many properties and functions with the scaffolding proteins found in other viruses (7,9,10,14,15,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). However, it does not physically construct the procapsid or control morphogenetic fidelity.…”

A Molecular Staple: D-Loops in the I Domain of Bacteriophage P22 Coat Protein Make Important Intercapsomer Contacts Required for Procapsid Assembly

“…Substitution at F170 leads to increased rigidity of the A domain, which decreases penton formation and results in tubes of coat protein arranged in arrays of hexons with altered orientation at the 3-fold symmetry axis (30,31). This region of coat protein is also thought to be important for interaction with scaffolding protein (7,29,30). However, the D244 and D246 residues are located near the exterior surface of coat protein, on the side of coat protein opposite to where scaffolding protein binds (7), so it is unlikely that scaffolding protein binding to coat protein is directly affected by these alanine substitutions.…”

“…Variants of coat protein were tested for the ability to produce infectious phage via complementation (7). Briefly, Salmonella strain DB7136 containing plasmid pMS11 was grown to mid-log phase and harvested, and the pellet was resuspended in a small volume of LB.…”

“…In bacteriophage P22, capsid assembly involves interaction of 415 coat protein monomers with ϳ100 molecules of scaffolding protein (5)(6)(7)(8)(9). This results in the formation of an intermediate structure called the procapsid (10), into which ejection proteins (11,12) as well as a portal complex are coassembled (13).…”