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. 2015 Oct;89(20):10569-79.
doi: 10.1128/JVI.01629-15. Epub 2015 Aug 12.

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

Nadia G D'Lima  1 , Carolyn M Teschke  2
Affiliations

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

Nadia G D'Lima et al. J Virol. 2015 Oct.

Abstract

Bacteriophage P22, a double-stranded DNA (dsDNA) virus, has a nonconserved 124-amino-acid accessory domain inserted into its coat protein, which has the canonical HK97 protein fold. This I domain is involved in virus capsid size determination and stability, as well as protein folding. The nuclear magnetic resonance (NMR) solution structure of the I domain revealed the presence of a D-loop, which was hypothesized to make important intersubunit contacts between coat proteins in adjacent capsomers. Here we show that amino acid substitutions of residues near the tip of the D-loop result in aberrant assembly products, including tubes and broken particles, highlighting the significance of the D-loops in proper procapsid assembly. Using disulfide cross-linking, we showed that the tips of the D-loops are positioned directly across from each other both in the procapsid and the mature virion, suggesting their importance in both states. Our results indicate that D-loop interactions act as "molecular staples" at the icosahedral 2-fold symmetry axis and significantly contribute to stabilizing the P22 capsid for DNA packaging.

Importance: Many dsDNA viruses have morphogenic pathways utilizing an intermediate capsid, known as a procapsid. These procapsids are assembled from a coat protein having the HK97 fold in a reaction driven by scaffolding proteins or delta domains. Maturation of the capsid occurs during DNA packaging. Bacteriophage HK97 uniquely stabilizes its capsid during maturation by intercapsomer cross-linking, but most virus capsids are stabilized by alternate means. Here we show that the I domain that is inserted into the coat protein of bacteriophage P22 is important in the process of proper procapsid assembly. Specifically, the I domain allows for stabilizing interactions across the capsid 2-fold axis of symmetry via a D-loop. When amino acid residues at the tip of the D-loop are mutated, aberrant assembly products, including tubes, are formed instead of procapsids, consequently phage production is affected, indicating the importance of stabilizing interactions during the assembly and maturation reactions.

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Figures

FIG 1
FIG 1
Alanine-scanning mutagenesis of the I domain D-loop. Shown are refined cryo-EM models of P22 coat protein in the procapsid (A) and phage (B), focusing on the icosahedral 2-fold axis of symmetry (19). Adjacent coat protein monomers are colored in blue and gray, and the D-loops of the I domains of these coat monomers are highlighted in orange and pink, respectively. (C) A telescopic view of the same model of the procapsid shown in panel A rotated slightly to show the D-loops, highlighted in black. Each individual amino acid in the D-loops that was replaced with an alanine is represented by a different color. (D) The effect of alanine substitutions on phage formation was tested by complementation of phage possessing an amber mutation in gene 5, which codes for coat protein, with variant coat proteins expressed from a plasmid. To guide the reader, relative titer data have been colored to match the respective residue in the D-loop shown in panel C.
FIG 2
FIG 2
Charged residues near the tips of the D-loop are important for procapsid assembly. (A) Cell lysates from phage-infected cells expressing variant coat proteins from a plasmid at 30°C were separated on a sucrose gradient, and fractions from the sucrose gradient were analyzed by SDS-PAGE. The peak of sedimenting procapsids centers about fraction 16, while phage or large misassembled products are found in fraction 23. Capsid fragments are found higher in the gradient, around fraction 10. The proteins found in procapsids that can be seen on this gel are indicated on the right side of the WT portion. (B) Transmission electron micrographs of particles present in fractions 10, 16, and 23, taken at ×68,000 but zoomed in to show the morphology of the particles. White arrows are used to indicate normal procapsids in fraction 16 and phage in fraction 23. (C) Additional micrographs of tubes formed by D246A showing a greater field of particles. The micrographs were taken at a magnification of ×18,500 (left) or ×68,000 (right). The micrographs were taken on material from fraction 23 of the sucrose gradient.
FIG 3
FIG 3
Analysis of mutants showing a cold-sensitive phenotype. Lysates from cells expressing variant coat proteins that exhibited a cold-sensitive phenotype by complementation, infected with 5−13− phage at 16°C, were separated on a sucrose gradient, and the fractions were analyzed by SDS-PAGE (A) and transmission electron microscopy at ×68,000 (B). A scale bar is shown in the bottom right of panel B.
FIG 4
FIG 4
Assembly products of D246A coat protein monomers in vitro. Urea-denatured D246A coat protein was refolded by dialysis. The particles produced were visualized by negatively stained microscopy (left). When D246A coat protein is refolded in the presence of excess scaffolding protein, spiral formation still occurs (right). The micrographs were taken at ×68,000, and a scale bar is shown at the bottom of the right side.
FIG 5
FIG 5
Aberrant assembly products of D244A and D246A variants are formed prior to DNA packaging. (A) Lysates from cells expressing variant coat proteins infected with phage carrying amber mutations in genes 2, 5, and 13 (to prevent DNA packaging, WT coat protein production, and cell lysis) were separated on a 5 to 20% sucrose gradient. Fractions from the sucrose gradient were analyzed by SDS-PAGE. (B) Samples from fractions 18 (procapsids) and 23 (primarily misassembled products) were analyzed by transmission electron microscopy (TEM). The magnification was ×68,000, and a scale bar is at the bottom right.
FIG 6
FIG 6
The tips of the D-loops in adjacent capsomers are in close proximity in procapsids. (A) The refined cryo-EM model showing the icosahedral 2-fold axis of symmetry in which the D-loops from adjacent coat monomers (shown in light blue and gray) are highlighted in black. Residues L243 and N245, shown in blue and tan, respectively, were replaced with cysteines. (B) The effect of substitutions C405S, L243C/C405S, and N245C/C405S on phage formation was tested by complementation of phage that possess an amber mutation in gene 5, which codes for coat protein, with variant coat proteins expressed from a plasmid.
FIG 7
FIG 7
The tips of the D-loop remain across from each other even after heat expansion. (A) C405S, L243C/C405S, and N245C/C405S procapsids were treated with the oxidizing agent copper phenanthroline (CuPhe) and subjected to nonreducing SDS-PAGE on a 10% SDS gel. Disulfide bonding dimeric coat protein is indicated by “Coat*,” and the molecular mass markers are indicated on the left side of the gels in both panels A and D. (B) Procapsids of each variant were incubated at 24°C or at 72°C and subjected to agarose gel electrophoresis. The positions of procapsids and heat-expanded heads are indicated on the right side of the 1% agarose gel, stained with Coomassie blue. (C) Electron micrographs of C405S and N245C/C405S procapsids showing release of pentons at 72°C along with an expansion in capsid volume. White arrows indicate gaps in the procapsid caused by release of pentons. L243C/C405S procapsids show the same results (data not shown). (D) The variant procapsids were incubated at 24°C or heat expanded at 72°C, followed by treatment with CuPhe, and separated on a 10% SDS gel.
FIG 8
FIG 8
The tips of the D-loop are across from each other in phage. (A) A 10% SDS gel showing sucrose gradient fractions after lysates from Salmonella cells expressing N245C/C405S coat protein, infected with 5−13− amber phage, were separated on a sucrose gradient. (B) Fractions 16 and 23 from the sucrose gradients of N245C/C405S lysates analyzed by TEM. (C) Fractions 16 and 23 of the N245C/C405S lysates, containing procapsids and phage, respectively, were treated with CuPhe and analyzed by SDS-PAGE after incubation in nonreducing or reducing SDS sample buffer. The molecular mass markers are indicated on the left, and “Coat*” indicates the position of disulfide-linked dimeric coat protein.
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