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,a Carolyn M. Teschkea,b Department of Molecular and Cell Biologya and Department of Chemistry,b University of Connecticut, Storrs, Connecticut, USA

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.

D

ouble-stranded DNA (dsDNA) viruses, such as the tailed phages and herpesviruses, have coat proteins that lack sequence homology but possess a common HK97 fold (1, 2). Herpesviruses infect vertebrate hosts and in humans are responsible for a wide spectrum of diseases causing conditions ranging from cold sores and genital sores to chicken pox and shingles. Thus, understanding the assembly of herpesviruses is crucial for identification of novel drug targets. Bacteriophage P22 is a dsDNA virus with a scaffolding protein-mediated assembly pathway, which is similar to that of herpesvirus. Therefore, P22 provides a good simple model system for studying the capsid assembly of dsDNA viruses (3, 4). In bacteriophage P22, capsid assembly involves interaction of 415 coat protein monomers with ⬃100 molecules of scaffolding protein (5–9). 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). DNA gets packaged through the portal complex (5), scaffolding protein exits, and the capsid expands in volume (14). The addition of plug, tail needle, and tailspike proteins results in a mature infectious virion (15). Some coat proteins having the HK97 fold are embellished with extra domains (Fig. 1) (16). P22 coat protein is one of these, as it has a nonconserved accessory domain inserted between the A and P domains (10, 17, 18). This domain, referred to as the insertion domain (I domain) (19), plays an important role in the folding of coat protein by acting as an intramolecular chaperone (20). The I

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domain is also involved in determination of capsid size (21) and is hypothesized to stabilize procapsids by forming intersubunit interactions between adjacent capsomers (10, 19, 20). A recent nuclear magnetic resonance (NMR) solution structure of the isolated I domain revealed a large flexible loop, referred to as the D-loop (18, 19, 22). Fits of the I domain NMR structure into the cryo-electron microscopy (cryo-EM) density map for both the procapsid and virion suggest that the D-loop could form electrostatic interactions with I domain residues of the adjacent coat protein subunit across an icosahedral 2-fold axis of symmetry. Specifically, residues D244 and D246 at the tip of the D-loop were suggested to form salt bridges with R299 and R269, respectively (19). Furthermore, the interactions between D-loops in the procapsid appear extensive (Fig. 1A). During capsid maturation, the

Received 23 June 2015 Accepted 8 August 2015 Accepted manuscript posted online 12 August 2015 Citation D’Lima NG, Teschke CM. 2015. A molecular staple: D-loops in the I domain of bacteriophage P22 coat protein make important intercapsomer contacts required for procapsid assembly. J Virol 89:10569 –10579. doi:10.1128/JVI.01629-15. Editor: R. M. Sandri-Goldin Address correspondence to Carolyn M. Teschke, [email protected]. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/JVI.01629-15

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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.

D-loops are modeled to pull away from one another, breaking some of these interaction while maintaining interactions just at the D-loop tip (Fig. 1B) (19). In this study, we tested our structural model and showed that residues in the D-loop do play a crucial role in coat protein assembly into procapsids. Amino acid substitutions near the tip of the D-loop that abolish negative charges cause a lethal phenotype due to the inability of the coat protein variants to correctly assemble into procapsids. Instead, these coat protein variants assemble into aberrant forms, such as spirals and tubes (polyheads). Further, disulfide cross-linking experiments confirm that the D-loop tips from adjacent coat monomers are in close proximity at the 2-fold axis in both procapsids and phage. MATERIALS AND METHODS Bacteria and bacteriophage. Bacteriophage P22 strains used in this study have amber mutations in gene 5 coding for coat protein (5⫺am N114), or amber mutations in genes 5 and 13 (13⫺am H101; prevents cell lysis) or in genes 5, 13, and 2 (2⫺am H200; prevents DNA packaging). All strains carried the c1-7 allele to prevent lysogeny. Phage strains were used to infect Salmonella enterica serovar Typhimurium strain DB7136 [leuA414(Am), hisC525(Am) sup0] (23). Plasmids. In vivo studies were performed using plasmid pMS11, which contains gene 5 (encoding coat protein gp5) cloned between sites

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BamHI and HindIII of plasmid vector pSE380 (Invitrogen). For generation of procapsids without phage infection, the genes for both coat protein (gp5) and scaffolding protein (gp8) were cloned into pET3a (assembler plasmid, a gift from Peter E. Prevelige). Coat protein variants were generated by introducing amino acid substitutions in gp5 by site-directed mutagenesis of gene 5 in pMS11 or assembler plasmids. Complementation assay. 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. 5⫺am phage were added to the bacteria in top agar, and isopropyl ␤-D-1-thiogalacatopyranoside (IPTG) was added to a final concentration of 1 mM to induce expression of coat protein from the plasmid and layered onto LB agar plates with 100 ␮g/ml of ampicillin. The plates were incubated overnight at different temperatures, the plaques were counted, and the phage titer was determined. The relative titer of each variant was calculated by dividing the phage titers at each temperatures by the titer produced by phage infection of Salmonella cells carrying wild-type (WT) coat gene cloned into pMS11 at 30°C. Production of particles in vivo. To assess phage production in vivo, Salmonella DB7136 cells containing plasmid pMS11 encoding coat protein variants were grown to mid-log phase at 30°C in 30 ml of LB broth. Cells were infected with either 5⫺13⫺ or 2⫺5⫺13⫺ amber phage at a multiplicity of infection (MOI) of 5. Concurrently, IPTG was added to a

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final concentration of 1 mM, and the infected cells were grown for an additional 4 h. Cells were harvested and resuspended in 1 ml of 10 mM sodium phosphate buffer (pH 7.6) containing 20 mM MgCl2 and frozen at ⫺20°C. After thawing of the cells on ice, DNase and RNase were added at a final concentration of 100 ␮g/ml. Phenylmethylsulfonyl fluoride (PMSF) and MgCl2 were added at final concentrations of 1 mM and 20 mM, respectively. Cells were lysed by repeated freeze-thaw cycles. Cell debris was removed by centrifugation and the supernatant was centrifuged at 60,000 rpm in an RP80-AT2 rotor (Sorvall) at 4°C for 20 min. Pellets were resuspended in 100 ␮l of 10 mM sodium phosphate buffer (pH 7.6) containing 20 mM MgCl2 on a shaker at 4°C overnight. Resuspended pellets were subjected to sucrose or CsCl density centrifugation. Sucrose density centrifugation. Resuspended pellets (100 ␮l) from the lysates of phage-infected cells were loaded on top of a 5 to 20% sucrose gradient prepared in 20 mM sodium phosphate buffer (pH 7.6). Gradients were formed using a Biocomp Instruments gradient maker by following the manufacturer’s instructions. Gradients were subjected to centrifugation at 105,000 ⫻ g in an RP55-S swinging-bucket rotor (Sorvall) at 20°C for 35 min in a Sorvall RC M120EX microultracentrifuge. Acceleration and deceleration settings were set to 1 and 9, respectively. Gradients were fractionated by collecting 100-␮l fractions from the top of the gradient using a positive-displacement micropipettor (Rainin). Fractions from the gradient were analyzed by SDS-PAGE using large-format 10% gels (Bio-Rad), as well as by electron microscopy. Cesium chloride density gradient centrifugation. Gradients were formed by layering solutions into an open-top, 16- by 52-mm, polyclear centrifuge tube (Seton). First, 2 ml of 1.6-g/ml CsCl was added to the bottom of the tube and then layered with 2 ml of 1.4-g/ml CsCl, 1 ml of 25% (wt/wt) sucrose, and finally 1 ml of particles pelleted from a lysate of phage-infected cells. All the solutions were made in 10 mM Tris (pH 7.6)–100 mM MgCl2. The gradients were centrifuged at 30,000 rpm for 1 h at 18°C in a Sorvall S50ST swinging-bucket rotor. The band corresponding to phages was harvested by puncturing the side of the tube with a 22.5-gauge needle. The solution containing the phages was dialyzed into 10 mM Tris (pH 7.6)–100 mM MgCl2 and then treated with 2.5 mM copper phenanthroline (see below). Disulfide bond formation was assessed by SDS-PAGE. Electron microscopy. Samples were prepared by spotting 3 ␮l from a sucrose gradient fraction onto mesh copper grids coated with a carbon film (Electron Microscopy Sciences). The samples were stained with a 1% (wt/vol) solution of uranyl acetate. The grids were dried and visualized by an FEI Tecnai G2 Spirit BioTwin transmission electron microscope equipped with an AMT 2k XR40 charge-coupled-device (CCD) camera. Preparation of procapsids in vivo with proteins expressed from a plasmid. Escherichia coli BL21 cells transformed with an assembler plasmid encoding coat protein variants and scaffolding protein were grown to mid-log phase at 30°C with aeration. Expression of coat and scaffolding proteins was induced by addition of 1 mM IPTG followed by growth of the culture for 4 h. Cells were harvested and resuspended in buffer B (50 mM Tris, 25 mM NaCl, 2 mM EDTA [pH 7.6]). Lysozyme (200 ␮g/ml) and 0.1% (wt/vol) Triton X-100 were added to the resuspended cells before freezing at ⫺20°C. The cells were thawed, and complete EDTA-free protease inhibitor cocktail (Roche) was added to a final concentration of 1⫻. Cells were lysed by four freeze-thaw cycles. DNase and RNase (50 ␮g/ml) were added to the cell suspension along with 2 mM MgCl2 and 0.5 mM CaCl2 and incubated at room temperature for 30 min. The lysate was subjected to centrifugation at 32,000 ⫻ g for 15 min at 4°C using a Fiberlite F-18 12X50 rotor (Thermo Scientific), followed by ultracentrifugation at 206,000 ⫻ g in a T-865 rotor (Sorvall) for 40 min at 4°C to pellet the procapsids. The pellets was resuspended overnight in buffer B on a shaker at 4°C and loaded onto a 150-ml Sephacryl S-1000 column (GE Healthcare), run at 0.2 ml/min and 4°C. Fractions containing pure procapsids were pooled and centrifuged at high speed to sediment the pure procapsids, as described above. The procapsid pellet was resuspended in buffer B overnight at 4°C on a shaker, and the protein concentration was deter-

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mined by UV absorbance at 280 nm after dilution of the procapsids (1:40) in 6 M guanidine HCl for 15 min to generate coat protein monomers. Generation of coat protein monomers for D246A. The assembled particles made of the coat protein variant D246A lacked scaffolding protein. Thus, these empty procapsids were unfolded in 9 M urea in 10 mM sodium phosphate buffer (pH 7.6) for 30 min either alone or with purified scaffolding protein (24) at room temperature. An equal volume of phosphate buffer was then added, and the unfolded protein(s) was subjected to dialysis in a 12,000-Da-cutoff dialysis membrane (Spectrum Labs) against 1 liter of phosphate buffer at 4°C changed three times for at least 3 h each time. The refolded protein(s) was centrifuged at 60,000 rpm in an RP80-AT rotor (Sorvall) at 4°C for 20 min to pellet assembled products. Disulfide cross-linking of coat proteins in procapsids. Procapsids generated using the assembler plasmid were diluted in 10 mM sodium phosphate buffer (pH 7.6) to a final concentration of 1 mg/ml. To allow for disulfide bond formation, 20 ␮l of dichloro(1,10-phenanthroline)copper(II) (Sigma-Aldrich) (CuPhe) was added to the procapsids to a final concentration of 2.5 mM. Oxidation was allowed to proceed for 1 h at room temperature before the addition of SDS sample buffer and heated at 60°C for 2 min. Disulfide bond formation was assessed by SDS-PAGE with staining with Coomassie brilliant blue. To assess disulfide bond formation in procapsids and phage generated from phage-infected cells, oxidizing agent was directly added to fractions 16 or 23 from the sucrose gradient. Heat expansion assay. Procapsids were diluted to 1 mg/ml in 20 mM phosphate buffer (pH 7.6) and split into 20-␮l aliquots, which were incubated at 24°C or 72°C for 15 min. The aliquots were split after incubation so that 10 ␮l was used for agarose gel electrophoresis, while 10 ␮l was used for electron microscopy. For electrophoresis, 10 ␮l of the incubated sample was mixed with 5 ␮l of agarose sample buffer (50% glycerol, 0.25% bromophenol blue, 1⫻ Tris glacial acetic acid EDTA buffer), and 5 ␮l was loaded on a 1% agarose gel run at 100 V for 45 min. The gel was stained with Coomassie blue to visualize the bands.

RESULTS

Amino acid substitutions of D-loop residues affect formation of infectious phage. The D-loop of the I-domain encompasses residues V239 to N254 (19). In order to assess the importance of residues in this region, alanine-scanning mutagenesis was performed throughout the D-loop (Fig. 1C). Of the D-loop residues, three charged amino acids, D244, D246, and K249, near the tip of the D-loop in the I domain of one coat protein monomer were suggested to form salt bridges with charged residues in the I domain of an adjacent coat protein and thus were of particular interest (19). To determine if substitutions in the D-loop would be detrimental to formation of mature virions as a consequence of improper folding or inability to assemble coat protein, complementation assays were performed. Salmonella cells transformed with a plasmid encoding coat protein were infected with phage carrying an amber mutation in gene 5 (codes for coat protein; 5⫺am). Phage would only be produced if plasmid-encoded variant coat proteins could complement the 5⫺am phage at various temperatures, expressed as titers relative to those of the 5⫺am phage complemented by WT coat expressed from a plasmid (Fig. 1D). In Fig. 1D, we show that coat proteins with amino acid substitutions at the N- and C-terminal ends of the D-loop (W241A, Q242A, and N251A) were able to support phage production at higher temperatures, but the titer dropped significantly at 16°C, indicating a cold-sensitive phenotype, suggestive of an assembly defect. Substitutions toward the middle of the D-loop (L243A and K249A) did not have a detrimental effect on phage production at any temperature tested. Substitution of N245 at the tip of the

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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.

D-loop with alanine resulted in a slight decrease in titer at 16, 25, and 30°C. However, substitutions of charged residues near the tip of the D-loop had a pronounced effect on phage formation: D244 and D246 replacement with alanine caused a lethal phenotype, as the titer at all temperatures was at or near the reversion frequency of the 5⫺am phage (Fig. 1D). The lethal phenotype caused by D244A and D246A coat proteins could be due to either disruption of the folding of coat protein, resulting in aggregation, or the inability of coat protein monomers to interact properly to assemble into procapsids. D244A and D246A variants form aberrant structures in vivo. In order to determine if the lethal phenotype caused by alanine substitutions of D244 and D246 was due to inability of these vari-

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ants to assemble into procapsids, Salmonella DB7136 cells with plasmids encoding the variant coat proteins were grown at 30°C and infected with phage carrying amber mutations in genes 5 and 13, which encode coat protein and a holin for cell lysis, respectively. Particles pelleted from lysates of infected cells expressing variant coat proteins were separated on a 5 to 20% linear sucrose gradient, and the products were analyzed by transmission electron microscopy (TEM) and SDS-PAGE (Fig. 2). In the case of WT coat protein, the sucrose gradient profile shows two peaks of coat protein (Fig. 2A). One peak corresponds to mature phages that have incorporated coat protein (gp5), portal protein (gp1), tailspike proteins (gp9), the ejection proteins (gp16 and gp20) and have packaged DNA. These are seen at the

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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.

bottom of the gradient (fraction 23), while procapsids run higher in the gradient, with a peak centered around fraction 16. Scaffolding protein (gp8) associates with coat protein in procapsids and comigrated with it in the gradient, as expected. In Fig. 2B, micrographs of negatively stained samples from sucrose gradient fractions are shown. With lysates containing WT coat protein, phages and a small number of procapsids were observed in fraction 23. Fraction 16 contained procapsids and empty heads, which are abortive assembly products. DNA-filled phages usually appear shiny in negative stain, while the stain can penetrate both procapsids and empty heads and therefore look dark on the inside. Empty heads can be identified by the presence of the tail machinery but have a dark interior to the head, indicating loss of DNA. The lethal coat protein variant D244A did not have much coat protein in fraction 23 (Fig. 2A), indicating that phages were not produced. Also, the peak of coat protein that normally sediments around fraction 16 was shifted further up the sucrose gradient near fraction 10 and did not have associated scaffolding protein. Electron micrographs of these fractions showed no procapsids in fraction 16 or mature phage in fraction 23 (Fig. 2B). Instead, the few particles seen in fraction 23 always appeared to have leaked DNA and had aberrant morphology. Fraction 10 contained incompletely formed particles, so these products migrated further up the gradient than completely formed procapsids. Thus, this amino acid substitution appears to destabilize procapsids and interfere with proper procapsid assembly (discussed further below). The D246A coat variant, which also caused a lethal phenotype, showed coat protein concentrated at the bottom of the sucrose gradient and no significant peak around fraction 16 (Fig. 2A). Electron micrographs of the corresponding fractions revealed that D246A, like D244A, formed aberrant particles and incomplete assembly products, seen in fraction 16, rather than procapsids.

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Interestingly, in fraction 23 of the sucrose gradient, which usually contains phages, electron micrographs revealed long tube-like structures (Fig. 2B and C). In most cases the tubes were open at one end (data not shown). In contrast to coat proteins D244A and D246A, coat protein N245A was capable of producing procapsids and mature phage, as shown by both sucrose gradient profiles and corresponding electron micrographs. The cause of the reproducible but slight decrease in relative titer at lower temperatures (Fig. 1D) is unclear. Thus, our data indicate that the aspartic acid residues at the tip of the I domain D-loop make critical interactions required for proper assembly. Altered residues near the N- and C-terminal ends of the D-loop caused a slight cold-sensitive phenotype, with the exception of the W241A mutant, which forms plaques at 16°C near the reversion frequency of the amber phage, indicating a more severe phenotype. To understand the cause of the phenotypes, an infection using 5⫺13⫺ amber phage was performed in liquid culture with cells expressing these coat protein variants at 16°C. Sucrose gradient analysis showed that with the exception of coat protein W241A, the variants were able to make procapsids and phages. In the case of W241A, procapsids of the correct size and shape were observed, but mature phage were not formed (Fig. 3), suggesting that substitution of the tryptophan did not affect the ability of the coat protein to assemble into procapsids with the minor proteins and scaffolding protein incorporated but did affect the ability of the W241A coat protein to undergo DNA packaging to form phages. The Q242A and N251A coat protein variants appear to have somewhat less coat protein in fraction 23, consistent with fewer phages being formed due to some defect in assembly. However, the phages formed by these variants were infectious. The amount of phage being generated in infected cells early in the infection must not be enough to cause many plaques on an agar

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plate, but phage were observed in the lysates, consistent with the minor phenotype. Thus, our data suggest that residues further from the D-loop tip are less important for assembly than the residues near the tip, which must make important intersubunit contacts. Assembly defects caused by D246A cannot be corrected by scaffolding protein. In P22, scaffolding protein is an assembly chaperone that promotes the assembly of coat protein monomers into correctly sized T ⫽ 7 procapsids. In vivo, the absence of scaffolding protein leads to assembly of coat protein into aberrant structures, including petite T ⫽ 4 capsids and spirals (25–28). In previous work, we showed the coat protein variant F170L, like D246A coat protein, formed tubes (29). The position F170 is located in the ␤-hinge region of the coat protein core and is a fourstranded ␤-sheet important for conformational switching during assembly and capsid maturation (21, 30, 31). This variant forms tubes in the absence of scaffolding protein both in vivo and in vitro (29). However, in the presence of scaffolding protein, tube formation of F170L coat protein can be decreased or eliminated (29). Since D246A coat protein formed tubes in infected cells in vivo (with scaffolding protein present), we asked if this coat protein variant was also able to generate tubes in vitro, as was observed with the F170L coat protein variant. Additionally, we asked if scaffolding protein plays a role in controlling the morphology of the assembled product. During purification of D246A particles, which were generated by coexpression of coat protein and scaffolding protein from a plasmid, we noticed that when the particles were run over a size exclusion column, there was very little association of scaffolding protein (data not shown). This is unlike particles formed from WT coat protein, which are coeluted with scaffolding protein as procapsids. Nevertheless, we pooled D246A coat protein-containing fractions from the S1000 column and purified them as usual for procapsids. In case of WT procapsids, scaffolding protein is extracted from the procapsids by guanidine treatment to generate empty procapsid shells, which are devoid of scaffolding protein. These shells are denatured with urea and used to generate coat protein monomers by refolding coat protein by dialysis against buffer. Since D246A particles had no associated scaffolding protein, we treated them as empty shells. When we refolded D246A to form coat protein monomers, over 90% of protein pelleted, instead of remaining soluble as observed for WT monomeric coat protein, indicating the presence of large assembled or aggregated products. Since the D246A coat protein variant formed tubes in phage-infected cells, we expected the in vitro-assembled products to contain tubes. However, on analysis of the products by electron microscopy, complex spirals and aggregates of spirals were seen (Fig. 4). Our data show that tube formation of D246A coat protein occurred in phage-infected cells, where scaffolding protein was present, while in vitro the D246A coat protein misassembled into spirals. However, there is no scaffolding present in our in vitro refolding procedure. Therefore, we refolded D246A coat protein in the presence of purified scaffolding protein to determine if it could prevent the spiral formation and promote tubes. Even when scaffolding protein was added at a 2-fold molar excess, D246A spiral formation still occurred (Fig. 4), showing that scaffolding protein cannot rectify the assembly defect of D246A coat protein in vitro. Of note, when D246A coat protein is coexpressed with scaffolding protein as described above, the assembly products are

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FIG 4 Assembly products of D246A coat protein monomers in vitro. Ureadenatured 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.

spirals and not tubes. Thus, in phage-infected cells another phage protein likely nucleates the improper tube formation. Nevertheless, these data show that D246A coat protein is able to assemble, but not form proper procapsids, indicating that interactions formed via residue D246 are crucial to establishing the correct assembly pathway. Capsid rupture is not responsible for incomplete assembly products. The presence of aberrant incompletely formed procapsids, especially in the case of D244A coat protein, made us question whether these products resulted from defective assembly or from rupture upon DNA packaging into an unstable capsid. We reasoned that if the broken procapsid particles generated from D244A and D246A coat proteins were a result of rupture of the capsid due to DNA packaging, lysates from a complementation experiment in which assembly is halted at the procapsid stage should show correctly sized procapsids. We used phage that contain an amber mutation in gene 2 that prevents DNA packaging, in addition to amber mutations in genes 5 and 13, and complemented this triple amber phage with WT or variant coat proteins expressed from a plasmid. Since DNA packaging is prevented, mature phage are not formed. From sucrose gradient analysis of lysates, WT coat protein made correctly sized procapsids observed in a peak near fraction 18, but no mature phage were seen in the bottom fraction, as expected (Fig. 5A). On the other hand, aberrant incompletely formed procapsids were still seen in case of both the D244A and D246A coat protein variants (Fig. 5B), as was observed from the 5⫺13⫺ phage infection (Fig. 2). In fact, the D246A variant still formed tubes. These results further verify that the aberrant forms seen in case of these variants are not due to defects in the later stages of capsid morphogenesis, such as capsid maturation. Rather, appropriate interactions between D-loop residues are required for proper assembly of coat protein into procapsids. The tips of the D-loop of adjacent coat monomers contact each other at the 2-fold axis, not only in phage but also in procapsids. Our recent structural investigations suggest that D-loops from coat monomers in adjacent capsomers are proximate in procapsids but during maturation the loops move apart such that only the tips of the loops appear to interact (Fig. 1A and B) (19). Thus, the D-loop was proposed to form interactions across 2-fold axes of symmetry important for stabilizing procapsids but less so for mature heads (18, 19). To test this hypothesis, single cysteine substitutions were made at position N245 or L243 in the D-loop of

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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.

coat protein (Fig. 6A) in a cysteine-free background (C405S). These sites were chosen because they are near the tip of the D-loop but did not cause a deleterious phenotype when changed to alanine (Fig. 1D). In order to verify that the cysteine substitutions did not perturb the assembly pathway or affect the folding or conformation of coat protein, these variants were tested by complementation of 5⫺am phage at various temperatures. Both C405S and N245C/C405S coat proteins were able to complement 5⫺am phage at all temperatures; however, L243C/C405S coat protein showed a slightly decreased ability to support phage growth at all temperatures (Fig. 6B). Additionally, both N245C/C450S and L243C/C450S coat monomers were able to assemble into procapsid-like particles in vitro upon addition of scaffolding protein, with kinetics similar to those of WT coat protein monomers (data not shown), and in vivo when expressed with scaffolding protein on the assembler plasmid (see below and Fig. 7). Thus, these substitutions do not affect the ability of coat protein to form procapsids or mature. Procapsids of the cysteine variant coat proteins were purified and the oxidizing agent copper phenanthroline (CuPhe) was used to induce intermolecular disulfide bond formation. Only cysteines within a short range of each other (⬃2 Å) will readily form disulfide bonds (32). On analysis of the coat protein cysteine variants by nonreducing SDS-PAGE, the coat protein variant N245C located near the tip of the D-loop formed an intermolecular disulfide bond when procapsids were incubated with CuPhe at room temperature (Fig. 7A). On the other hand, coat protein L243C, which is modeled to be on the side of the D-loop, did not show disulfide bond formation in the procapsid. The same results were obtained when we used empty procapsid shells (data not shown). Coat protein with its sole native cysteine mutated to serine

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(C405S) was used as a control and did not show any disulfide cross-links (Fig. 7A). Electron micrographs were used to verify that CuPhe-treated N245C procapsids remained intact and did not have distorted morphology after reaction with the oxidizing agent and subsequent disulfide bond formation. The cross-linked N245C procapsids resemble untreated procapsids or WT procapsids in size, shape, and morphology by TEM (data not shown). Contrary to our expectations, our data provide direct evidence that the tips of the D-loops are in close proximity at the 2-fold axis between coat subunits, even in the procapsid form, in which the distance between adjacent N245 residues is modeled to be ⬃16 Å (19). Coat proteins in P22 undergo significant conformational changes during expansion of the procapsid into the mature capsid. During the maturation process, the capsid diameter increases by about 10% (14, 33). This expansion process can be mimicked in vitro by the addition of heat or destabilizing agents (34, 35). Heat-expanded procapsids release coat proteins in penton positions from the capsid (35, 36). The expansion process also results in changes in intersubunit contacts between coat subunits (35, 37). In order to test if the D-loop positions change during expansion, we subjected C405S, L243C/C405S and N245C/C405S procapsids to heat expansion at 72°C. The expansion process was followed by monitoring a shift in the mobility of procapsids in an agarose gel (Fig. 7B), as well as by transmission electron microscopy (Fig. 7C). Our results show that the variant procapsids were able to expand and release pentons. These expanded procapsids were treated with CuPhe and subjected to nonreducing SDS-PAGE. After expansion, N245C was able to form an intermolecular disulfide bond, while L243C was not able to do so (Fig. 7D), suggesting that the

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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 The tips of the D-loop remain across from each other even after heat

tips of the D-loops, which are in close proximity in the procapsid, remain in proximity after capsid expansion. In order to verify that our results seen with heat-expanded procapsids would be reproducible in genuine phage, we infected Salmonella cells carrying a plasmid encoding either L243C/C405S or N245C/C405S coat protein with 5⫺13⫺ amber phage and separated the resulting particles on CsCl gradients. These coat proteins were able to produce both procapsids and phage, further confirming that the substitutions did not dramatically affect coat protein assembly. Phages purified by CsCl gradients were treated with CuPhe, and only those with N245C/C405S coat protein formed disulfide-linked dimers (data not shown). We also analyzed the lysates from phage-infected cells expressing the N245C/C405S variant by sucrose gradients and electron micrographs (Fig. 8A and B). When procapsids (fraction 16) and phages (fraction 23) were treated with CuPhe, disulfide crosslinks of coat protein were seen at equal amounts in case of both

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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.

procapsids and mature phages (Fig. 8C). In the presence of 2-mercaptoethanol in the SDS sample buffer, the disulfidebonded products were reduced (Fig. 8C). These data verify that the tips of the I domain D-loops in coat protein are unexpect-

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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.

edly in close proximity (⬃2 Å) in the procapsid form and remain in close proximity even after expansion. DISCUSSION

The presence of a nonconserved accessory domain in the structurally conserved HK97 protein fold of bacteriophage P22 coat pro-

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tein raises several questions regarding its significance. Numerous studies have shown that the I domain is important for capsid stability, folding, and size determination (10, 18, 20, 38, 39). Our recent NMR solution structure of the isolated I domain revealed the presence of two large loops, referred to as the D- and S-loops (19, 22). These loops, though flexible in monomeric coat protein, gain appreciable electron density in the procapsid and mature virion (19), suggesting that they become more constrained once assembled. Fits of the NMR structure into the cryo-EM density of the procapsid and mature virion indicated that the D-loop of one coat monomer could form potential intermolecular salt bridges with I domain residues in the adjacent coat monomer across the 2-fold axes in the procapsid. Based on the models, the D-loops were thought to move apart in the mature virion and be positioned directly across from each other (19). Important contacts are made by the I domain D-loop. Our data directly show that D-loop residues, particularly at the tip of the loop, form important interactions necessary for proper capsid assembly, as substitutions of aspartic acid residues with alanines did not support coat protein assembly and phage formation and therefore led to a lethal phenotype. Alanine substitutions further away from the tip of the D-loop had less dramatic effects. For instance, K249, located along the side of the D-loop, was suggested to make a salt bridge with negatively charged residues in the I domain of the neighboring coat subunit (19). However, substitution with alanine at this position did not have a deleterious effect on procapsid assembly or phage formation. This result further highlights the importance of residues near the tip of the D-loop rather than residues located along its sides. Comparison of tube formation by P22 coat protein variants. The lethal phenotype caused by the D244A and D246A substitutions at the tip of the D-loop is due to the inability of these coat protein variants to correctly assemble into procapsids. D244A coat protein generates particles that are unable to complete assembly. Thus, disruption of this D-loop interaction across the 2-fold axis of symmetry seems to destabilize procapsids in vivo. In contrast, D246A coat protein lost the ability to assemble into proper procapsids, but rather, it assembles into tubes in vivo. Tube formation of P22 coat protein has been seen previously only when substitutions of phenylalanine at position 170 were made (29, 30). Position F170 is located in the ␤-hinge of coat protein’s A domain, a region shown to be important for conformational switching during maturation (29, 37). 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. In addition, the D-loop does not appear to make contact with the A domain and so is not likely to affect its flexibility. Interestingly, tubes of D246A coat protein are irregularly shaped and appear open at one end. This is markedly different from F170A, K, or L tubes, which are more regular and can close (29, 30). Since our data show that D-loops staple coat protein monomers together at the 2-fold symmetry axis, we propose that D-loop contacts must be very important for stabilizing the pen-

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ton-hexon interface, in addition to stabilizing coat proteins in adjacent hexons. Thus, when we disrupt these crucial intersubunit contacts, pentons do not get incorporated correctly during assembly from coat protein monomers, resulting in irregular tubes with ends that do not close. Structural studies would be required to probe the nature of this unique aberrant form, but unfortunately, the irregularity of the tubes precludes cryo-EM reconstruction. In vitro, coat protein D246A undergoes uncontrolled assembly of aberrant spiral forms at concentrations well below that required for WT coat protein to assemble in an uncontrolled, scaffolding protein-independent fashion (28) This result may indicate that interactions between D-loops during assembly could help control proper procapsid assembly via correct positioning of coat proteins into hexons and pentons, and perhaps there are repulsive interactions at the D-loop tip that decrease the propensity of WT coat protein to assemble without scaffolding protein. Residues D244 and D246 were proposed to form salt bridges with R299 and R269, respectively (19). We generated an alanine substitution at R269, but this variant resulted in coat protein aggregates, suggesting that this substitution was deleterious to folding. Residue R299 resides in the I domain in a region where four temperature-sensitive-folding mutants were previously identified (between residues 294 and 302), indicating that this region is critical for coat protein folding. In addition, residue R299 makes a stabilizing intramolecular salt bridge with E307 (C. Harprecht, K. Robbins, C. Teschke, and A. Alexandrescu, unpublished data), so we did not attempt replacement of this amino acid. We also introduced cysteines on the side of the D-loop opposite to L243, by making substitutions at positions 247, 248, and 249 in the cysteine-free background. However, these variant coat proteins were not soluble, even though alanine substitutions at these positions resulted in soluble coat protein. Since many of the temperaturesensitive-folding mutants of coat protein are found in the I domain, this is perhaps not a surprising result (40). Nevertheless, the aberrant products seen in D244A and D246A coat proteins directly show that D-loop residues make important intersubunit contacts between coat monomers required to assemble into procapsids. The D-loop must be in a different conformation than modeled. In the recent cryo-EM models, the D-loops from adjacent coat proteins make electrostatic bonds with sites in the apposing I domain (19). Since the length of a disulfide bond is 2.08 Å, disulfide bond formation between cysteines introduced at the tip of the D-loop further confirm that these residues of adjacent coat monomers are in extremely close proximity. These results are not consistent with the procapsid cryo-EM model in which N245 residues are suggested to be ⬃16Å apart (19). When we generated a cysteine at position 243, which is modeled to be along the side of the D-loops and about 14 Å apart, this mutant is not able to form a disulfide bond, suggesting that the D-loops must be located directly opposite each other, rather than being apposed as suggested previously(19). Further, our data reveal that the proximity of the D-loops in the procapsid form is maintained in the capsid even after maturation. In conclusion, our data confirm an additional role for the P22 coat protein I domain. Here we present evidence that D-loop interactions effectively staple capsomers together across 2-fold axes of symmetry and are critical to formation of proper T ⫽ 7 procapsids having the stability to withstand DNA packaging. In the absence of chainmail cross-links found in HK97, P22 has evolved a

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different mechanism by which it can assemble and stabilize its capsids. ACKNOWLEDGMENTS This work was supported by NIH grant GM076661 to C.M.T. We thank Marie Cantino and Xuanhao Sun of the University of Connecticut Bioscience Electron Microscopy Laboratory for assistance with electron microscopy and Kunica Asija for assistance with some experiments.

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