P22 virus capsid

Although P22 maturation has been investigated for decades, a unified model that incorporates thermodynamic and biophysical analyses is not available. A general and specific model of icosahedral capsid maturation is of significant interest to theoreticians searching for fundamental principles as well as virologists and material scientists seeking to alter maturation to their advantage.

To address this challenge, we have combined the results from orthogonal biophysical techniques including differential scanning fluorimetry, atomic force microscopy, circular dichroism, and hydrogen-deuterium exchange mass spectrometry.

Source parameters were as follows: drying gas 6. The resulting multiple charge state distributions for protein were deconvoluted using a maximum entropy deconvolution algorithm in the Bruker Compass Analysis software. To test for binding of the capsids with 6xHis tags, approximately 0. Bound capsids were eluted from the column at a flow rate of 0.

Gels were run at a constant current of 35 mA for approximately 1 hour. Subsequently, a , dilution of anti-mouse HRPO conjugate in blocking solution was added for 30 min at room temperature. The membrane was washed with TBS, 0. A dilution of a commercially available mouse anti-6xHis tag antibody was made into a 50 mM Carbonate buffer, pH 9.

Fifty microliters of this antibody solution was loaded into each well on a coated 96 well ELISA plate Nunc and incubated for approximately 2. A PBS control containing no protein and each P22 capsid sample at 0. The plate was subsequently incubated for 3 hrs at room temperature with a rabbit antibody recognizing the P22 coat protein in the same buffer used for the capsid incubation. A background absorbance value for the PBS control was subtracted from the each average recorded absorbance to yield the data presented in Figure 2C.

C Enzyme Linked Immunabsorbent Assay displaying the relative amount of each assembled P22 capsid that interacts with an anti-His antibody. Twenty five microliters of each capsid at approximately 1. Astra 5. FluoresceinMaleimide Pierce was reacted with 2 mg 3. A 20 fold molar excess of F5M per P22 subunit was added dropwise from a stock solution in DMSO, and the reaction was stirred vigerously for 2 hours at room temperature.

Pelleting of the procapsids through a sucrose cushion, followed by resuspension and an additional pelleting by ultracentrifugation in PBS buffer removed excess F5M from the samples.

The UV-visible absorbance of a series of F5M concentrations in the denaturing buffer was measured to calculate the extinction coefficient of the F5M in these conditions. Controls for the labeling experiments were carried out by the addition of a corresponding volume of neat DMF. After stirring rapidly for 3 hours at room temperature, P22 capsids were diluted into pH 7. The frequency and dissipation values were recorded using QSoft software program version 1.

To probe the location of the structurally unresolved C-terminus of the coat protein CP in the bacteriophage P22 capsid, a series of genetic mutations were made and biochemically characterized. In the structural model of the P22 procapsid coat protein, the last 5 amino acids are not resolved and their location is ambiguous Fig 1. However, the published models of the P22 capsid 22 — 23 suggest that the C-terminus of the CP is directed towards the exterior of the capsid S2.

A representation of the assembled P22 procapsid A from the published cyroEM structure, which includes residues 1— of the amino acid P22 coat protein. The residues — are highlighted in the zoomed versions of the pentamer B and hexamer C units. In both, it appears that the CP C-terminus extends toward the capsid exterior. Genetic fusion of short peptides to the C-terminus of the CP was used to test the accessibility of this region to the exterior environment of the P22 procapsid. The genetic manipulation was confirmed by DNA sequencing.

The novel procapsids were purified using the same methodology as used for purification of the wtP22 procapsids 19 , which included ultracentrifugation through a sucrose cushion, followed by size exclusion chromatography Sephacryl S, S3.

Analysis of these materials by SDS page gel electrophoresis confirms the presence of both P22 coat protein and scaffold protein S4. No significant changes in particle assembly were observed upon the addition of amino acids to the C-terminus. By size exclusion chromatography SEC , all constructs eluted at approximately 65 mL, which is consistent with the elution volume of the assembled wtP22 procapsid S3. The fractions from this peak were pooled and subjected to HPLC-size exclusion chromatography coupled to multi-angle light scattering MALS and refractive index detection.

Analysis of the light scattering revealed packaged procapsids with particle diameters reflecting that of assembled capsids for each sample S7 and all of the capsids had particle RMS radii in the 23—30 nm range and hydrodynamic radii between 25 and 33nm.

The mass of each procapsid calculated from multi-angle light scattering was observed to be between 22 and 30 MDa. In this work, bacteriophage P22 virus-like particles VLPs containing CYP activity, immunologically inert and functionalized in order to be recognized by human cervix carcinoma cells and human breast adenocarcinoma cells were designed.

The CYP was encapsulated inside the virus capsid obtained from the bacteriophage P Peptide masses were estimated from both migration distance in the SDS gel for the 43 kDa product and the predicted mass for a residue peptide.

Subtraction of this reconstruction from that of un-digested ExH particles rendered at the same resolution produced a difference map showing the location of the N-arm in the subunit closest to the missing penton hole Figure 2. The CP of mature, tail-less but genome-containing, heads is known to be trypsin-resistant Lanman et al.

Left Cutaway view along two-fold direction of digested ExH reconstruction computed to 7. The ExH and digested ExH reconstructions were both computed at An enlarged view of one vertex appears in the insert. Right Same as left panel but showing that difference densities corresponding to nano-gold labels in reconstructions of three shell variants pinpoint the locations of residues blue , green , and red in the WT procapsid shell computed to 9.

Difference map analyses were all performed with gold-labeled and WT shell maps computed to As a further guide to accurate modeling of the CP structure, we used cryo-reconstruction methods to study shells that were engineered to include cysteine residues so electron-dense, 1.

All mutant specimens behaved similarly to WT as monitored by standard phage assays that test for the ability of variant proteins to fold and assemble in vivo into infectious particles under physiological temperatures data not shown. Candidates for nanogold labeling and cryo-TEM imaging were identified on the basis of a quantitative fluorescence assay, in which tetramethylrhodamine TMRM was used to determine the molar ratio of cysteines available for modification Experimental Procedures, Table 3.

Cryo-reconstructions of purified shells derived from three cysteine variants FC, AC, and TC were compared via difference map analysis to an unlabeled, WT shell reconstruction Figure 2 , Table 2. None of the variants exhibited any obvious changes in CP secondary or tertiary structure compared to WT CP data not shown. Neither cysteine substitution nor gold-labeling appear to cause structural perturbations, which raises the level of confidence that individual residue locations were identified and could be modeled accurately.

The AC reconstructed density map showed extra density was clearly present, but only on the penton surface Figure 2. Though the TMRM labeling experiment indicated that not all hexon subunits receive label, those that do must occur randomly within the six, quasi-equivalent subunits in each asymmetric unit Johnson, The averaging inherent in producing the icosahedral reconstruction would therefore reduce any signal attributed to labeled hexon subunits.

In FC, label accumulated at the centers of the pentons and hexons, whereas in TC, all subunits at the icosahedral and quasi three-fold axes of symmetry received label on the inner surface of the shell. Consistent with this, a difference map computed between reconstructed maps of nano-gold treated and untreated i.

Modeling of the P22 CP subunit was further guided by the results of limited proteolysis of soluble CP. N-terminal a. We used time-course trypsin digestion to produce proteolytic fragments from samples of the refolded, C-terminal half and analyzed these by SDS-PAGE and mass spectrometry Supplemental Data.

The presence of a well-defined, prominent band in the SDS gel suggested that a contiguous, folded domain existed within the CP C-terminal half black arrow, Figure 3. SDS gel showing trypsin digestion of the C-terminal region a. The black arrow points to the band corresponding to residues — telokin domain. The presence of an Ig-like domain in the P22 CP is not surprising since Ig domains often occur in viruses and phage Fraser et al.

Structure-based sequence alignment provides a more accurate means to align sequences of structurally homologous proteins that exhibit low sequence similarity Zhang and Skolnick, The top two rows show secondary structure depictions of firstly, the final P22 CP model yellow helices and cyan b-strands as identified with the program Stride Frishman and Argos, and secondly, as predicted orange helices and violet b-strands using the Jpred3 server Cole et al.

Next, the a. Regions corresponding to the HK97 and telokin folds are shaded in green and pink, respectively. Sequence shading is based on the Jalview Blosum62 coloring. Briefly, the residue is colored dark blue HK97 or dark purple telokin if it matches the consensus sequence not shown Clamp et al.

The residue is colored light blue HK97 or light purple telokin if it does not match the consensus sequence, but has a positive Blosum62 score Clamp et al. Colored circles beneath the aligned sequences identify residues blue , green , and red , where nanogold-labeling occurred in P22 shell variants studied by cryo-TEM.

Regions of the CP identified by trypsin digestion are highlighted with yellow and pink bars, corresponding to the N-terminal arm and telokin domain, respectively. The P22 protrusions in the ExH and shell reconstructions were both modeled using the same telokin template Holden et al.

The residues in P22 CP predicted by FFAS03 to adopt a telokin fold correspond to those that span the trypsin-resistant, C-terminal half described above, and highlight the notion that the ED is a stable, contiguous domain.

The HK97 and telokin templates served as guides to manually segment out respective density volumes from the two reconstructed maps, corresponding to the core and ED portions of the P22 CP in ExH and shell particles.

The X-ray templates were then fitted independently as rigid bodies into the segmented densities, and each was adjusted as needed to produce a backbone structure onto which the P22 sequence was threaded. This threading was greatly guided by knowledge gained from the gold-labeling residue positions and digestion N-arm location results described above.

The final two P22 CP models Figure 5A were derived through an exhaustive, iterative process involving several cycles of manual threading, energy minimization, molecular dynamics-driven flexible fitting, and density segmentation Experimental Procedures.

The reliability of each CP model was independently assessed and found to be consistent with the results of cross-linking Kang et al. Even though the models do not provide a full atomic description of the P22 CP, they do reliably represent the bulk of the CP structure before and after capsid maturation. Full asymmetric unit models for the ExH and shell particles were constructed by separately fitting and refining the respective CP models within the remaining unmodeled, reconstructed density for five ExH or six shell subunits Figure 5B.

These were then used to generate complete, icosahedral capsid models that accounted for and reliably represented all experimentally observed cryoTEM density Figure 6. A Final, refined models of the P22 CP in ExH left and shells right , aligned with respect to their telokin domains magenta. Structural landmarks previously identified in HK97 Gertsman et al.

B Modeled asymmetric units of ExH one hexon and the shell hexon plus one penton subunit , viewed along the hexon axis from outside the capsid. Segmented density for one subunit in each cryo-reconstruction is included to illustrate the quality of match between model and experimental data. C Same as B but for side views hexon axes vertical with only telokin domains colored D Same as B but viewed from inside the capsid with only N-arms a.

Boxes draw attention to the N-arms of F-subunits, which lie closest to the missing penton. E Enlarged views of N-arms show that in five ExH subunits A—E they adopt one helix-loop-helix conformation whereas in all shell subunits A—G they adopt another, with the primary difference confined to the loop and N-terminal helix. Furthermore, this loop-helix motif in the ExH F-subunit is more similar to that observed in all shell subunits.

Large gaps between capsomers are apparent. Final ribbon model of the P22 shell subunit, which shows no large gaps between capsomers. Notably, the Prohead II model does not account for any of the shell density corresponding to the ED domain.

F Cryo-reconstruction of the P22 procapsid shell computed to 9. The P22 shell and ExH CP models suggest that two structural components primarily drive capsid stabilization during maturation. These include the N-arm and P-loop structures. However, five of the six N-arms in ExH adopt a second, distinctly different helix-loop-helix structure.

Since heat treatment leads to penton release and formation of expanded particles, the penton:hexon interface likely represents the region of weakest intersubunit association in capsids. Loss of pentons removes the stabilizing interactions previously present between the penton and the N-arm of the nearby hexon subunit Figure 7B,C and this probably allows the N-arm of this one subunit to refold into the immature procapsid state.

This therefore solidifies the notion that trimeric N-arm interactions contribute to capsid stability Figure 7B,C. A An ExH CP model superimposed with corresponding segmented density from the ExH cryo-reconstruction with enlarged, stereo view of region in box containing N-arm shown at right.

B ExH cryo-reconstruction with three hexon ribbon models docked into the density. The icosahedron line drawing shows the view direction is along a strict three-fold symmetry axis. The region of the model surrounded by a dashed envelope is shown in stereo at the right, which gives a closer view of stabilizing interactions formed by symmetry-related subunits. The P-loops are all in black, subunits are colored differently, and the N-arms corresponding to Figure 3D are shown in darker shades to highlight intersubunit interactions.

Similar sets of interactions occur at the quasi three-fold axes not shown. C Same as B , but for the shell CP models and reconstruction. The modeled P-loop in P22 is 17 residues longer than the P-loop in HK97 and forms extensive interactions with portions of adjacent three-fold- and local three-fold-related subunits, such as the N-arm Figure 7B,C.

The reconstructed densities corresponding to the P-loops in both maps are equally as strong as the remaining CP density, indicating that the P-loops are rigid components that contribute to the structural integrity of all subunits. This common core provides the blueprint for building icosahedral capsids of all these viruses. Conservation of a CP core appears to be a recurring theme in icosahedral virus assembly Bamford et al.

Insertions and other additions to capsid cores provide adaptive functions such as occur in cell recognition and receptor binding Kontou et al.