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An unexpected twist in viral capsid maturation

Abstract

Lambda-like double-stranded (ds) DNA bacteriophage undergo massive conformational changes in their capsid shell during the packaging of their viral genomes. Capsid shells are complex organizations of hundreds of protein subunits that assemble into intricate quaternary complexes that ultimately are able to withstand over 50 atm of pressure during genome packaging1. The extensive integration between subunits in capsids requires the formation of an intermediate complex, termed a procapsid, from which individual subunits can undergo the necessary refolding and structural rearrangements needed to transition to the more stable capsid. Although various mature capsids have been characterized at atomic resolution, no such procapsid structure is available for a dsDNA virus or bacteriophage. Here we present a procapsid X-ray structure at 3.65 Å resolution, termed prohead II, of the lambda-like bacteriophage HK97, the mature capsid structure of which was previously solved to 3.44 Å (ref. 2). A comparison of the two largely different capsid forms has unveiled an unprecedented expansion mechanism that describes the transition. Crystallographic and hydrogen/deuterium exchange data presented here demonstrate that the subunit tertiary structures are significantly different between the two states, with twisting and bending motions occurring in both helical and β-sheet regions. We also identified subunit interactions at each three-fold axis of the capsid that are maintained throughout maturation. The interactions sustain capsid integrity during subunit refolding and provide a fixed hinge from which subunits undergo rotational and translational motions during maturation. Previously published calorimetric data of a closely related bacteriophage, P22, showed that capsid maturation was an exothermic process that resulted in a release of 90 kJ mol-1 of energy3. We propose that the major tertiary changes presented in this study reveal a structural basis for an exothermic maturation process probably present in many dsDNA bacteriophage and possibly viruses such as herpesvirus, which share the HK97 subunit fold4.

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Figure 1: HK97 assembly and morphology.
Figure 2: P-loops located at three-fold axes act as invariant pivot points.
Figure 3: Spine helix bends during maturation.
Figure 4: A working hypothesis for the formation, meta-stability and subsequent maturation of HK97, represented with a single hexamer.

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Primary accessions

Protein Data Bank

Data deposits

The sequence for W336F, E-loop truncated prohead II has been deposited in the Protein Data Bank under accession number 3E8K.

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Acknowledgements

We thank V. Reddy for assistance with crystallographic studies and for discussions. We thank R. Huang for providing HK97 capsomer samples and for discussions, and T. Matsui for help with X-ray data collection. We thank B. Firek and C. Moyer for mutagenesis of the HK97 constructs used in the study. We also thank B. Szymczyma for material used in the study. We also thank I. Wilson for discussions. We thank the staffs at beamlines 14-BMC and 23-ID-D of the Advanced Photon Source for assistance in data collection. This work was supported by NIH grants RO1 AI40101 (to J.E.J), RO1 GM47795 (to R.W.H) and NIH Training Grant GM08326.

Author Contributions I.G. was the lead investigator that crystallized the prohead II particles, collected the X-ray data and determined and refined the structure. He also collected and interpreted the hydrogen/deuterium exchange data and prepared the first draft of the paper. L.G. helped with the initial crystallography of the prohead II particles. M.G. helped with the initial collection and interpretation of the hydrogen/deuterium exchange data. K.L. characterized the kinetics and parameters associated with the prohead II to EI transition facilitating the hydrogen/deuterium exchange studies of the EI intermediate. J.A.S. made important contributions during the refinement of the prohead II structure. R.L.D. and R.W.H. developed the HK97 expression system that allowed the studies to be performed, prepared the prohead II mutations that facilitated the production of crystals that diffracted to high resolution, contributed valuable advice for handling the particles and helped in writing the manuscript. E.A.K. supervised the hydrogen/deuterium exchange studies that were all performed in her laboratory. J.E.J. supervised the crystallography aspect of the project, coordinated the overall project and helped in writing the manuscript.

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Correspondence to John E. Johnson.

Supplementary information

Supplementary Information

This file contains Supplementary Figure 1 with Legend, Supplementary Table 1, Supplementary Methods, Supplementary References and Supplementary Movie Legends 1-3 (PDF 204 kb)

Supplementary Movie 1

This file shows the transition of subunit F between Prohead II and Head II. (see file s1 for full legend). (MOV 4681 kb)

Supplementary Movie 2

This file shows the isolated view of hexon expansion. (see file s1 for full legend). (MOV 5698 kb)

Supplementary Movie 3

This file shows isolated helix transitions. (see file s1 for full legend). (MOV 4475 kb)

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Gertsman, I., Gan, L., Guttman, M. et al. An unexpected twist in viral capsid maturation. Nature 458, 646–650 (2009). https://doi.org/10.1038/nature07686

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