Structural basis for scaffolding-mediated assembly and maturation of a dsDNA virus

Formation of many dsDNA viruses begins with the assembly of a procapsid, containing scaffolding proteins and a multisubunit portal but lacking DNA, which matures into an infectious virion. This process, conserved among dsDNA viruses such as herpes viruses and bacteriophages, is key to forming infectious virions. Bacteriophage P22 has served as a model system for this study in the past several decades. However, how capsid assembly is initiated, where and how scaffolding proteins bind to coat proteins in the procapsid, and the conformational changes upon capsid maturation still remain elusive. Here, we report Cα backbone models for the P22 procapsid and infectious virion derived from electron cryomicroscopy density maps determined at 3.8- and 4.0-Å resolution, respectively, and the first procapsid structure at subnanometer resolution without imposing symmetry. The procapsid structures show the scaffolding protein interacting electrostatically with the N terminus (N arm) of the coat protein through its C-terminal helix-loop-helix motif, as well as unexpected interactions between 10 scaffolding proteins and the 12-fold portal located at a unique vertex. These suggest a critical role for the scaffolding proteins both in initiating the capsid assembly at the portal vertex and propagating its growth on a T = 7 icosahedral lattice. Comparison of the procapsid and the virion backbone models reveals coordinated and complex conformational changes. These structural observations allow us to propose a more detailed molecular mechanism for the scaffolding-mediated capsid assembly initiation including portal incorporation, release of scaffolding proteins upon DNA packaging, and maturation into infectious virions.

[1]  J. King,et al.  Nucleation and growth phases in the polymerization of coat and scaffolding subunits into icosahedral procapsid shells. , 1993, Biophysical journal.

[2]  D. Baker,et al.  Refinement of protein structures into low-resolution density maps using rosetta. , 2009, Journal of molecular biology.

[3]  P. Weigele,et al.  Structure of the coat protein-binding domain of the scaffolding protein from a double-stranded DNA virus. , 2000, Journal of molecular biology.

[4]  S. Casjens,et al.  Nucleotide sequence of the bacteriophage P22 genes required for DNA packaging. , 1991, Virology.

[5]  P. Prevelige,et al.  Functional domains of bacteriophage P22 scaffolding protein. , 1998, Journal of molecular biology.

[6]  Matthew L. Baker,et al.  Backbone structure of the infectious ε15 virus capsid revealed by electron cryomicroscopy , 2008, Nature.

[7]  John E. Johnson,et al.  The P22 tail machine at subnanometer resolution reveals the architecture of an infection conduit. , 2009, Structure.

[8]  W. Chiu,et al.  Identification of the sites of interaction between the scaffold and outer shell in herpes simplex virus-1 capsids by difference electron imaging. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[9]  Matthew L. Baker,et al.  Backbone structure of the infectious Epsilon15 virus capsid revealed by electron cryomicroscopy , 2008 .

[10]  J. King,et al.  Three-dimensional transformation of capsids associated with genome packaging in a bacterial virus. , 1993, Journal of molecular biology.

[11]  B L Trus,et al.  Molecular architecture of bacteriophage T4 capsid: vertex structure and bimodal binding of the stabilizing accessory protein, Soc. , 2000, Virology.

[12]  P. Prevelige,et al.  A pilot protein participates in the initiation of P22 procapsid assembly. , 1991, Virology.

[13]  J. King,et al.  Scaffolding protein regulates the polymerization of P22 coat subunits into icosahedral shells in vitro. , 1988, Journal of molecular biology.

[14]  T. Dokland,et al.  Structural transitions during maturation of bacteriophage lambda capsids. , 1993, Journal of molecular biology.

[15]  Wah Chiu,et al.  Cryo-EM of macromolecular assemblies at near-atomic resolution , 2010, Nature Protocols.

[16]  Kristin N. Parent,et al.  'Let the phage do the work': using the phage P22 coat protein structures as a framework to understand its folding and assembly mutants. , 2010, Virology.

[17]  M. Rossmann,et al.  Structural and functional similarities between the capsid proteins of bacteriophages T4 and HK97 point to a common ancestry. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[18]  M. Rossmann,et al.  Conservation of the capsid structure in tailed dsDNA bacteriophages: the pseudoatomic structure of phi29. , 2005, Molecular cell.

[19]  P. Prevelige,et al.  Kinetic and calorimetric evidence for two distinct scaffolding protein binding populations within the bacteriophage P22 procapsid. , 2001, Biochemistry.

[20]  J. King,et al.  In vitro folding of phage P22 coat protein with amino acid substitutions that confer in vivo temperature sensitivity. , 1995, Biochemistry.

[21]  W. Chiu,et al.  Mechanism of scaffolding-directed virus assembly suggested by comparison of scaffolding-containing and scaffolding-lacking P22 procapsids. , 1999, Biophysical journal.

[22]  L. Black.  DNA packaging in dsDNA bacteriophages. , 1989, Annual review of microbiology.

[23]  Simeon S. Andrews,et al.  A conformational switch in bacteriophage p22 portal protein primes genome injection. , 2008, Molecular cell.

[24]  J. King,et al.  Structural studies of bacteriophage lambda heads and proheads by small angle X-ray diffraction. , 1979, Journal of molecular biology.

[25]  J. King,et al.  Structure of phage P22 coat protein aggregates formed in the absence of the scaffolding protein. , 1978, Journal of molecular biology.

[26]  John E. Johnson,et al.  Topologically linked protein rings in the bacteriophage HK97 capsid. , 2000, Science.

[27]  J. King,et al.  Binding of scaffolding subunits within the P22 procapsid lattice. , 1994, Virology.

[28]  Kevin Cowtan,et al.  research papers Acta Crystallographica Section D Biological , 2005 .

[29]  W Chiu,et al.  Role of the scaffolding protein in P22 procapsid size determination suggested by T = 4 and T = 7 procapsid structures. , 1998, Biophysical Journal.

[30]  J. King,et al.  Visualization of the maturation transition in bacteriophage P22 by electron cryomicroscopy. , 2000, Journal of molecular biology.

[31]  M. Heel,et al.  Exact filters for general geometry three dimensional reconstruction , 1986 .

[32]  J. King,et al.  Purification of the coat and scaffolding proteins from procapsids of bacteriophage P22. , 1981, Virology.

[33]  S Kundhavai Natchiar,et al.  Crystal Structure of Human Adenovirus at 3.5 Å Resolution , 2010, Science.

[34]  John E. Johnson,et al.  An unexpected twist in viral capsid maturation , 2009, Nature.

[35]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[36]  J. King,et al.  Assembly in vitro of bacteriophage P22 procapsids from purified coat and scaffolding subunits. , 1982, Journal of molecular biology.

[37]  D. Botstein,et al.  Structure and assembly of the capsid of bacteriophage P22. , 1976, Philosophical transactions of the Royal Society of London. Series B, Biological sciences.

[38]  J. King,et al.  Catalytic head assembling protein in virus morphogenesis , 1974, Nature.

[39]  D. Stuart,et al.  What does structure tell us about virus evolution? , 2005, Current opinion in structural biology.

[40]  Albert J R Heck,et al.  A Docking Model Based on Mass Spectrometric and Biochemical Data Describes Phage Packaging Motor Incorporation* , 2010, Molecular & Cellular Proteomics.

[41]  Alasdair C. Steven,et al.  Dynamics of herpes simplex virus capsid maturation visualized by time-lapse cryo-electron microscopy , 2003, Nature Structural Biology.

[42]  W Chiu,et al.  EMAN: semiautomated software for high-resolution single-particle reconstructions. , 1999, Journal of structural biology.

[43]  M. Baker,et al.  Coat protein fold and maturation transition of bacteriophage P22 seen at subnanometer resolutions , 2003, Nature Structural Biology.

[44]  Matthew L. Baker,et al.  Structural Changes in a Marine Podovirus Associated with Release of its Genome into Prochlorococcus , 2010, Nature Structural &Molecular Biology.

[45]  P. Prevelige,et al.  Bacteriophage P22 scaffolding protein forms oligomers in solution. , 1997, Journal of molecular biology.

[46]  P. Bradley,et al.  Toward High-Resolution de Novo Structure Prediction for Small Proteins , 2005, Science.

[47]  S. Casjens,et al.  Molecular genetics of bacteriophage P22 scaffolding protein's functional domains. , 2005, Journal of molecular biology.

[48]  Ivo Atanasov,et al.  Atomic Structure of Human Adenovirus by Cryo-EM Reveals Interactions Among Protein Networks , 2010, Science.

[49]  J. King,et al.  Temperature-sensitive mutations in the phage P22 coat protein which interfere with polypeptide chain folding. , 1993, The Journal of biological chemistry.

[50]  L. Black,et al.  Portal control of viral prohead expansion and DNA packaging. , 2009, Virology.

[51]  John E. Johnson,et al.  P22 coat protein structures reveal a novel mechanism for capsid maturation: stability without auxiliary proteins or chemical crosslinks. , 2010, Structure.

[52]  A. Klug,et al.  Physical principles in the construction of regular viruses. , 1962, Cold Spring Harbor symposia on quantitative biology.

[53]  R. Hendrix,et al.  Assembly in vitro of bacteriophage HK97 proheads. , 1995, Journal of molecular biology.

[54]  E. Orlova,et al.  Structural framework for DNA translocation via the viral portal protein , 2007, The EMBO journal.

[55]  P. Prevelige,et al.  A P22 Scaffold Protein Mutation Increases the Robustness of Head Assembly in the Presence of Excess Portal Protein , 2002, Journal of Virology.