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• W2024025050 abstract "•CryoEM structure of the 1,300 Å capsid of gammaherpesvirus RRV at 7.2 Å resolution•Domain organizations and secondary structures of the four capsid proteins resolved•Noncovalent chainmail achieved via a hierarchy of four levels of organization•Triplex heterotrimers likely driving putative spherical to angular capsid maturation Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization. Like many double-stranded DNA viruses, tumor gammaherpesviruses Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus withstand high internal pressure. Bacteriophage HK97 uses covalent chainmail for this purpose, but how this is achieved noncovalently in the much larger gammaherpesvirus capsid is unknown. Our cryoelectron microscopy structure of a gammaherpesvirus capsid reveals a hierarchy of four levels of organization: (1) Within a hexon capsomer, each monomer of the major capsid protein (MCP), 1,378 amino acids and six domains, interacts with its neighboring MCPs at four sites. (2) Neighboring capsomers are linked in pairs by MCP dimerization domains and in groups of three by heterotrimeric triplex proteins. (3) Small (∼280 amino acids) HK97-like domains in MCP monomers alternate with triplex heterotrimers to form a belt that encircles each capsomer. (4) One hundred sixty-two belts concatenate to form noncovalent chainmail. The triplex heterotrimer orchestrates all four levels and likely drives maturation to an angular capsid that can withstand pressurization. Gammaherpesviruses constitute a group of double-stranded DNA (dsDNA) tumor viruses that collectively form one of the three subfamilies of the Herpesviridae family (Roizman et al., 2007Roizman B. Knipe D.M. Whitley R.J. Herpes simplex viruses.in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2007: 2502-2601Google Scholar). These viruses are of significant medical relevance, and the two known human gammaherpesviruses, Kaposi’s sarcoma-associated herpesvirus and Epstein-Barr virus, are associated with lymphomas and other malignancies (Chang et al., 1994Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D.M. Moore P.S. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4973) Google Scholar, Ganem, 2007Ganem D. Kaposi’s sarcoma-associated herpesvirus.in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2007: 2847-2888Google Scholar, Rickinson and Kieff, 2007Rickinson A.B. Kieff E. Epstein-Barr Virus.in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2007: 2656-2700Google Scholar). Alphaherpesvirus and betaherpesvirus subfamilies include the well-studied herpes simplex virus type 1 (HSV-1) (Roizman et al., 2007Roizman B. Knipe D.M. Whitley R.J. Herpes simplex viruses.in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2007: 2502-2601Google Scholar) and human cytomegalovirus (Mocarski et al., 2007Mocarski E.S. Shenk T. Pass R.F. Cytomegaloviruses.in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2007: 2702-2772Google Scholar), respectively. We previously showed at ∼25 Å resolution that the overall structural arrangement of the capsid of rhesus monkey rhadinovirus (RRV), a model gammaherpesvirus (O’Connor and Kedes, 2007O’Connor C.M. Kedes D.H. Rhesus monkey rhadinovirus: a model for the study of KSHV.Curr. Top. Microbiol. Immunol. 2007; 312: 43-69PubMed Google Scholar, Orzechowska et al., 2008Orzechowska B.U. Powers M.F. Sprague J. Li H. Yen B. Searles R.P. Axthelm M.K. Wong S.W. Rhesus macaque rhadinovirus-associated non-Hodgkin lymphoma: animal model for KSHV-associated malignancies.Blood. 2008; 112: 4227-4234Crossref PubMed Scopus (63) Google Scholar), is similar to those of alphaherpesviruses and betaherpesviruses (Yu et al., 2003Yu X.K. O’Connor C.M. Atanasov I. Damania B. Kedes D.H. Zhou Z.H. Three-dimensional structures of the A, B, and C capsids of rhesus monkey rhadinovirus: insights into gammaherpesvirus capsid assembly, maturation, and DNA packaging.J. Virol. 2003; 77: 13182-13193Crossref PubMed Scopus (26) Google Scholar), even though the protein sequence identities across these subfamilies are only ∼20%. Herpesviruses are highly complex, with a dsDNA genome of about 200 kilobases, encoding about 100 genes (Roizman et al., 2007Roizman B. Knipe D.M. Whitley R.J. Herpes simplex viruses.in: Knipe D.M. Howley P.M. Griffin D.E. Lamb R.A. Martin M.A. Roizman B. Straus S.E. Fields Virology. Lippincott-Williams & Wilkins, Philadelphia2007: 2502-2601Google Scholar). To house this much dsDNA, herpesvirus capsids are very large, ∼1,300 Å in diameter. Because of the limited depth of focus in current electron microscopes, this large diameter presents a technical challenge to achievement of high-resolution structure by cryo electron microscopy (cryoEM) (Leong et al., 2010Leong P.A. Yu X. Zhou Z.H. Jensen G.J. Correcting for the ewald sphere in high-resolution single-particle reconstructions.Methods Enzymol. 2010; 482: 369-380Crossref PubMed Scopus (23) Google Scholar, Zhang and Zhou, 2011Zhang X. Zhou Z.H. Limiting factors in atomic resolution cryo electron microscopy: no simple tricks.J. Struct. Biol. 2011; 175: 253-263Crossref PubMed Scopus (51) Google Scholar). Indeed, despite recent progress in near atomic-resolution structural studies of smaller viruses (e.g., Jiang et al., 2008Jiang W. Baker M.L. Jakana J. Weigele P.R. King J. Chiu W. Backbone structure of the infectious epsilon15 virus capsid revealed by electron cryomicroscopy.Nature. 2008; 451: 1130-1134Crossref PubMed Scopus (180) Google Scholar, Liu et al., 2010Liu H. Jin L. Koh S.B. Atanasov I. Schein S. Wu L. Zhou Z.H. Atomic structure of human adenovirus by cryo-EM reveals interactions among protein networks.Science. 2010; 329: 1038-1043Crossref PubMed Scopus (281) Google Scholar, Veesler et al., 2013Veesler D. Ng T.S. Sendamarai A.K. Eilers B.J. Lawrence C.M. Lok S.M. Young M.J. Johnson J.E. Fu C.Y. Atomic structure of the 75 MDa extremophile Sulfolobus turreted icosahedral virus determined by CryoEM and X-ray crystallography.Proc. Natl. Acad. Sci. U S A. 2013; 110: 5504-5509Crossref PubMed Scopus (60) Google Scholar, Yu et al., 2008Yu X. Jin L. Zhou Z.H. 3.88 A structure of cytoplasmic polyhedrosis virus by cryo-electron microscopy.Nature. 2008; 453: 415-419Crossref PubMed Scopus (240) Google Scholar, Zhang et al., 2008Zhang X. Settembre E. Xu C. Dormitzer P.R. Bellamy R. Harrison S.C. Grigorieff N. Near-atomic resolution using electron cryomicroscopy and single-particle reconstruction.Proc. Natl. Acad. Sci. U S A. 2008; 105: 1867-1872Crossref PubMed Scopus (294) Google Scholar, Zhang et al., 2010Zhang X. Jin L. Fang Q. Hui W.H. Zhou Z.H. 3.3 A cryo-EM structure of a nonenveloped virus reveals a priming mechanism for cell entry.Cell. 2010; 141: 472-482Abstract Full Text Full Text PDF PubMed Scopus (267) Google Scholar), the highest resolution structure obtained thus far among herpesviruses is about 9 Å for the capsid of HSV-1, an alphaherpesvirus (Zhou et al., 2000Zhou Z.H. Dougherty M. Jakana J. He J. Rixon F.J. Chiu W. Seeing the herpesvirus capsid at 8.5 A.Science. 2000; 288: 877-880Crossref PubMed Scopus (272) Google Scholar). For human gammaherpesvirus capsids, poor sample quantity and quality have further hindered progress (Germi et al., 2012Germi R. Effantin G. Grossi L. Ruigrok R.W. Morand P. Schoehn G. Three-dimensional structure of the Epstein-Barr virus capsid.J. Gen. Virol. 2012; 93: 1769-1773Crossref PubMed Scopus (13) Google Scholar, Wu et al., 2000Wu L. Lo P. Yu X. Stoops J.K. Forghani B. Zhou Z.H. Three-dimensional structure of the human herpesvirus 8 capsid.J. Virol. 2000; 74: 9646-9654Crossref PubMed Scopus (61) Google Scholar). Fortunately, RRV provides an excellent source of gammaherpesviruses, as it grows to high titer in rhesus fibroblasts and allows for purification to obtain uniform capsids (Chang et al., 1994Chang Y. Cesarman E. Pessin M.S. Lee F. Culpepper J. Knowles D.M. Moore P.S. Identification of herpesvirus-like DNA sequences in AIDS-associated Kaposi’s sarcoma.Science. 1994; 266: 1865-1869Crossref PubMed Scopus (4973) Google Scholar, Desrosiers et al., 1997Desrosiers R.C. Sasseville V.G. Czajak S.C. Zhang X. Mansfield K.G. Kaur A. Johnson R.P. Lackner A.A. Jung J.U. A herpesvirus of rhesus monkeys related to the human Kaposi’s sarcoma-associated herpesvirus.J. Virol. 1997; 71: 9764-9769PubMed Google Scholar, O’Connor et al., 2003O’Connor C.M. Damania B. Kedes D.H. De novo infection with rhesus monkey rhadinovirus leads to the accumulation of multiple intranuclear capsid species during lytic replication but favors the release of genome-containing virions.J. Virol. 2003; 77: 13439-13447Crossref PubMed Scopus (17) Google Scholar). The icosahedral capsid shell of herpesviruses is comprised of four abundant proteins (Newcomb et al., 1993Newcomb W.W. Trus B.L. Booy F.P. Steven A.C. Wall J.S. Brown J.C. Structure of the herpes simplex virus capsid. Molecular composition of the pentons and the triplexes.J. Mol. Biol. 1993; 232: 499-511Crossref PubMed Scopus (222) Google Scholar, Rixon, 1993Rixon F.J. Structure and assembly of herpesviruses.Semin. Virol. 1993; 4: 135-144Crossref Scopus (165) Google Scholar, Trus et al., 1995Trus B.L. Homa F.L. Booy F.P. Newcomb W.W. Thomsen D.R. Cheng N. Brown J.C. Steven A.C. Herpes simplex virus capsids assembled in insect cells infected with recombinant baculoviruses: structural authenticity and localization of VP26.J. Virol. 1995; 69: 7362-7366Crossref PubMed Google Scholar, Zhou et al., 1995Zhou Z.H. He J. Jakana J. Tatman J.D. Rixon F.J. Chiu W. Assembly of VP26 in herpes simplex virus-1 inferred from structures of wild-type and recombinant capsids.Nat. Struct. Biol. 1995; 2: 1026-1030Crossref PubMed Scopus (130) Google Scholar). In gammaherpesviruses, these proteins are (1) the major capsid protein (MCP/ORF25), the monomeric subunit of both hexon and penton capsomers; (2) the triplex monomer protein (TRI-1/ORF62); (3) the triplex dimer protein (TRI-2/ORF26); and (4) the small capsomer interacting protein (SCIP/ORF65) (O’Connor et al., 2003O’Connor C.M. Damania B. Kedes D.H. De novo infection with rhesus monkey rhadinovirus leads to the accumulation of multiple intranuclear capsid species during lytic replication but favors the release of genome-containing virions.J. Virol. 2003; 77: 13439-13447Crossref PubMed Scopus (17) Google Scholar, Yu et al., 2003Yu X.K. O’Connor C.M. Atanasov I. Damania B. Kedes D.H. Zhou Z.H. Three-dimensional structures of the A, B, and C capsids of rhesus monkey rhadinovirus: insights into gammaherpesvirus capsid assembly, maturation, and DNA packaging.J. Virol. 2003; 77: 13182-13193Crossref PubMed Scopus (26) Google Scholar). The limited resolution of the capsid structures of all herpesviruses has hindered our understanding of the molecular interactions among these proteins. These interactions are essential for capsid assembly, in particular for orchestration of the extensive conformational changes required for capsid maturation from the immature spherical shape to the mature angular shape (Newcomb et al., 2000Newcomb W.W. Trus B.L. Cheng N. Steven A.C. Sheaffer A.K. Tenney D.J. Weller S.K. Brown J.C. Isolation of herpes simplex virus procapsids from cells infected with a protease-deficient mutant virus.J. Virol. 2000; 74: 1663-1673Crossref PubMed Scopus (106) Google Scholar). Bacteriophage HK97, also a dsDNA virus that must withstand very high internal pressure, up to 50 atmospheres (Gelbart and Knobler, 2009Gelbart W.M. Knobler C.M. Virology. Pressurized viruses.Science. 2009; 323: 1682-1683Crossref PubMed Scopus (76) Google Scholar), with just one capsid protein of 280 amino acid (aa) residues in the mature virus, also undergoes extensive conformational changes, leading to a chainmail topology in its capsid upon maturation (Bamford et al., 2005Bamford D.H. Grimes J.M. Stuart D.I. What does structure tell us about virus evolution?.Curr. Opin. Struct. Biol. 2005; 15: 655-663Crossref PubMed Scopus (298) Google Scholar, Wikoff et al., 2000Wikoff W.R. Liljas L. Duda R.L. Tsuruta H. Hendrix R.W. Johnson J.E. Topologically linked protein rings in the bacteriophage HK97 capsid.Science. 2000; 289: 2129-2133Crossref PubMed Scopus (555) Google Scholar). The special fold that enables HK97 to build chainmail, the “Johnson” fold, is found in the capsid protein of other dsDNA bacteriophages (Dai et al., 2010Dai W. Hodes A. Hui W.H. Gingery M. Miller J.F. Zhou Z.H. Three-dimensional structure of tropism-switching Bordetella bacteriophage.Proc. Natl. Acad. Sci. U S A. 2010; 107: 4347-4352Crossref PubMed Scopus (48) Google Scholar, Jiang et al., 2006Jiang W. Chang J. Jakana J. Weigele P. King J. Chiu W. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus.Nature. 2006; 439: 612-616Crossref PubMed Scopus (250) Google Scholar) and is also evident in the floor region of the MCP in HSV-1 (Baker et al., 2005Baker M.L. Jiang W. Rixon F.J. Chiu W. Common ancestry of herpesviruses and tailed DNA bacteriophages.J. Virol. 2005; 79: 14967-14970Crossref PubMed Scopus (214) Google Scholar). However, how this ancient fold and the chainmail strategy are adapted in the assembly and maturation of the much more complex mammalian herpesvirus capsid remains a puzzle in the absence of knowledge about the folds of all capsid proteins at the secondary structure level. Here, we report a 7.2 Å structure of the RRV capsid by cryoEM and single particle reconstruction. The 1,378 aa long MCP of RRV is organized into six domains that include one forming a fold homologous to the Johnson fold and another interacting with SCIP. Each triplex heterotrimer is comprised of a TRI-1 monomer (with three domains) and a dimer of TRI-2 subunits (with three domains each). The structure also reveals how the MCP and triplex domains (1) interact to bind MCP subunits within a capsomer, (2) bind capsomers to each other, (3) form noncovalently linked belts of capsomers that encircle each individual capsomer, and (4) concatenate the belts to form noncovalent chainmail. Two distinct conformations of the Johnson fold are identified, suggesting a role of conformational switching in orchestrating the global architectural changes required for herpesvirus capsid maturation. Because of the large size of the RRV capsid, we used higher accelerating voltage (300 kV) for cryoEM imaging (Figure 1A) to partially alleviate the depth-of-field limitation (Zhang and Zhou, 2011Zhang X. Zhou Z.H. Limiting factors in atomic resolution cryo electron microscopy: no simple tricks.J. Struct. Biol. 2011; 175: 253-263Crossref PubMed Scopus (51) Google Scholar, Zhou and Chiu, 2003Zhou Z.H. Chiu W. Determination of icosahedral virus structures by electron cryomicroscopy at subnanometer resolution.Adv. Protein Chem. 2003; 64: 93-124Crossref PubMed Scopus (41) Google Scholar) and achieved an effective resolution of 7.2 Å on the basis of the 0.143 “gold-standard” Fourier shell correlation (FSC) criterion (Figure S1A available online). At this resolution, molecular boundaries (Figure 1B; Movie S1) and secondary structure elements (Figure 1C) can be resolved (Nakagawa et al., 2003Nakagawa A. Miyazaki N. Taka J. Naitow H. Ogawa A. Fujimoto Z. Mizuno H. Higashi T. Watanabe Y. Omura T. et al.The atomic structure of rice dwarf virus reveals the self-assembly mechanism of component proteins.Structure. 2003; 11: 1227-1238Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, Zhang et al., 2003Zhang X. Walker S.B. Chipman P.R. Nibert M.L. Baker T.S. Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A.Nat. Struct. Biol. 2003; 10: 1011-1018Crossref PubMed Scopus (143) Google Scholar, Zhou et al., 2001Zhou Z.H. Baker M.L. Jiang W. Dougherty M. Jakana J. Dong G. Lu G. Chiu W. Electron cryomicroscopy and bioinformatics suggest protein fold models for rice dwarf virus.Nat. Struct. Biol. 2001; 8: 868-873Crossref PubMed Scopus (119) Google Scholar), permitting computational segmentation of individual proteins for structural comparison and identification of molecular interactions (Figure 1D). Indeed, sausage-shaped densities, corresponding to helices, are particularly visible in slices of the capsid shell (Figure 1C; Figures S1B and S1C) and from the inside of the capsid (Figure 1D, right; Movie S2). The validity of the interpretation of α helices and putative β sheets can also be gauged by the excellent agreement of such elements in the upper domain with a homology model derived from HSV-1 (Figure S1C) and the Johnson-fold domains between RRV and bacteriophage HK97 (Figures S1D–S1F). The RRV capsid proteins at the innermost radius of the capsid shell (Figure 1D, inside view) are organized in a T = 16 icosahedral lattice (with 12 pentons and 150 hexons that share edges) that is common to the capsids of other herpesviruses. (The putative DNA-translocating portal [Deng et al., 2007Deng B. O’Connor C.M. Kedes D.H. Zhou Z.H. Direct visualization of the putative portal in the Kaposi’s sarcoma-associated herpesvirus capsid by cryoelectron tomography.J. Virol. 2007; 81: 3640-3644Crossref PubMed Scopus (33) Google Scholar] that replaces one of the pentons is not visible here because of icosahedral averaging.) Both penton and hexon capsomers are cylindrical (Figure 1D, side view) with central channels (Movie S2), three of which are visible in the center of Figure 1B and in the inside and outside views in Figure 1D. At the outermost radius of the capsid shell, the icosahedrally averaged capsid also contains 320 triangular components (Figure 1B), each a triplex heterotrimer that interdigitates among the hexons and pentons (Figure 1D, outside view). An asymmetric unit contains six triplex heterotrimers, Ta through Tf (Figure 1D, outside view), each spanning half of the height of the hexon and residing at the inner half of the capsid shell (Figure 1D, side view). Hexagons and pentagons outlining hexons and pentons at the outermost capsid (Figure 1B) are rotated approximately 30° and 36°, respectively, from their orientations in a standard T = 16 lattice. Thus, the radially oriented MCP subunits appear to contribute hexagon/pentagon corners at the radius of the outermost capsid. (See also dashed outlines in the outside view in Figure 1D.) The difference between the outer lattice and the standard T = 16 lattice is similar to the difference between an icosidodecahedron (with 20 triangles and 12 pentagons) and a dodecahedron (with just 12 pentagons). To facilitate the following description of the complex capsid structure, we show a small region of the floor of the capsid shell, a region containing three colored MCP capsomers (orange, blue, and green) (Figures 2A and 2B ). These happen to be three hexons, but they could be two hexons and a penton. At the lowest level of organization (Figure 2A), the six MCP subunits in a hexon capsomer are held together by (“intracapsomer”) interactions between neighboring MCP subunits. At the second (“intercapsomer”) level (Figure 2B), every pair of neighboring capsomers is linked together by binding of an MCP subunit in one capsomer to an MCP subunit in the other. The binding site sits on each and every local two-fold axis (Figure 2B, inset, “2”). In addition, three capsomers are “stapled” together by interactions with a triplex heterotrimer at each and every local and global three-fold position centered among the three capsomers (Figure 2B, “3”). The triplex proteins play another role at the third (“belts”) level by noncovalently connecting belts of MCP subunits (e.g., the yellow, magenta, and red belts in Figure 2C and Movie S3), six around a central hexon or five around a central penton. Comprising a fourth level, the belts concatenate (Figures 2D and 2E). If chainmail is an extended fabric composed of concatenated belts (Figure 2E), then the capsid of RRV may be described as chainmail. Pentons and hexons contain five and six MCP subunits, respectively. With 7.2 Å resolution, we can see that each subunit contains a radially elongated MCP monomer and a V-shaped SCIP monomer on top (Figure 3A). Each MCP subunit has six domains distributed in three regions (Figure 3A; Movie S4): in the upper region the upper domain (Figures 3B–3D); in the middle region the channel, buttress (Figures 3E and 3F) and helix-rich (Figure 3G) domains; and in the floor region the dimerization domain (Figure 3H) and a domain that contains a fold similar to the gp5 structure of bacteriophage HK97 (Figures 3I and 3J) (Wikoff et al., 2000Wikoff W.R. Liljas L. Duda R.L. Tsuruta H. Hendrix R.W. Johnson J.E. Topologically linked protein rings in the bacteriophage HK97 capsid.Science. 2000; 289: 2129-2133Crossref PubMed Scopus (555) Google Scholar). (Also compare the structures of HK97 and RRV MCP in the superimposition of Movie S4 and Figure S1D with the RRV MCP Johnson-fold domain in Figures S1E and S1F.) This Johnson fold was subsequently seen in other viruses (e.g., Baker et al., 2005Baker M.L. Jiang W. Rixon F.J. Chiu W. Common ancestry of herpesviruses and tailed DNA bacteriophages.J. Virol. 2005; 79: 14967-14970Crossref PubMed Scopus (214) Google Scholar, Bamford et al., 2005Bamford D.H. Grimes J.M. Stuart D.I. What does structure tell us about virus evolution?.Curr. Opin. Struct. Biol. 2005; 15: 655-663Crossref PubMed Scopus (298) Google Scholar, Dai et al., 2010Dai W. Hodes A. Hui W.H. Gingery M. Miller J.F. Zhou Z.H. Three-dimensional structure of tropism-switching Bordetella bacteriophage.Proc. Natl. Acad. Sci. U S A. 2010; 107: 4347-4352Crossref PubMed Scopus (48) Google Scholar, Jiang et al., 2006Jiang W. Chang J. Jakana J. Weigele P. King J. Chiu W. Structure of epsilon15 bacteriophage reveals genome organization and DNA packaging/injection apparatus.Nature. 2006; 439: 612-616Crossref PubMed Scopus (250) Google Scholar). The dimerization domain (Figure 3H) extends along the side of the Johnson-fold domain and reaches beneath it (Figure 3A). The MCP upper domain (MCPud) is rich in helices and loops (Figure 3B). To build a pseudoatomic model of RRV MCPud, we used these secondary structure elements as constraints for comparative modeling with the crystal structure of HSV-1 MCPud (Bowman et al., 2003Bowman B.R. Baker M.L. Rixon F.J. Chiu W. Quiocho F.A. Structure of the herpesvirus major capsid protein.EMBO J. 2003; 22: 757-765Crossref PubMed Scopus (85) Google Scholar) as a template. Ramachandran statistics of the MCPud homology model (Figures S1G and S1H) indicate model quality on par with the original HSV-1 VP5 upper domain structure. The differences and similarities of the RRV MCPud (red) and the HSV-1 MCPud (green) are clear in the superimposed ribbon models (Figure 3C). Whereas the interior structures, which are mostly helical, are highly conserved, structures on and near the external surfaces show some differences, especially at the uppermost tip (magnified in the top box at the upper right in Figure 3C), where the red and green loops do not superimpose. Indeed, a proline-rich, 12 aa segment within the HSV-1 (green) loop (green highlighted portion of aa sequence in Figure 3D) is absent in the RRV (red) loop. The middle region of MCP encompasses the channel, buttress (Figure 3E), and helix-rich domains (Figure 3G). We divided the middle region into these three domains because they are spatially separated and have distinctive structural roles (e.g., channel and buttress domains form the channel and provide support, respectively) (Movie S4) and because the conformation of the helix-rich domain differs greatly between MCP in hexon and penton. The channel domain contains a large, twisted, central, putative β sheet (Figure 3E). The helix-rich domain of hexon MCP contains three long helices, one of which adopts a different conformation in penton MCP. For the RRV Johnson-fold domain (Figure 3I), we have followed the HK97 terminology to describe structural features (named subdomains) that are homologous to domains established in the HK97 gp5 (Wikoff et al., 2000Wikoff W.R. Liljas L. Duda R.L. Tsuruta H. Hendrix R.W. Johnson J.E. Topologically linked protein rings in the bacteriophage HK97 capsid.Science. 2000; 289: 2129-2133Crossref PubMed Scopus (555) Google Scholar) (Figure 3J). The RRV Johnson-fold domain contains an extended loop (E loop) and a peripheral (P) subdomain with a 58 Å long “spine” helix that together represent the contribution of each MCP subunit to the belt described in Figure 2C. On the basis of sequence analysis, we assign the spine helix of RRV MCP to aa 147 to 184 (Figure S2), the only segment of the MCP sequence that contains a helix of more than 30 aa. The spine helix and its connected, putative β sheet in the P subdomain contribute to the long tongue-shaped density in the hexon MCP in Figures 3A and 4A and in the penton MCP in Figure 4B, which are similar but not identical (Figure 4C). The axial (A) subdomain has a central, putative β sheet flanked by two helices. The long N-terminal arm, which is not clearly resolved in our RRV map, can adopt multiple conformations in HK97 (Gertsman et al., 2009Gertsman I. Gan L. Guttman M. Lee K. Speir J.A. Duda R.L. Hendrix R.W. Komives E.A. Johnson J.E. An unexpected twist in viral capsid maturation.Nature. 2009; 458: 646-650Crossref PubMed Scopus (104) Google Scholar, Veesler et al., 2012Veesler D. Quispe J. Grigorieff N. Potter C.S. Carragher B. Johnson J.E. Maturation in action: cryoEM study of a viral capsid caught during expansion.Structure. 2012; 20: 1384-1390Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar). Both pentons and hexons have an axial channel running from the upper region through the floor region of the MCP, which constitutes the floor of the capsid shell (Figures 1B–1D). Side views of a pair of neighboring MCPs in a hexon (Figure 4D) and in a penton (Figure 4E) show the interior walls of their axial channels. The most striking differences between the penton and hexon channels are the sites of interaction or lack thereof. Specifically, adjacent MCPs in a hexon have four interaction sites (Figure 4D; Movie S5), two between their upper domains (#1 and #2), one between the small, putative β sheet of their channel domains (#3), and one between their Johnson-fold domains in the floor (#4). Only #4 is present between subunits in pentons (Figure 4D versus Figure 4E). Even though the limited resolution of our cryoEM map has precluded modeling of side chains of aa residues, we are confident about the assignment of the aa segments contributing to these interactions. We suggest in the Discussion that these interaction sites offer insight into capsid maturation. Interaction site #1 is located between helices in the interfacial region of neighboring MCPud, near the interior of the channel (Figure 4F). The densities arising from six interaction sites #1 inside each hexon form a bonded ring near the top of the channel. A fit of our pseudoatomic model of MCPud into hexon cryoEM density reveals the details of these interactions, specifically a region containing polar aa residues from two neighboring MCPud (Figure 4F; Movie S5). On one subunit, the most likely contributors to this interaction are Ser670, Lys671, and Asp672, which are located at the end of a helix (aa 670–694). On the other subunit, the most likely contributors are Asn643 and Asn644, located in a kink between two helices (aa 624–645 and 654–665) (Figure 4F; Figure S2). Interaction site #2, also located in the interfacial region between two neighboring MCPud but close to the outer surface of the hexon, rather than near the interior of the channel (Movie S5), likely involves charged residues, Arg973 of one MCPud and Glu707 of its neighboring MCPud (Figure 4F; Figure S2). Interaction site #3, which lies in the channel domain of each MCP, is mediated through the small, putative β sheets on neighboring MCP monomers near the interior of the channel (Figure 4G); thus, six interaction sites #3 comprise a second, lower constricting ring inside each hexon channel. Although MCP monomers in both hexons and pentons show interaction site #4 in the floor region, the absence of interaction sites #1 to #3 among pentons, including the absence of the two constricting rings, is accompanied by a splaying outward of the middle and upper regions of MCP monom" @default.
• W2024025050 created "2016-06-24" @default.
• W2024025050 creator A5003807322 @default.
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• W2024025050 date "2014-10-01" @default.
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• W2024025050 title "Four Levels of Hierarchical Organization, Including Noncovalent Chainmail, Brace the Mature Tumor Herpesvirus Capsid against Pressurization" @default.
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