During FY14, we focused mainly on the following four subprojects: (1) Hepatitis B Virus Capsid Assembly. We study the HBV capsid protein which presents two of the three clinically important antigens - core antigen (cAg, capsids) and e-antigen (eAg, unassembled protein) - of this major human pathogen. After first showing that capsid protein self-assembles from dimers into shells of two different sizes, we obtained, in 1997, a cryo-EM density map in which we visualized the 4-helix bundle that forms the dimerization motif. This was the first time that such detailed information was achieved by cryo-EM. We went on to investigate the antigenic diversity of HBV by using cryo-EM and molecular modeling to characterize the epitopes of a total of eight monoclonal antibodies. The epitopes lie in two regions on the capsid surface: on the protruding spikes and on the l 'floor'region. We have also used x-ray crystallography to determine the structure of the e-antigen which has at its N-terminus an additional ten residues. The eAg structure revealed a monomer with a similar fold to the cAg monomer but an entirely different mode of dimerization. This switch accounts for the profound differences in assembly properties and antigenicity between the two proteins. These results were published in FY13. In FY13, we also published a study in which native mass spectrometry was combined with hydrogen-deuterium exchange to investigate the effects on HBV capsids of binding two antibodies. In FY14, we extended this line of investigation as reported in two publications. In these studies (1,2), we addressed the effects of capsid binding by antibodies bind near the spike apices (the alfa-determinant) or in the floor regions (the beta-determinant). These studies also drew on a new technique called gas-phase electrophoretic mobility molecular analysis (GEMMA). We found that both methods readily distinguished Fab binding to the two capsid morphologies (T=3 and T=4) and could provide accurate masses and dimensions for these large immune complexes, up to 8MDa. As such, native MS and GEMMA provide valuable alternatives to a more time-consuming cryoelectron microscopy analysis for preliminary characterization of capsid-antibody complexes. We are currently investigating the dependence of capsid structure on incorporated nucleic acid and the structure of the T=3 capsid. (2) Assembly and Maturation of Bacteriophage Capsids. Our interest in capsid assembly lies in the massive conformational changes that accompany their maturation. These transitions afford unique insights into allosteric regulation. We study maturation of several phages to exploit expedient aspects of each system. The tailed phages afford an excellent model for herpesvirus capsids, reflecting common evolutionary origins. In FY14, we pursued the following investigations. (2a) Coupling of genome packaging to capsid maturation in the cystovirus system. The capsids of double-stranded RNA viruses serve as compartments for the replication and transcription of the viral genomes. We investigate the structural basis of this remarkable phenomenon for phage phi6, which has a tripartite genome. In FY08, we located the P2 polymerase inside the viral procapsid. In FY10, we completed a study using cryo-electron tomography to map the distributions of P2-occupied sites and of the external sites occupied by P4, the packaging ATPase. In FY11, we investigated the expansion transformation undergone by the procapsid and identified two structural intermediates. In FY12, we located the P7 packaging facilitator, which turned out to compete with the P2 polymerase for sites on the interior surface of the capsid (5). In FY13, we determined the crystal structure of the capsid protein P1, revealing a flattened trapezoid subunit with a novel alfa-helical fold (14). We also solved the procapsid by cryo-electron microscopy to 0.45 nm resolution. We found that maturation proceeds via two intermediate states and involves some remodeling, besides huge rigid-body rotations. These observations set the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine. In FY14, we have focussed on the binding of the cellular protein YazQ to the outer surface of the mature phi6 capsids. This study is ongoing. (2b) Packaging and injection of internal proteins. While many structural approaches can be used to localize protein subunits, domains, or epitopes that are exposed on the surface of a nucleocapsid or other macromolecular complex, there have been few that are applicable to proteins that are buried in the interior. We have been exploring the potentiality of radiation damage under cryo-EM conditions for this purpose. Sustained radiation eventually elicits the formation of bubbles of hydrogen gas in proteins that are embedded in DNA. Their location can be exploited in reconstructions calculated from earlier images of the same (undamaged) specimen in which the internal proteins are unseen. Our first success was with PhiKZ, a large and complex virus that infects the pathogenic bacterium Pseudomonas aeruginosa. We detected a cylindrical """""""" inner body"""""""" 105 nm long, consisting of stacked tiers with 6-fold symmetry. Its shape and position suggest that the inner body plays a role of organizer in the DNA packaging process. In FY14, we went on to characterize the internal structure of bacteriophage T7, viewed as a partially defined model system (3). T7 is known to have a barrel-shaped protein core under the portal vertex. Here, we detected two classes of bubbles. Initially, 1 to 4 bubbles nucleate in the core: subsequently, these bubbles grow and merge. 3D reconstruction depicted a bipartite cylindrical gas cloud in the core. Its portal-distal half is occupied by a 3 nm-wide dense rod. We propose that it represents an end of the packaged genome, poised for injection into a host cell. Single bubbles appearing later at other sites may represent residual scaffolding protein. (3) Over the past 20 years, we have studied many aspects of herpesvirus assembly, focussing mainly on the nucleocapsid. In FY14, we completed a study to map the various epitopes of the gB glycoprotein, a fusogenic component of the viral envelope (4). gB presents as a rod with three lobes (base, middle, crown) and it has 4 functional regions (FR), defined by the reactivities of different monoclonal antibodies (MAbs). We characterized FR1 epitopes by visualizing Fab-gB complexes, confirming the locations of several epitopes and localizing for the first time those of two other Mabs. We are also pursuing structural studies of capsid maturation. (4) Papillomavirus maturation. The Papillomaviridae are a family of DNA viruses that inhabit the skin or mucosal tissues of their vertebrate hosts. Unlike other non-enveloped viruses, papillomaviruses are released into the environment through a gradual process called desquamation. During this process, disulfide cross links are formed between neighboring molecules of the major capsid protein, L1. This is thought to stabilize the maturing virion. In FY14, we completed a study in which time-lapse cryo-EM was used to study the maturation of HPV16 (5). Initially, the virion is a loosely connected prolapsed that condenses into the mature papillomavirus capsid. In this process, the procapsid shrinks by 5% in diameter, and its pentameric capsomers change in structure (most markedly in their axial region), and the interaction surfaces between adjacent capsomers are consolidated. Biowulf has been used in some of the HBV and HSV studies.

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Eren, Elif; Watts, Norman R; Dearborn, Altaira D et al. (2018) Structures of Hepatitis B Virus Core- and e-Antigen Immune Complexes Suggest Multi-point Inhibition. Structure 26:1314-1326.e4
Mata, Carlos P; Mertens, Johann; Fontana, Juan et al. (2018) The RNA-binding protein of a double-stranded RNA virus acts like a scaffold protein. J Virol :
Zhuang, Xiaolei; Watts, Norman R; Palmer, Ira W et al. (2017) Chimeric rabbit/human Fab antibodies against the hepatitis Be-antigen and their potential applications in assays, characterization, and therapy. J Biol Chem 292:16760-16772
Newcomb, William W; Fontana, Juan; Winkler, Dennis C et al. (2017) The Primary Enveloped Virion of Herpes Simplex Virus 1: Its Role in Nuclear Egress. MBio 8:
Wu, Weimin; Leavitt, Justin C; Cheng, Naiqian et al. (2016) Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging. MBio 7:
Wu, Weimin; Newcomb, William W; Cheng, Naiqian et al. (2016) Internal Proteins of the Procapsid and Mature Capsids of Herpes Simplex Virus 1 Mapped by Bubblegram Imaging. J Virol 90:5176-86
Pérez-Berná, Ana J; Marion, Sanjin; Chichón, F Javier et al. (2015) Distribution of DNA-condensing protein complexes in the adenovirus core. Nucleic Acids Res 43:4274-83
Aksyuk, Anastasia A; Newcomb, William W; Cheng, Naiqian et al. (2015) Subassemblies and asymmetry in assembly of herpes simplex virus procapsid. MBio 6:e01525-15
Fontana, Juan; Steven, Alasdair C (2015) Influenza virus-mediated membrane fusion: Structural insights from electron microscopy. Arch Biochem Biophys 581:86-97
Cardone, Giovanni; Duda, Robert L; Cheng, Naiqian et al. (2014) Metastable intermediates as stepping stones on the maturation pathways of viral capsids. MBio 5:e02067

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