During FY12, 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. These studies indicated, on statistical sampling grounds, that the total number of distinct epitopes is in the range of 15-20. They lie in two regions on the capsid surface: on the protruding spikes and on the low-lying 'floor'region (15). Similar analyses of polyclonal Fabs from a human patient indicated the presence of epitopes in both regions. We also investigated by x-ray crystallography the structure of the e-antigen which differs from cAg in having at its N-terminus an additional ten residues (1). eAg and cAg are antigenically distinct but are cross-reactive. 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 (11). We also completed and 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. By focusing on the capsid protein, we were able to acquire deuterium uptake profiles covering its entire 149-residue sequence and to reveal, in localized detail, the changes in H/D exchange rates that accompanied antibody binding. We observed protection of two epitopes upon Fab binding, but also found that regions distant from the epitopes are also affected, i.e allosterically transmitted effects on conformation. Even at sub-stoichiometric Fab binding, an overall increase in the rigidity of the capsid structure was detected, as well as a general damping of the breathing motions of the capsid (10). (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 FY13, 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. Fitting the crystal structure into the procapsid disclosed substantial conformational differences between the two P1 conformers. Maturation via two intermediate states involves remodeling on a similar scale, besides huge rigid-body rotations. The capsid structure and its stepwise maturation which correlates with sequential packaging of three RNA segments sets the cystoviruses apart from other dsRNA viruses as a dynamic molecular machine. (2b) Packaging and injection of internal proteins. Infection by double-stranded DNA phages involves the injection of proteins as well as genomic DNA into the host cell. PhiKZ is a large and complex virus that infects the pathogenic bacterium Pseudomonas aeruginosa. The virion has a large icosahedral capsid containing densely packed DNA (280kbp) as well as a proteinaceous "inner body" which is invisible in cryo-electron micrographs because of contrast-matching with the surrounding DNA. We found that the inner body is exceptionally sensitive to electron irradiation and explodes into bubbles of gaseous products at doses that leave the surrounding capsid only slightly blurred. In conventional cryo-EM, internal proteins are hard to detect because they are contrast-matched with the DNA, but they can be visualized by bubblegram imaging (3). This technique revealed that that the inner body is 24 nm wide, 105 nm long, and consists of stacked tiers with 6-fold symmetry. Biochemical analysis indicated that the inner body has five major proteins as well as a number of minor proteins (4). The shape and position of the inner body suggest that it plays a role of organizer in the DNA packaging process. We then went on to apply the same approach to define the corresponding structure in the genetically better defined T7 coliphage (work in progress). (3) Herpesviruses have an icosahedral nucleocapsid surrounded by an amorphous tegument and a lipoprotein envelope. The tegument comprises at least 20 proteins destined for delivery into the host cell. As the tegument does not have a regular structure, the question arises of how its proteins are recruited. The HSV-1 tegument is known to contact the capsid at its vertices and two proteins, UL36 and UL37, had been identified as candidates for this interaction. We showed by cryo-EM that capsids with and without UL37 exhibit a vertex-associated density that represents the ordered portion of UL36 (336 kDa).These observations (2) support the hypothesis that UL36 provides a flexible scaffold to which other tegument proteins, including UL37, bind. They also indicate how sequential conformational changes in the maturing nucleocapsid control the ordered binding of UL36 and UL37. (4) While much of the research described above was performed with icosahedral capsids which may be imaged by high-resolution cryo-EM, many viruses do not conform to icosahedral symmetry. Nevertheless, their structures may be studied by cryo-electron tomography. We published the first such analysis of a pleiomorphic virus - herpes simplex virus type 1 - in 2003 and have gone on to make numerous other applications. In FY12, we completed such studies of Rubella virus (8) and Newcastle disease virus (7). In FY12 we studied how influenza virus responds to acidic pH (9). The low pH of endosomes triggers conformational changes in hemagglutinin (HA) that mediate fusion of the viral and endosomal membranes. At pH 4.9, we observed dramatic changes in morphology: elongated particles were no longer observed (6);and larger particles representing fused virions appeared to shed their layer of matrix protein, which we infer makes the particle more pliable and conducive to membrane fusion. In a more detailed analysis we also found that shedding is preceded by a conformational change in M1 protein (12).

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National Institute of Arthritis and Musculoskeletal and Skin Diseases
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Cairns, Tina M; Fontana, Juan; Huang, Zhen-Yu et al. (2014) Mechanism of neutralization of herpes simplex virus by antibodies directed at the fusion domain of glycoprotein B. J Virol 88:2677-89
Bereszczak, Jessica Z; Watts, Norman R; Wingfield, Paul T et al. (2014) Assessment of differences in the conformational flexibility of hepatitis B virus core-antigen and e-antigen by hydrogen deuterium exchange-mass spectrometry. Protein Sci 23:884-96
Bereszczak, Jessica Z; Havlik, Marlene; Weiss, Victor U et al. (2014) Sizing up large protein complexes by electrospray ionisation-based electrophoretic mobility and native mass spectrometry: morphology selective binding of Fabs to hepatitis B virus capsids. Anal Bioanal Chem 406:1437-46
Cardone, Giovanni; Moyer, Adam L; Cheng, Naiqian et al. (2014) Maturation of the human papillomavirus 16 capsid. MBio 5:e01104-14
Cheng, Naiqian; Wu, Weimin; Watts, Norman R et al. (2014) Exploiting radiation damage to map proteins in nucleoprotein complexes: the internal structure of bacteriophage T7. J Struct Biol 185:250-6
Wu, Weimin; Chen, Zhaochun; Cheng, Naiqian et al. (2013) Specificity of an anti-capsid antibody associated with Hepatitis B Virus-related acute liver failure. J Struct Biol 181:53-60
Bereszczak, Jessica Z; Rose, Rebecca J; van Duijn, Esther et al. (2013) Epitope-distal effects accompany the binding of two distinct antibodies to hepatitis B virus capsids. J Am Chem Soc 135:6504-12
Nemecek, Daniel; Boura, Evzen; Wu, Weimin et al. (2013) Subunit Folds and Maturation Pathway of a dsRNA Virus Capsid. Structure 21:1374-83
DiMattia, Michael A; Watts, Norman R; Stahl, Stephen J et al. (2013) Antigenic switching of hepatitis B virus by alternative dimerization of the capsid protein. Structure 21:133-42
Fontana, Juan; Steven, Alasdair C (2013) At low pH, influenza virus matrix protein M1 undergoes a conformational change prior to dissociating from the membrane. J Virol 87:5621-8

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