We aim to elucidate the molecular mechanisms that control the assembly of viruses with the ultimate goals of defining targets for antiviral compounds, characterizing viral antigenicity, and establishing precedents for understanding the assembly of macromolecular complexes in general. Our research focuses on the large-scale conformational changes that accompany capsid maturation and on the interaction of viruses with host cells. To this end, we pursue five subprojects. (1) Hepatitis B Virus Capsid Assembly. Background. We have been studying the HBV capsid protein which carries two major antigens, core antigen and e-antigen. After our first studies showed that the protein forms dimers capable of self-assembly into particles of two different sizes and its fold is predominantly alpha-helical, in 1997, we calculated a cryo-EM density map at 0.9nm resolution, in which much of the secondary structure was visible, including the 4-helix bundle that forms the dimerization motif. Our subsequent research helped delineate the path of the polypeptide chain, which has since been confirmed by crystallography. We are now investigating the antigenic diversity (distinct epitopes simultaneously present) of the HBV capsid. Results. We used cryo-EM of Fab-labelled capsids to study the capsid binding of three more monoclonals with conformational epitopes bringing the total to six. The availability of atomic models of the capsid and prototypic Fabs allows us to identify the discontinuous peptides that make up these epitopes. All monoclonals so far characterized have quite different epitopes. We published two papers describing them and are analyzing the remaing two. Conclusions. An extensive range of antigenic diversity is presented by the capsid spike a simple motif of two helix-loop-helix elements, and other epitopes are presented on the capsid floor (2) Capsid Assembly and Maturation of Herpesviruses. Herpesviruses constitute an extensive family of large, complex, DNA viruses. Eight members cause diseases in humans, including skin diseases. We have studied herpesvirus capsid assembly for several family members, including herpes simplex (HSV), and cytomegalovirus (CMV). Initially we defined the molecular anatomy of the capsid and then characterized the roles of its six major proteins in assembly. These roles are distinct from any other known family of animal viruses but share numerous points of resemblance with DNA bacteriophages. One major finding was our discovery, in 1996, of the HSV procapsid - a normally short-lived precursor that differs radically from the mature capsid in structure, stability, and composition. Maturation of the procapsid is controlled by the viral protease and is a potential target for antiviral drugs. We have been investigating this process of time-resolved cryo-EM. Results and Conclusions. We completed and published the work described last year on dynamic visualization of procapsid maturation. The basic molecular mechanism involves relative rotations and translations of domains in the surface lattice. Switches at different quasiequivalent sites are thrown at different times. We also started work on the portal/connctor complex which represents the channel through which DNA pases into the capsid, and performed tomographic studies of intact virions. (3) Assembly and Maturation of Bacteriophage Capsids. Our primary interest in bacteriophage capsid assembly lies in the monumental conformational changes that accompany maturation of theirprocapsids. These changes are irreversible, involve partial refolding, and are stringently controlled. As such, they afford unique opportunities for insight into large-scale regulatory conformational changes in protein complexes. Although phages vary widely in size, sequence, and other properties, maturation is a universal feature. We study this event in several systems to exploit expedient aspects of each. Over the past year we continued to clarifying the maturation pathway of the HK97 capsid and to investigate the packing of DNA in T4 heads. Results. In order to study the energetics of capsid maturation in a quantitative way, we initiated calorimetric comparisons of each distinct structural state in conjunction with cryo-EM analyses. The results to date indicate that, unlike T4 in which the major stabilizing event is the surface lattice expansion, this transition has little effect for HK97 which relies mainly on the formation of a network of covalent crosslinks. The packing of T4 DNA is highly complex and apparently different from that of T7 which we described earlier in terms of the orientation of the spool relative to the portal axis. (4) Electron Microscopic Studies of HIV- and AIDS-related Proteins. We participate in the NIH Targeted Antiviral Program by pursuing relevant projects in which our expertise in EM-based structural analysis may be applied. We performed electron microscopic investigations of mutant HIV capsids which are produced by certain mutants in the capsid (CA) protein. The capsids were found to be extremely disordered. This observation has been written up as part of a larger study. The main thrust of our IATAP work has now shifted towards applications of electron tomography. We successfully completed an analysis of herpes simplex by this technique (see above) and are now shifting our attention to retroviral specimens. At least two herpesviruses - Kaposis Sarcoma-associated Herpesvirus (KSHV) and cytomegalovirus (CMV) - pose significant threats to immunocompromized AIDS patients. Our studies of these viruses (see above) fall partly under the aegis of the IATAP program.
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