Peter E. Prevelige - US grants

DESCRIPTION (provided by applicant): Viral capsids assemble inside infected cells through a staged series of structural transformations. The initial structure that is assembled is seldom the biologically active form. To gain insight into this process considerable effort has been put into characterizing the structures of the initial and final states of the capsids. A missing piece of the picture is a characterization of the dynamic pathway of transformation. As a class, the dsDNA containing viruses such as the bacteriophage and Herpesviridae initially assemble a procapsid into which the DNA is packaged. DNA packaging requires an ATP dependent motor protein termed a portal or connector protein, and the process of packaging itself results in a structural transformation from an unstable to stable capsid. In this proposal we will obtain dynamic information about the process of DNA packaging and capsid expansion. The morphogenetic similarities of these dsDNA viruses allow us the luxury of selecting the system best suited for studying each step with the reasonable expectation that we will be discovering underlying general principles. Therefore, we will study the structural transformation accompanying DNA packaging in bacteriophage P22 and the portal or connector motor protein complex function in bacteriophage Phi-29. The specific aims of the proposal are: 1) To determine if there is a conserved structural motif in the portal protein motor complexes of these dsDNA viruses. 2) To characterize the motions that enable the P22 procapsid to capsid transformation. and 3) To identify the dynamic motions in the Phi-29 portal motor complex during DNA packaging. These studies will elucidate the fundamental mechanisms underlying the assembly of DNA viruses and may suggest novel targets for the development of antivirals.

[unreadable] DESCRIPTION (provided by applicant): The objective of this program is to provide high quality training at the predoctoral and postdoctoral levels and to prepare outstanding individuals for careers in research and teaching in Basic Mechanisms of Virology. We have selected from among the more than 30 virologists at UAB those whose research programs focus on Basic Mechanisms in Virology to develop a multidisciplinary, multi-departmental, group of eighteen investigators with proven expertise and research interests in molecular and structural biology of viruses. The ongoing research interests of these faculty members include investigations of the structure and assembly of viruses and viral components, the genetics and replication of RNA and DNA vaccines, structure-function relationships in viral proteins, and the development of novel antiviral agents and vaccines. The faculty members in this proposal occupy laboratories that are well equipped and well funded for all phases of research. The participating faculty, from the Departments of Biochemistry, Microbiology, Pediatrics, and Medicine, believe that we have at UAB an excellent environment for providing outstanding predoctoral and postdoctoral training in modern molecular Virology. Predoctoral trainees are selected after their first year of study from the pool of highly qualified students entering a multi-departmental program entitled "the Cellular and Molecular Biology (CMB) Graduate Program" or a highly selective M.D./Ph.D. program. Specific requirements for predoctoral and postdoctoral trainees enrolled on the Basic Mechanisms of Virology Program include participation in advanced level courses in virology, a journal club, the weekly Virology research seminars as well as attendance at Departmental seminars. Additional advanced courses in related fields are required for students and available to postdoctoral trainees. Trainees are encouraged to apply for individual fellowships and awards, and are instructed in how to write research proposals. In addition to research activities and courses, the training program includes participation in local, regional, and national scientific meetings. Recruitment of both predoctoral and postdoctoral trainees will be at the national level through a variety of recruitment programs and will encourage recruitment of minority participants. [unreadable] [unreadable]

Viruses have shells made of repeated protein subunits surrounding their genetic information. Many viruses, including polio, herpes, and influenza, have icosahedraloshaped shells. It is not understood how these shells self-assemble from hundreds of similar protein subunits. A resolution to this question is important because it allows us to better understand biological phenomena in general and provides a paradigm for nano- and meso-scale self- assembling systems which will become increasingly important in materials and manufacturing. It might also eventually result in mechanisms for interrupting shell formation and interfering with the infection process. Drs. Berger (MIT) and Prevelige (Univ. of Alabama at Birmingham) are using cutting edge high performance computing and biotechnology to design a realistic computer "toolkit" that will allow biologists to study virus shell assembly on a computer screen more easily and less expensively than comparable laboratory work. The synergy of computational and biochemical approaches promises rapid advances in our understanding of virus assembly and self-assembling systems in general. This work is supported by the Computational Biology Activity (BIO), the Computational Mathematics Program (MPS), and the Office of Multidisciplinary Activities (MPS).

[unreadable] DESCRIPTION (provided by applicant): HIV assembles in a two step process in which an immature virion composed of the Gag polyprotein assembles at the plasma membrane, acquires the envelope glycoprotein, and buds from the infected cell. In the second step, a viral encoded protease cleaves the Gag polyprotein into its constituent matrix (MA), capsid (CA), and nucleocapsid (NC) structural domains. Cleavage results in a profound morphological rearrangement of th structural domains marked by the formation of a conical core of CA surrounding a complex of the NC and viral RNA. If the core is not well formed, either through blocked cleavage or the introduction of mutations, the resultant virus is non-infectious. This suggests that blocking the structural rearrangement is a potential therapeutic approach. To do so requires a detailed understanding of the immature and mature virions as well as the sequence of events driving the transformation. We have developed a mass spectrometry based hydrogen/deuterium exchange and crosslinking approach that allows us to the structural rearrangements that accompany maturation at the molecular level. In this application we propose to use that technology to: Aim 1 - Determine whether CA exists in a domain swapped configuration in either the immature or mature virion Aim 2- Obtain a detailed understanding of the process of HIV maturation at the molecular level Aim 3 - Assemble stable hexamers of HIV-1 CA and peform a detailed biophysical and structural analysis to fully characterize the N-terminal domain/C-terminal domain interaction that is formed upon maturation. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The objective of this proposal is to develop a general methodology for the rapid identification of intersubunit interfaces in protein complexes, and utilize it to identify the subunit/subunit interfaces in immature and mature HIV-1 capsids, The strategy will be to synthesize a family of biotin-tagged chemical cross-linkers with both specific and non-specific cross-linking activity and use them as distance constraints to position subunits of known atomic structure (in this case the structural proteins of HIV) relative to one another in space, To achieve the necessary structural resolution the cross-linked amino acid residue pairs will be identified by mass spectrometry. This project extends the parent grant (AI44626) whose mission is to utilize hydrogen/deuterium exchange studies to identify intersubunit interfaces and dynamic motions in HIV capsids. The novel component of this proposal is the synthesis of biotin tagged cross-linkers, which can be rapidly purified using strepavidin affinity techniques coupled with mass spectrometry for identification of the cross-linked peptides. Such cross-linkers are not commercially available. Our preliminary data indicates that while the cross-linking data can be used to pack subunits, its widespread applicability is limited by the inherent difficulty of detecting a small number of cross-linked peptides against a background of a large number of uncross-linked peptides. Biotin tagged cross-linkers can be rapidly purified from the complex digests and analyzed by mass spectrometry resulting in increases in sensitivity and throughput. As a result the technique will become a generally applicable tool for merging data from the structural genomics and proteomics initiatives.

Abstract Not Provided.

A grant has been awarded to the University of Alabama at Birmingham (UAB) under the direction of Dr. David Chaplin for partial support of the purchase of a 200 kV field-emission gun cryo-electron microscope (cryo-EM). Contemporary research in a broad range of scientific disciplines ranging from nanotechnology, to biochemistry, virology, and cell biology relies on defining molecular structures to understand biophysical and biochemical mechanisms. Electron microscopy is one of the most important techniques in modern life science research and the only one that is able to directly image structures ranging in size from individual atoms to whole cells and tissues. Cryo-electron microscopy is a technique that allows biological material to be imaged in its native, hydrated form, in the absence of the damaging stains and fixatives required by conventional electron microscopy. Due to its high quality imaging system, the cryo-EM can achieve a resolution that approaches that of X-ray crystallography, allowing structure determination of proteins and protein/nucleic acid complexes. Using tomographic approaches, the three-dimensional organization of whole cells and organelles can be visualized in their native, functional states. The acquisition of this cryo-EM is a central component of campus-wide structural biology initiatives that link the UAB School of Medicine and School of Natural Sciences & Mathematics. The instrument will be available on a shared basis to researchers throughout the southeast United States, expanding its impact to that of a regional resource.

Establishment of the 200 kV cryo-EM at UAB will dramatically accelerate research and promote teaching, training, and learning at UAB. Several research programs, ranging from the structure and assembly of bacteriophages and animal viruses to cell wall pore proteins and the analysis of nanoparticles used in preparation of prosthetic joints will benefit from the availability of this instrument. In addition to these ongoing research projects, the cryo-EM will stimulate the development of new research initiatives and will support the expansion of training programs leading to careers in structural biology research. These training initiatives will include laboratory courses available to both undergraduate and graduate students at UAB. The cryo-EM will also be used as part of the BioTeach program offered through UAB's Center for Community Outreach and Development. BioTeach provides basic knowledge and laboratory skills to high school science teachers.

The acquisition of this 200 kV cryo-EM will dramatically advance the scientific discovery process and will promote teaching, training, and learning at UAB. Local availability of this cryo-EM will accelerate progress in research focused on structural aspects of virus assembly and maturation, and in nanostructures for bioengineering and biomedicine. Lastly, the cryo-EM will be used in an educational program to exposes Alabama K-12 teachers to high-end instruments and modern approaches used in scientific research. This experience is expected to stimulate their students, particularly underserved minorities and females, to consider post-secondary educational training and careers in science.

[unreadable] DESCRIPTION (provided by applicant): This application seeks partial funding for a biannual conference series entitled "Virus Assembly" to be held under the sponsorship of the Federation of American Societies of Experimental Biology (FASEB). The next meeting will be held in Saxton's River, Vermont, June 17-22, 2006, with subsequent meetings[unreadable] being held in June/July 2008, and June/July 2010. This conference is structured to provide an optimal opportunity for cross-disciplinary interaction between scientists that share interests in all aspects of virus assembly including virus structure, particle formation and maturation, genome packaging, virus entry and exit, virus trafficking within cells and host interactions. These topics are discussed within the context of fundamental issues in virology and as they relate to novel approaches for antiviral intervention. We also seek to encourage discussion of relatively unconventional applications of virology, such as the use of viruses in nanotechnology and materials science. The conference promotes[unreadable] the involvement of young female and minority scientists in these research areas. Among the invited 45 speakers and session chairs there will be 12 women. 9 additional presentations will be solicited from young scientists who submit abstracts. The major themes will be 1) structures of virions and viral proteins, 2) packaging of the viral genome, 3) viral entry, 4) viral fusion and uncoating, 5) trafficking of viruses in the cell, 6) viral protein folding and chaperone function, 7) dynamics of viral assembly, 8) virus-host interactions and 9) viruses in biotechnology and materials science.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The assembly of HIV-1 is a multi-step process in which several individual structural proteins undergo a substantial morphological rearrangement. The capsid (CA) protein plays a crucial but poorly understood role in this assembly. CA contains two-independently folded domains, the N-terminal (NTD) and C-terminal (CTD) domains, connected by a flexible linker. The CTD of HIV-1 has been shown to dimerize in solution both in the mature capsid protein and in the isolated domain (Gamble et al. 1997). The current structural model of the mature virion indicates the fundamental building block of the mature core is attributed to a CA hexamer formed by the self-association of the NTD tied together by dimerization of the CTD (Gamble et al. 1997). Additionally, CTD dimerization has been shown to be required for this hexamer formation (Lanman et al. 2003). The CTD of HIV contains the major homology region (MHR), a sequence of 20 amino acids that is highly conserved across different genera of retroviruses (Wills and Craven 1991). It is well documented that viral assembly is highly sensitive to MHR mutations but from a structural perspective it is not clear why MHR mutations are so deleterious (Cairns and Craven 2001). Recent studies have shown that a structural homology exists between the CTD of CA and the dimeric zinc finger associated domain SCAN (Ivanov et al. 2005). However, the SCAN domain dimer is domain swapped in comparison to the CTD of CA. In a domain swapped dimer, a structural element from one subunit is exchanged with the corresponding structural element from another subunit (Liu and Eisenberg 2002). The domain swapped region of the SCAN dimer corresponds to the MHR of the CTD. A domain swapped dimer would provide an explanation for the importance of the MHR because hydrogen bonding in this region would occur across the dimer interface. To determine whether CA can assemble when the C-terminal domain is domain swapped a fusion construct was engineered where the C-terminal domain of HIV-1 CA was replaced with the SCAN domain. These interactions will be studied using H/D exchange and fast photochemical oxidation.

Intellectual Merit
The project focuses on deciphering the sequence of protein/protein interactions which occur during the assembly of viral capsids using the bacteriophage phi29 as a model system. Viral capsids are protective protein shells that self assemble from hundreds of chemically identical protein molecules. In the final capsid these molecules are precisely positioned in space with an overall spherical form. The protein shell surrounds and protects the viral nucleic acid and in one class of capsid the shell assembles first and the nucleic acid is subsequently pumped into the shell through a conduit known as a portal. Despite the ubiquity of this architectural theme, little is known about the pathway or sequence of protein/protein interaction through which the proteins self-assemble. Recent experimental data suggests that assembly nucleates from a complex composed of multiple copies of two proteins, a scaffolding protein and the conduit forming portal protein. The project will use chemical cross-linking/mass spectrometry, hydrogen/deuterium exchange studies and mutational analysis in concert with computational docking to determine the detailed structure of this nucleation complex, and use this information to chemically stabilize the nucleation complex. The stabilized nucleation complexes will then be used to seed assembly reactions both in bulk solution and in single molecule experiments to probe the sequence and kinetics of subunit addition to growing capsids and derive a molecular level understanding of the assembly pathway.

Broader Impact
A detailed quantitative description of the molecular pathway of viral capsid assembly is required by the cadre of physicist and mathematicians who are developing models of self-assembly using viruses as a paradigm and a description at this level of detail is not currently available for any virus. The research project itself employs a wide variety of biophysical and biochemical tools and serves as an ideal training platform for the graduate and undergraduate students who will be carrying out the experiments. Finally, because detailed molecular pathways are best appreciated and understood when illustrated by animation the assembly pathway defined by this project will be animated for a lay audience by students in the Department of Art and the Art History Department exposing them to frontline science while allowing them to refine their animation skills and develop a portfolio.