The infections virion is composed of a glycoprotein-containing envelope, a tegument layer, and an icosahedral capsid enclosing its double-stranded DNA (dsDNA). We have isolated the HCMV virus and its intermediate particles at different stages of assembly and maturation from HCMV infected cells. These include the intranuclear A-, B-, and C-capsids, the tegument containing cytoplasmic capsids, and the infectious and non-infectious virus particles.The long-term objective of this project is to understand the structural basis of HCMV capsid maturation and pathogenesis at high resolution and provide useful structural information for developing therapeutic strategies against HCMV infections.

Central to pathogenesis and lytic KSHV infection is the viral replication, including the well-coordinated assembly process leading to subviral particles (pro-, A-, B-, C-capsids) and infectious virions. Using electron cryomicroscopy (cryoEM) techniques well established during his previous structural studies of other herpesviruses, the applicant has published the first three-dimensional (3D) structure of KSHV B-capsids purified from BCBL-1 cells. This structure, while revealing a capsid shell almost identical to those of other herpesviruses, nevertheless exhibited striking differences at the outermost region of the capsid. This observation suggests that KSHV and other herpesviruses share a similar mechanism of capsid assembly, but virion proteins situated at the outer radial regions, such as pORF65 and the tegument proteins, may have KSHV-specific assembly/functional roles. Our studies are directed at testing this hypothesis by determining and understanding the molecular interactions essential to KSHV capsid and tegument assembly. The goals are (1) to determine the structural basis of KSHV assembly and molecular interactions by determining a high-resolution (~7 \u0169 structure of the KSHV B-capsid using cryoEM reconstruction techniques well established by the applicant?s group; (2) to locate pORF65 and other capsid proteins and visualize their antibody epitopes; (3) to elucidate the mechanisms whereby capsid proteins interact with both tegument proteins and viral DNA by determining the structure of the intact KSHV virion and comparing it with other herpesviruses; (4) to determine the structural basis for KSHV viral assembly and morphogenesis by structural comparisons of KSHV B-capsid and protease-minus capsids (procapsids), which are generated by ribozyme-mediated, KSHV protease-specific inhibition. The results of these experiments will lead to a better understanding of the process of KSHV lytic replication and the identification of potential targets for therapeutic intervention.

The pyruvate dehydrogenase complex (PDC), one of the largest and most complex multienzyme systems, catalyzes the conversion of pyruvate to acetyl CoA for energy generation in the carbohydrate metabolism pathway. Human PDC (hPDC), located in the mitochondria of muscle cells, plays a central role in regulating the myocardial ATP production in the heart. Our research aims at understanding how the major components of hPDC perform their functions by determining the structural basis of their interactions.

Determination of the three-dimensional (3D) organization of PDC is key to understanding its highly regulated multi-functional roles and molecular mechanisms of fuel selection in heart muscle. While much progress has been made in the 3D structures of microbes and yeast PDCs through x-ray crystallography, NMR spectroscopy, and electron cryomicroscopy (cryoEM), 3D structural studies of the hPDC complexes have been hampered both by the sample unavailability and by the increased complexity of additional regulatory components. However, it has now become feasible to study hPDC by cryoEM, owing to the recent development of a recombinant expressing and in vitro assembly system for hPDC in Dr. Roche?s group and new methods developed in Our group both in preserving PDC complexes for cryoEM imaging and in computationally classifying their images. Taking advantages of these new opportunities, our goals are (1) to determine the location of the linker region to the E1 binding domain of human E2 by 3D structural comparison of cores formed by full length and truncated E2; (2) to determine the structural organization of the four major hPDC components in the intact hPDC complex, including E2 core bound with E1 (E2/E1), E2 core with E3BP and E3 (E2/E3BP/E3) and the entire complex E1/E2/E3BP/E3; (3) to visualize the binding of pyruvate kinase (PDK) by difference electron imaging of E1/E2 with or without PDK, or PDK complexed with chemical labels; and (4) to use the 3D structures obtained above as a framework into which the atomic structures of PDC components from other species are fit to deduce an atomic model for the hPDC complex. Our long-term goal is to determine how the multi-components of hPDC and their cofactors interact with each other to understand their dynamic, multi-functional roles in myocardial energy production.

CPV is a single-shelled insect virus 800Å in diameter and is the structurally simplest member of the Reoviridae, a diverse family including the human rotavirus, which causes severe gastroenteritis. The structural simplicity and stability make CPV an ideal model system for developing new reconstruction method and for studying reovirus replication at the highest possible resolution. First, we will advance the reconstruction software so that the 3D structures of large virus particles can be determined to 4-Šresolution. A distributed database and access tools will be designed to handle the increased data complexity for high-resolution studies. A novel refinement procedure and parallel 3D merging program will be developed to improve accuracy and speed. Subsequently, these techniques will be applied to study the protein-RNA interactions by structural comparisons of full, empty, and active transcribing CPV at 4-Šresolution. As an independent test of our new procedures, we will compare the structure with the blue-tongue virus (BTV) sub-core structure from x-ray crystallography. This comparison will also unambiguously resolve the protein-RNA interactions as yet unresolved by x-ray due to the lack of an empty BTV structure.

Viruses in the 11 genera of this family all contain a characteristic segmented dsRNA genome and highly conserved transcriptional enzyme complexes (TEC). Up to 12 TECs can be enclosed in a protein shell made up of 120 molecules of the inner capsid shell protein (CSP). These viruses are capable of endogenous messenger RNA (mRNA) transcription and 5’ capping within an intact virus particle, using virally-encoded enzymes. The detailed molecular mechanisms and structural basis of many steps of the dynamic process of mRNA transcription, processing, and release are yet to be determined. Cytoplasmic polyhedrosis virus (CPV) is an ideal system for studying the structural basis of viral RNA transcription because of its stability, ease of purification and its simplistic single-shell composition as compared to other dsRNA viruses. The goal of the research activities proposed in this renewal application is to determine the high-resolution structures of quiescent and transcribing CPV particles that are associated with several key steps of the RNA transcription process. A unique strength of this application is that we are applying the high-resolution electron cryomicroscopy (cryoEM) and related methods, which have been well established through the support of our current Welch Foundation funding, to address a fundamentally important biochemical question. These methods include biochemistry, high-resolution cryoEM reconstruction, and integrative, cryoEM-constrained, sequence-based comparative modeling. We will determine the structures of the quiescent CPV and three key intermediates of transcribing CPV at ~4-Å and 5-7 Å resolutions, respectively. Structural comparisons of these particles will reveal the various structural changes associated with different transcriptional stages: activation, initiation, and mRNA release, as well as how these structural changes are coordinated sequentially to fulfill the transcription process. The successful outcome of this project will provide a wealth of much-needed structural information leading to a better understanding of the dynamic process of viral RNA transcription by describing how chemical interactions among the multiple subunits of large macromolecular machines facilitate the execution of such process.