The goal of this proposal is to understand the structure and assembly of the vaccinia virion core. The core contains the viral DNA and at least 50 viral proteins. Core substructures can be distinguished by electron microscopy, including lateral bodies, a core wall, and a tubular structure in the lumen of the core, however very little is known about the precise protein composition of each substructure, the function of the individual proteins within the core, or the role of individual core proteins in core assembly during virus morphogenesis. The project will focus on a subset of nine core proteins that break down into four categories: 1) a cysteine protease required for cleavage of the major structural virion proteins (I7L), 2) four putative core wall proteins (A3, A4, A10, F17), 3) two putative tube proteins (H4, L4), and two putative core lumenal proteins (E6 and E8). In one aim the assembly of the pox virion core will be investigated by performing infections with wild type virus or virus bearing mutations in specific core structural proteins, and analyzing the infections by electron microscopy and immunofluorescence confocal microscopy. Infections will be monitored not only for the specific structures formed but also for the distribution within virus factories of each of the proteins and the viral DNA. These studies will reveal how individual components of the core are trafficked during morphogenesis, and how each component contributes to the evolution of the final core structure. In a second aim the structure of the pox virion core will be investigated by performing controlled degradation of wild type virions or virions bearing mutations in specific core structural proteins, and analyzing the virions and their degradation products by electron microscopy, gel electrophoresis and western blotting. Virion substructures will be probed with a panel of antibodies specific for proteins hypothetically contained in each of the major virion substructures, including lateral bodies, core wall, tube and lumen. Analysis of wild type virions in this fashion will reveal the localization of specific proteins within specific core substructures. Analysis of the mutant virions will reveal defects in specific core substructures, thus reinforcing assignments made from studies of wild type virions, and providing information on the function of each protein within the core structure.

Public Health Relevance

This project is relevant to public health on three levels. First, it advances our understanding of the basic mechanisms of protein trafficking and assembly of macromolecular structures, which in turn is a central part of the normal framework on which our understanding of disease is built. Second, it advances our understanding of the molecular mechanisms of virus replication and virus cell interactions, which has implications in general for treatment of virus induced disease. Third, the project provides insight specifically into the replication of poxviruses, which are of particular interest to public health as research tools, as a source of therapeutic proteins, as oncolytic vectors, and as potential bioterrorist weapons.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Study Section
Virology - A Study Section (VIRA)
Program Officer
Challberg, Mark D
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University of Florida
Schools of Medicine
United States
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Jesus, Desyree Murta; Moussatche, Nissin; Condit, Richard C (2016) An improved high pressure freezing and freeze substitution method to preserve the labile vaccinia virus nucleocapsid. J Struct Biol 195:41-8
Moussatche, Nissin; Condit, Richard C (2015) Fine structure of the vaccinia virion determined by controlled degradation and immunolocalization. Virology 475:204-18
Jesus, Desyree Murta; Moussatche, Nissin; McFadden, Baron B D et al. (2015) Vaccinia virus protein A3 is required for the production of normal immature virions and for the encapsidation of the nucleocapsid protein L4. Virology 481:1-12
Condit, Richard C; Moussatche, Nissin (2015) The vaccinia virus E6 protein influences virion protein localization during virus assembly. Virology 482:147-56
Jesus, Desyree Murta; Moussatche, Nissin; Condit, Richard C (2014) Vaccinia virus mutations in the L4R gene encoding a virion structural protein produce abnormal mature particles lacking a nucleocapsid. J Virol 88:14017-29
Afonso, Priscila P; Silva, PatrĂ­cia M; Schnellrath, Laila C et al. (2012) Biological characterization and next-generation genome sequencing of the unclassified Cotia virus SPAn232 (Poxviridae). J Virol 86:5039-54
McFadden, Baron D H; Moussatche, Nissin; Kelley, Karen et al. (2012) Vaccinia virions deficient in transcription enzymes lack a nucleocapsid. Virology 434:50-8
Boyd, Olga; Turner, Peter C; Moyer, Richard W et al. (2010) The E6 protein from vaccinia virus is required for the formation of immature virions. Virology 399:201-11
Boyd, Olga; Strahl, Audra L; Rodeffer, Carson et al. (2010) Temperature-sensitive mutant in the vaccinia virus E6 protein produce virions that are transcriptionally inactive. Virology 399:221-30
Moussatche, Nissin; Damaso, Clarissa R; McFadden, Grant (2008) When good vaccines go wild: Feral Orthopoxvirus in developing countries and beyond. J Infect Dev Ctries 2:156-73

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