The goals of this proposal are to complete the physical mapping and phenotypic analysis of a combined set of 132 temperature sensitive vaccinia virus mutants from two different mutant collections, Dales and Condit, and to study in detail a defined subset of these mutants in order to refine our understanding of virion morphogenesis.
In Aim 1, mutants from each of the unmapped or partially mapped complementation groups in the combined Dales-Condit collection of vaccinia virus temperature sensitive mutants will be mapped to a single gene using marker rescue.
In Aim 2, heretofore uncharacterized representatives from each complementation group in the combined Dales-Condit collection will be subjected to a first level phenotypic analysis, consisting of assays for viral DNA replication, viral protein synthesis, and virion morphogenesis.
In Aim 3, representatives from selected complementation groups in the combined Dales- Condit collection will be subjected to detailed analysis for defects in virion morphogenesis. Characterization will include assays for virion temperature sensitivity, post-translational cleavage of viron proteins, DNA packaging, synthesis, virion localization and intracellular localization of the protein product of the affected gene, detailed electron microscopic analysis of cells infected under non-permissive conditions, and biochemical analysis of accumulated aberrant intermediate particles. The successful completion of this project will enhance our understanding of the functional organization of the poxvirus genome, it will provide for the scientific community an extensive permanent collection of vaccinia virus conditional lethal mutants for future use in poxvirus functional genomics, and it will elucidate a genetic and biochemical pathway for virus morphogenesis. Historically, studies of virus morphogenesis have impacted significantly on our understanding of self assembly systems, membrane biology, intracellular protein trafficking, and virus cell interactions. We fully expect that our studies will have a similar impact. Importantly, the value of this type of research in basic poxvirology to public health has been significantly increased recently given the potential for use of smallpox as a bioterrorist weapon. ? ?
|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, Patrícia 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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