The Orthopoxvirus genus of the family Poxviridae includes both the causative agent of smallpox, variola virus, and the vaccine used in its eradication, vaccinia virus. Recent events have renewed interest in Orthopoxviruses because of concerns that variola virus or one of its close relatives could be used as a biological weapon. Orthopoxviruses produce both enveloped and unenveloped virions and remarkably both types are infectious. While non-enveloped virions make up the majority of progeny virions the enveloped form (EV) is required for cell-to-cell spread, systemic infection and virulence. The major objective of work is to determine the molecular mechanism employed by envelope virus specific proteins to coordinate the intracellular envelopment of Orthopoxviruses. Our hypothesis is that the lumenal domain of three EV specific proteins, A33, A34 and B5, interact and that interaction is required for exit from the ER and subsequently proper sub-cellular targeting of B5, which is the main target of neutralizing antibodies, to the site of intracellular envelopment and subsequently into infectious enveloped virions.
The specific aims are: 1) Mapping residues of A33, A34 and B5 required for proper targeting and interaction. The studies proposed here will use an innovative live trans complementation microscopy assay and co-IP to map regions of interaction. 2) Characterization of the relationship between A33, A34 and B5 in EV production. Recombinant viruses will be created that express A3R, A3R and BR mutations defined in Aim1 to characterize the role the interaction has during viral morphogenesis. 3) Temporal and spatial characterization of A33, A34 and B5 interaction. The innovative fluorescent assays FRET and BiFC will be used to track the interaction in live cells. Mapping sites of interaction on these proteins will help define functional domains and provide for a better understanding of their structure and function. The B5R protein is required for the formation of enveloped poxviruses. We will investigate how this important protein is incorporated into and coordinates the formation of newly made viruses. A better understanding of this process will provide new targets for antivirals directed against poxviruses.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI067391-05
Application #
8012854
Study Section
Virology - A Study Section (VIRA)
Program Officer
Challberg, Mark D
Project Start
2007-02-15
Project End
2013-01-31
Budget Start
2011-02-01
Budget End
2013-01-31
Support Year
5
Fiscal Year
2011
Total Cost
$333,153
Indirect Cost
Name
University of Rochester
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
041294109
City
Rochester
State
NY
Country
United States
Zip Code
14627
Baker, Jonathon L; Ward, Brian M (2014) Development and comparison of a quantitative TaqMan-MGB real-time PCR assay to three other methods of quantifying vaccinia virions. J Virol Methods 196:126-32
Hollenbaugh, Joseph A; Gee, Peter; Baker, Jonathon et al. (2013) Host factor SAMHD1 restricts DNA viruses in non-dividing myeloid cells. PLoS Pathog 9:e1003481
Chan, Winnie M; Ward, Brian M (2012) Increased interaction between vaccinia virus proteins A33 and B5 is detrimental to infectious extracellular enveloped virion production. J Virol 86:8232-44
Chan, Winnie M; Ward, Brian M (2012) The A33-dependent incorporation of B5 into extracellular enveloped vaccinia virions is mediated through an interaction between their lumenal domains. J Virol 86:8210-20
Ward, Brian M (2011) The taking of the cytoskeleton one two three: how viruses utilize the cytoskeleton during egress. Virology 411:244-50
Chan, Winnie M; Ward, Brian M (2010) There is an A33-dependent mechanism for the incorporation of B5-GFP into vaccinia virus extracellular enveloped virions. Virology 402:83-93
Chan, Winnie M; Kalkanoglu, Aja E; Ward, Brian M (2010) The inability of vaccinia virus A33R protein to form intermolecular disulfide-bonded homodimers does not affect the production of infectious extracellular virus. Virology 408:109-18
Ward, Brian M (2009) Using fluorescent proteins to study poxvirus morphogenesis. Methods Mol Biol 515:1-11
Johnston, Sara C; Ward, Brian M (2009) Vaccinia virus protein F12 associates with intracellular enveloped virions through an interaction with A36. J Virol 83:1708-17
Earley, Amalia K; Chan, Winnie M; Ward, Brian M (2008) The vaccinia virus B5 protein requires A34 for efficient intracellular trafficking from the endoplasmic reticulum to the site of wrapping and incorporation into progeny virions. J Virol 82:2161-9