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Tracking Vaccinia Virus Recombination using Live Cell Imaging Open Access


Other title
Vaccinia Virus
DNA Recombination
Live Cell Imaging
Viral Replication
Gene Expression
Type of item
Degree grantor
University of Alberta
Author or creator
Paszkowski, Patrick
Supervisor and department
Evans, David (Medical Microbiology and Immunology)
Examining committee member and department
Marchant, David (Medical Microbiology and Immunology)
Lewis, John (Oncology)
Hendzel, Michael (Oncology)
Department of Medical Microbiology and Immunology
Date accepted
Graduation date
2016-06:Fall 2016
Master of Science
Degree level
Recombination between co-infecting poxviruses provides an important mechanism for generating genetic diversity in the face of selection pressures. However, poxviruses replicate in membrane-bound enclosed cytoplasmic structures known as factories or virosomes that could impede DNA mixing between co-infecting viruses; and mixing would seem to be an essential early step in recombination. It is hypothesized that virosome fusion events would be a prerequisite for recombination between co-infecting poxviruses. Moreover, the need to do so could delay or limit viral recombination. By engineering vaccinia virus (VACV) to express overlapping portions of a mCherry fluorescent protein fused to a cro DNA-binding element, this permits for live tracking of virus DNA and genetic recombination via spinning disc confocal microscopy in cells also expressing an EGFP-cro fusion protein. My studies show that different types of poxvirus recombination events exhibit distinct timing patterns, depending upon the relative locations of the recombining elements. Recombination between partly duplicated sequences is detected soon after post-replicative genes are expressed, as long as the reporter gene sequences are located in cis within an infecting genome. The same kinetics are also observed when the recombining elements are divided between VACV and transfected DNA. In contrast, recombination is significantly delayed when the recombining sequences are located on different co-infecting viruses, and mature recombinants are not detected until well after late gene expression is established. The delay is consistent with the hypothesis that virus factories create an impediment to inter-viral recombination, but this research suggest that even after factories merge there remain further constraints limiting virus DNA mixing and recombinant gene assembly. This delay could be related to the continued presence of ER-derived membranes within the fused virosomes, membranes that may once have wrapped individual factories.
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