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Baculovirus FP25K localization and transposition during insect cell infection

Garretson, Tyler A
http://rave.ohiolink.edu/etdc/view?acc_num=miami1476185925822047

Abstract Details

2016, Doctor of Philosophy, Miami University, Microbiology.
Baculoviruses are insect-specific, large DNA viruses known for their promise and versatility as gene expression platforms, biopesticides, and gene therapy vectors. They follow a uniquely biphasic life cycle that produces a budded virus (BV) and occlusion-derived virus (ODV). This switch from BV to ODV is partially carried out by a viral gene product, FP25K. However, the fp25k gene is unstable in cell culture infections, and mutations within this locus negatively impact gene expression and biopesticide production platforms. Despite its importance, FP25K protein function and the mechanism of gene instability are not completely understood. In Chapter 1, we examined FP25K localization and the domains involved in order to obtain FP25K functional information. This was accomplished by using an Autographa californica multiple nucleopolyhedrovirus (AcMNPV) bacmid virus that produces a functional AcMNPV FP25K-GFP fusion protein during insect cell infection to determine localization and functionality of this virus and its mutants through confocal microscopy and real-time PCR-based budding assays, respectively. A protein structure prediction search shows strong similarity between FP25K and a nucleic acid chaperone LINE-1 ORF1p. AcMNPV FP25K primarily localized to cytoplasmic amorphous structures, although a small amount was nuclear. Because LINE ORF1p also localizes with amorphous structures identified as stress granules, these FP25K cytoplasmic amorphous structures may represent stress granules. We also found that a conserved putative N-terminal coiled-coil domain present in alphabaculovirus but absent in betabaculovirus FP25Ks was critical for its cytoplasmic retention. In Chapter 2, we investigated mechanisms of fp25k gene instability. Previously, we reported a specific fp25k transposition event during Sf21 cell infection. To characterize this event and determine its sequence requirements, a combination of viral genome mapping and fp25k mutational analysis was employed. However during passage of fp25k mutants, homologous recombination occurred between viral constructs. Based on the previously published evidence that high molecular weight DNA concatemers are present during baculovirus replication, this may also indicate that replication is partially recombination-dependent. Overall, these data provide evidence towards better understanding FP25K protein function and gene stability through localization studies and characterization of transposition in Sf21 cells, respectively. Moreover, the protein structure prediction search and localization data support a mechanism of FP25K as a regulator of biphasic switch possibly through association with stress granules.
Xiao-Wen Cheng (Advisor)
100 p.
Cellular Biology; Virology
Baculovirus, FP25K, Localization, Transposition

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Citations

  • Garretson, T. A. (2016). Baculovirus FP25K localization and transposition during insect cell infection [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1476185925822047

    APA Style (7th edition)

  • Garretson, Tyler. Baculovirus FP25K localization and transposition during insect cell infection. 2016. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1476185925822047.

    MLA Style (8th edition)

  • Garretson, Tyler. "Baculovirus FP25K localization and transposition during insect cell infection." Doctoral dissertation, Miami University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=miami1476185925822047

    Chicago Manual of Style (17th edition)

miami1476185925822047
331
© 2016, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.