Open Collections

Andrews, Daniel Donald Thomas

University of British Columbia

Viruses must co-opt host cellular functions to facilitate virus infection in cells. To accomplish this, they have evolved diverse strategies to hijack the host cell for its own benefit. One such strategy is through the activity of virus-encoded proteases, which are not only essential for viral polyprotein processing, but also for the cleavage of host cell proteins to modulate cellular pathways such as translation and innate immunity. While enterovirus proteases such as 2A and 3C have been well-studied and target many host proteins, the flaviviral NS2B-NS3 protease is poorly characterized and only a few host protein targets have been identified to date. In this thesis, I hypothesized that the cleavage of currently unidentified host proteins promotes the modulation and hijacking of host cellular processes. The N-terminomics based approach terminal amine isotopic labeling of substrates (TAILS) was employed to identify protease-generated N-termini and host substrates cleaved during virus infection. Three primary aims were addressed: first, characterizing the cleavage of the adapter protein 14-3-3ε by the enteroviral 3Cᴾʳº; next, identifying substrates of the Zika virus (ZIKV) NS2B-NS3ᴾʳº in human A549 cells during infection; finally, to conduct a global analysis of protease-generated N-termini, including both viral and cellular protease substrates, in human and mosquito cell lines. Characterizing the cleavage of 14-3-3ε by 3Cᴾʳº resulted in two key observations: (i) cleavage of 14-3-3ε promotes virus infection and (ii) the newly formed N-terminal fragment represses RIG-I signalling. TAILS analysis of ZIKV-infected A549 cells also uncovered host proteins cleaved during virus infection, including the RNA binding protein HNRNPH3 which was found to be cleaved by NS2B-NS3ᴾʳº. Moreover, TAILS provided insights into the proteolytic activity of viral and cellular proteases during ZIKV infection in human (A549) and mosquito (C6/36) cell lines, shedding light on protease specificity and subcellular localization of these targets. In summary, TAILS identified novel targets of viral and cellular proteases during infection, enhancing our understanding of the biological pathways disrupted during picornavirus and flavivirus infection and offering potential therapeutic or antiviral targets against viral protease activity.

Text

2023-10-05

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/86074

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/