Xu, Amadeus Musheng; (2022) Using Vaccinia virus as a model system to understand microtubule-based transport. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Vaccinia virus is a great model system for studying the cell cytoskeleton as it efficiently hijacks both the actin and microtubule network during infection. Intracellular mature virions (IMV) are the first infectious form of the virus produced during replication and are released when the cell undergoes lysis. A subset of IMV undergo envelopment at the Golgi to become intracellular enveloped virions (IEV) which recruit kinesin-1 to undergo microtubule-based transport from their perinuclear site of assembly to the plasma membrane. Limited work indicates that IMV can also be transported on microtubules. However, we have little or no molecular understanding of how this is achieved. This understudied area of vaccinia virus cell biology is surprising given that IMV comprise the majority of infectious virions formed during infection. I have now used a combination of cell-based and in vitro approaches to gain insights into IMV motility. Unexpectedly, I found that IMV also recruit kinesin-1 to move on microtubules in infected cells, although at a much lower levels compared to IEV. Using CRISPR/Cas9 genome edited cells expressing endogenously GFP-tagged kinesin-1, I have determined the number of kinesin-1 motors on virions and find that IMV recruit on average ~50% fewer motors than IEV. Additionally, for the first time, I have shown that these motors remain stably attached to the virus for long periods without undergoing turnover. I have also reconstituted IMV motility on purified microtubules in vitro using extracts from infected cells. In this system, IMV virions are very processive, typically moving to the very ends of the microtubule. Using polarity-marked microtubules, I found that IMV are exclusively plus-end directed, moving at an average velocity of ~0.65 μm/s. Furthermore, there is near complete loss of IMV motility in vitro in the absence of kinesin-1. Consistent with this, there is also a defect in intracellular IMV spread in cells lacking kinesin-1. My observations now demonstrate that kinesin-1 transports IMV as well as IEV during vaccinia infection. Moreover, reconstitution of vaccinia virus microtubule-based motility in vitro provides a useful new tool to investigate kinesin-1 driven transport of IMV and IEV in a system that is both biochemically accessible and tuneable to the user requirements.

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