Virus Infection in Motion

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Nov 12, 2018

The paper in Nature Microbiology is here:

Seeing is believing

This blog is dedicated to anyone who has spent countless hours in the dark staring down an eyepiece waiting for something…anything…miraculous to happen. This has only happened twice in my scientific career. The first time, I was fortunate to catch the entry of a single vaccinia virus (VACV) particle which triggered systemic membrane blebbing. Following this observation, we discovered that VACV uses a novel mechanism of virus internalization termed apoptotic mimicry.  

The second time, while going through a set of live-cell movies of infected cell behavior, I found a gem (Figure 1). I watched as an infected cell detached from the coverslip, and to my amazement crawled towards and under another cell before pausing and crawling away. Needless to say, the target cell was none-to-pleased about this. I was instantly fascinated! I must have watched this movie 50 times. Was this a mechanism used by the virus to mediate spread? I wanted to know how this worked, what viral proteins were driving this and most of all how were they turning innocent cells into virus-spreading zombies! 

Figure 1. Movie of infected cells targeting and crawling under another cell. The intimate contact made suggested that motility-based cell-cell contact could be a mechanism of virus spread.

Vaccinia-induced motility: Heads versus tails 

In 1998 Geoffrey Smith and Michael Way showed that VACV infection could induce cell motility and that early VACV gene expression was necessary and sufficient to trigger this. The Way lab subsequently identified F11 as a virus protein that modulates RhoA activity to facilitate tail retraction. Viruses lacking F11 are defective for motility, virus spread and are attenuated in mice. As F11 was responsible for motile cell tail retraction, I reasoned that there must be at least one additional VACV gene that activates RTKs or Rho GTPases to trigger motility and direct the leading edge. 

Needle in a haystack 

I listed the properties that a VACV-encoded activator of cell motility must have: 1- The protein must be an early gene product, 2- It must be essential for virus spread, 3- Its deletion should not affect virus production, 4- Its loss should attenuate VACV lethality and 5- It should have the ability to activate RTKs or GTPases. To my surprise, of the >200 proteins encoded by VACV, only 1 met all of these criteria, the VACV growth factor (VGF).

VGF is a virus-encoded EGF/TGFahomolog that is expressed early and secreted from host cells. Its deletion results in a small plaque phenotype, no reduction in virus production and a virus which is attenuated even in an intercranial injection model. Most important, VGF has been shown to bind and activate the epidermal growth factor receptor (EGFR), a known activator of cell migration. 

The perfect candidates

The VGF was the perfect candidate, yet without live cell imaging no link between VGF and virus induced cell motility had ever been established or investigated. I obtained a VGF-deletion virus, applied live cell imaging to the problem, and was convinced that I had found the right protein.  

After a few false starts, the second perfect candidate joined the project in 2012. A master’s student, Corina Beerli, with initial guidance by a PhD student, Samuel Kilcher, ran with this project. After a brief hiatus, Corina joined my lab as a PhD student in 2015 and made it her mission to figure out how VGF was triggering cell motility. She built an array of fluorescently labeled deletion viruses compatible with live cell tracking and in vivoimaging and applied a battery of assays to work out VGF shedding, signaling and its role in virus spread. 

 You must crawl straight before you can walk

The acquisition of live cell time-lapse datasets of virus spread was critical to this study. While in the absence of VGF we could see motility defects at the single cell level,we wanted to determine how these impart a global defect in virus spread. Enter our very own virologist/computational biologist Artur Yakimovich. Employing Trackmate, single infected cells were tracked overtime for speed and directionality from the origin of infection (Figure 2). Analysis of these large datasets showed that the inability of VGF-, F11-, and VGF/F11-deleted viruses to spread infection and form virus plaques was directly related to defects in single cell velocity and directionality.

Figure 2. The movie displays the trajectories of vaccinia virus-infected cells as they crawl away from the epicentre of infection. The white dots that appear are the infected cells and the different color tracks correspond to the time in which the cells were infected *Note that infection spreads in waves*

In vivo veritas

As a true benchmark of its importance we wanted to determine the impact of VGF-mediated cell motility in vivo. In collaboration with Heather Hickmanat the NIH NIAID we compared lesion formation in wild-type and VGF-deleted viruses using 2-photon intravital imaging. Virus lacking VGF was highly attenuated in its ability to form a lesion, the classic hallmark of poxvirus infection. As viruses deleted for VGF had been reported to have attenuated lethality in mice, our data suggested that this is due to their inability to activate cell motility and efficiently spread the infection. 

The sum of all parts 

We found that vaccinia virus, a member of the poxvirus family and a relative of smallpox virus, encodes a protein that activates motility in infected cells. It does this by hijacking the cells normal motility activation pathways. We show that these infected cells act as mobile virus carriers which help spread the virus to surrounding cells through cell-cell contact. This in turn, enables the virus to spread infection further and faster than it would by simply releasing virus particles and letting them land where they may.

What’s really interesting is the pathway the virus takes over is the same one turned on during cancer metastasis, a process that intimately relies on cell motility. So now we can use VACV to study these pathways in a very targeted fashion, and on the flip side we might be able to repurpose drugs that target cancer metastasis to block the spread of infection. 

  • Jason Mercer is a principal investigator & MRC group leader at the University College London, United Kingdom
Go to the profile of Jason Mercer

Jason Mercer

Associate Professor of Virus Cell Biology, MRC LMCB UCL

Virus Cell Biology

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