I thought writing about working on LY6E for the last time would be a lot easier. This is, after all, a protein that I've somehow managed to study for about ten years. Fun fact- LY6E was my rotation project in the nascent Schoggins lab. LY6E, Part I and a tenth of LY6E, Part II made up my PhD dissertation. Four years into my postdoctoral career, and just 14 months removed from the Schoggins lab and Dallas, I have mixed feelings (mostly good!) about sending LY6E, Part III off into the world. Everything I know how to do as a scientist now is all thanks to years working on this tiny, three-finger protein, but it was only while writing up this most recent paper when I realized how LY6E has also shaped the questions I want to answer in the future.
Like many contemporary, 'hot' interferon stimulated genes (ISGs), LY6E was identified as an interesting gene to study through a high-throughput screen of hundreds of different genes. In the particular case of LY6E, it was a gain-of-function screen in a STAT1-deficient human fibroblast cell line with yellow fever virus (YFV). In supplementary figure 1 of LY6E, Part I, I show that I was lucky that the initial screen was performed in the cell line that it was, because overexpression of LY6E doesn't enhance YFV infection in certain other common cell lines used in virology. Oddly enough, LY6E overexpression in the same cell lines without a YFV phenotype inhibits coronavirus infection (unpublished, unless you want to scroll through my dissertation). Now, this isn't the problem that I set out to solve, but it did get me thinking about what the right cellular environment for any ISG is.
Fast forward to LY6E, Part II, figure 4, where I mashed up mouse spleens and tried to determine what immune cells are protected from murine coronavirus (MHV) infection by LY6E. While macrophages, neutrophils and dendritic cells could be infected ex vivo, B cells were surprisingly the only cell type significantly protected from coronavirus infection by LY6E expression. This result is what made John and I very confident that knocking LY6E out in CD19-expressing cells would result in mice that were as sensitive to MHV infection as mice lacking LY6E in the entire hematopoietic compartment (or Vav1-expressing cells).
I still vividly remember the look on John's face in the lab meeting where I showed him that LY6EΔCD19 mice survived MHV infection just as well as their non-knockout littermates. It seemed like such a good hypothesis... but biology just happened to be a little more complex than we thought. I won't delve into the MHV data from LY6E, Part III too much more here, other than to highlight the 'negative' result that loss of LY6E in T cells also didn't make mice more susceptible to MHV. This was interesting to me, as a LY6E historian, because in the mid-90's (back when LY6E was called SCA-2 or TSA-1), LY6E was shown to modulate T cell receptor signaling so I expected loss of LY6E in T cells to.. do something. Nope.
The LY6E project (but not humanity) was lucky when SARS-CoV-2 mutated enough to bind to the murine homolog of the SARS-CoV-2 receptor, ACE2. Despite this 'evolution,' wildtype C57BL/6J mice are still rather resistant to the gamma variant of concern (VOC), as are IFNAR-/- and IFNLR-/- mice. We've known for a while that LY6E is constitutively expressed in many cell types without exposure to type I interferons, so it was quite exciting to see that LY6EΔ/Δ mice were more sensitive to infection with the gamma VOC than their LY6E-wildtype littermates.
Whole lung RNA-sequencing unexpectedly gave us a hint as to where LY6E was protecting against SARS-CoV-2. I sent off RNA samples expecting just to see an exaggerated inflammatory response in lungs of LY6EΔ/Δ mice, but instead we saw similar inflammatory signatures to LY6E-wildtype littermates and a decrease in expression of ciliated and secretory cell-associated genes. Hm!
Unfortunately, by the time we realized that LY6E in certain epithelial cell subsets was maybe important through the RNA-Seq data, I was already settled into my new postdoctoral position in New York. I have endless amounts of gratitude and appreciation for Dr. Alex Wells, the supremely talented postdoc who did the RNA-FISH experiments to show that ciliated and club cells become susceptible to SARS-CoV-2 in the absence of LY6E. I also must absolutely shout out Dr. Marley Van Dyke, who took over managing my mouse colony and did all of the remaining animal experiments like a total pro after I moved away. Last, but far from least, I have to thank John for everything: letting me work on LY6E, trusting me to bring animal work to the lab, and giving me the opportunity to push my career forward by taking over an unfinished project and wrapping it up with a perfect bow.
Perhaps the reason why writing this was so hard is because goodbyes are hard, but I hope that LY6E, Part I through Part III, will be a useful map for someone in the future.