|Zika virus neuropathogenesis|

My scientific career began in the summer of 1991 in a small, poorly ventilated playroom in the 1930s starter home where I spent the first decade of my life. I was 5-years-old and had just stuttered my way through the children’s book The Value of Believing in Yourself, which told the story of Dr. Louis Pasteur’s struggle to develop the first rabies vaccine. I was enthralled with the idea that one person sheltered away in a lab could produce something that would save a countless number of lives. I incessantly repeated the phrase from the first page of the book: I believe I can, I believe I can.

By the time I entered graduate school, my research interest had shifted to the brain and in particular the genetic origins of pervasive developmental disorders. Viruses still seemed interesting, but as tools for circuit tracing and gene introduction. At the time, I paid little attention to the overlap between infectious disease and human brain development. 

This changed in early January 2016. I was working several hours a day in my graduate lab at MIT trying to wrap up a paper, while simultaneously building new projects in my postdoctoral lab at Harvard. I started receiving text messages from my mom asking about this new pathogen called the Zika virus that was causing babies to be born with small heads, a condition known as microcephaly. A vast majority of our family still lives in El Salvador, and she was concerned that this mosquito-borne illness was going to add to the burden of her motherland. I wasn’t paying much attention to the news—I had few answers for her.

The paper was finally accepted, so I moved full time to Harvard. Two days into this new stage of my career, I flew to San Francisco to meet my boss at a small conference. When I arrived, he was nowhere to be found. He showed up a few minutes into the first talk, looking pale and exhausted—some problem with a connecting flight or something. In his sleep-deprived state, he asked me if I had any interest in using our human neural cultures to study Zika. Before he could change his mind, I said yes and started ordering as many strains of the virus that were available. I had no background in virology and I barely knew the protocols for generating the cultured human cells that I would be using for all of my experiments.

Working alongside my colleague and future groomsman Dr. Max Salick, a postdoc at Novartis at the time, we set out to determine exactly how this virus was gaining entry into developing brain cells known as neural progenitors. It was the earliest months of Zika neurobiology, but the hypothesis that the transmembrane protein AXL was the primary entry route for this virus was gaining popularity. To formally test this theory, we removed AXL from the genomes of human stem cell lines, differentiated them into neural progenitors, and then blasted them with Zika. If this protein was indeed the front door for this virus, then the knockouts should have some protection from infection. Sadly, such was not the case and the Nobel committee had to choose someone else that year.

Instead of repeating the process ad nauseum until we found something that worked, we decided to perform a genome-wide CRISPR-Cas9 Zika survival screen in a new model of neural progenitor cells that I had created known as Stem cell-derived NGN2-accelerated Progenitors or SNaPs. This screen detected hundreds of genes that upon removal conferred some level of protection from viral death. We also found a couple dozen that made SNaPs more likely to die when they were removed, suggesting that these genes play a role in cell immunity.  

Two of the hits from our screen were ITGAV and ITGB5, which are important transmembrane integrin proteins that form a protein complex. Interestingly, ITGB5 has been identified as an entry factor for other viruses in non-brain cell types. It seemed like a promising target for validation experiments, so we created ITGB5 knockout SNaPs and infected with Zika. The results were staggering—infection decreased 80%. We then ran an experiment where we pre-treated the SNaPs with antibodies that blocked the ITGAV/B5 protein complex. Again, we saw protection from infection. This was it. This was the answer. Zika virus enters human neural progenitor cells through ITGAV/B5. My mother was so proud.

I had just sent the finished manuscript to my boss and was boarding a plane to New Orleans when I received a text from Max. Two Zika papers published today showing ITGB5 as entry factor for brain cells. We had been scooped. Two other labs were working on the same thing and came to the same conclusion. Their work was undeniably phenomenal. 

Unfortunately, we were not the first to report this exciting discovery. I felt like Pierre LeBlanc, a 19th century French scientist I just made up who narrowly lost out to Louis Pasteur in the race for a rabies vaccine. Oh well, so it goes. 

All was not lost, however. I thought about my favorite Pasteur quote: Let me tell you the secret that has led me to my goal. My strength lies solely in my tenacity. Stumbling around Bourbon Street that night, I came up with the idea of using the CRISPR screen results to help explain the differential infectivity levels we saw across cell lines. It ended up being a big step forward in that project, and has launched a series of non-Zika experiments I am working on now. 

I guess the moral of this story is best personified by another ageless Pasteur line: Sometimes you have to drink “hurricanes” and eat half a pound of fried shrimp at 4 am with your friends to zap your brain into spitting out good ideas.