“The SPARK Awards are a boost of energy, and really motivate me to give the best of myself to continue my fight against infectious diseases.”
Combating emerging Ebola virus threats with affordable cures
FUNDED: JANUARY 2020
FUNDED BY: the generosity of Rachel & Bob Perlmutter, Raydene &
Peter St. Clair, and 2019-20 Various Donors
What was the goal of your SPARK project?
Ebola virus relies on a viral protein called the polymerase protein to infect cells. This protein makes copies of the viral genetic material needed to form new virus particles that go on to infect other cells or other people. Polymerase is also a potential target for antiviral drugs. To understand precisely where these drugs should attack the polymerase, scientists need to be able to see the protein structure at the atomic level. However, there are no atomic structures of polymerases, largely because these proteins are difficult to produce, purify, and image with techniques like cryo-electron microscopy (cryo-EM) or crystallography. I proposed purifying Ebola polymerase protein for use in structure work, and succeeding meant overcoming several major technical hurdles.
Pivoting during a pandemic
The urgency generated by the COVID-19 pandemic in spring 2020 meant my lab had to quickly prioritize SARS-CoV-2 projects and I was asked by my Principal Investigator, Dr. Saphire, to support that critically sensitive work. However, thanks to the extensions granted to my SPARK cohort as a result of the pandemic, I was still able to work on my SPARK project and apply the tools I generated for my project with success.
SPARK project results:
I designed a platform that allows the rapid production and screening of variants of Ebola virus polymerase, that are engineered, to be more stable and produce more protein. I modified a battery of Ebola polymerases from different key pathogenic species of Ebola virus (Zaire and Sudan), the related Marburg virus, the original strain from bats (Mayinga), as well as the non-pathogenic Reston virus with a tag that allows more specific purification of the protein. Interestingly, I found that the expression levels of the various ebolavirus polymerases differed significantly among virus strains. Sudan and Mayinga ebola viruses expressed the highest amount of polymerase complex in human cells. I also stabilized the polymerase complex by co-producing it in cells with another viral protein called VP35. I rapidly upscaled the purification of the most promising engineered candidates using liters of insect cells. It was important to use insect cells for this step because they offer a high level of protein expression.
Preliminary purifications of the polmerase-VP35 complex had low protein yield, but by modifying our purification method, we increased the amount of protein we could get by 50%. From among the purification strategies, I selected those that showed both better yield and purity of the polymerase-VP35 complex. For the first time I can visualize the polymerase-VP35 complex clearly in a SDS-PAGE gel, but there are other lower abundant proteins in the mixture. We hypothesize that these proteins are contributing to the stability of the complex. In order to locate the polymerase-VP35 complex in the Electron Microscope grid we are labeling the complex with gold.
What’s next for this project?
I am now testing different sample preparation techniques to find conditions that will allow me to image this protein complex using cryo-EM, a high-resolution imaging technique that can shed light on atomic-level details of viral proteins.
I plan to further optimize the purification process and the stability of the Ebola virus polymerase-VP35 complex to pursue a high-resolution structure of the Ebola polymerase-VP35 complex. The interaction between the polymerase and VP35 is essential to fix the polymerase in one of its multiple functional states, but during this project, we learned that it is transient. Capturing this shifting interaction will affect the structural homogeneity required for getting high-resolution models by cryo-EM. We plan to increase the stability of the interaction through the binding with viral RNA and through a new artificial interface generated by self-complementing methods. We are also mapping the polymerase domains that serve as interaction partners for VP35. With this knowledge, we can engineer a construct that binds covalently both proteins and stabilizes their interaction. We are ready to start imaging the Ebola polymerase-VP35 complex using LJI’s Titan Halo Electron Microscope. Once we get a low-resolution model of the complex, we will start the imaging of the Ebola polymerase-VP35 complex using LJI’s Titan Krios Cryo-Electron Microscope. If conformational heterogeneity remains a problem, we will use computational methods to finely sort particles into class averages representing a single conformation. After processing the images, we aim to generate a threedimensional model that will be further refined and validated.
What’s next for Sara?
I see myself eventually leading a team focused on medicines to cure diseases caused by highly pathogenic human viruses. The global pandemic reminded me why I am committed to virology and also the relevance of our research to society. Among the practical experiences SPARK provided in project management and execution, it also honed my communication skills and comfort in being more visible inside and outside the scientific community. I’m grateful to have developed these essential skills for my career, and to have experienced first-hand how private philanthropic support can be a critical bridge between having an idea and executing it.