Interview Series: Cristea Lab Graduate Students Explore the Dynamic Regulation of Innate Immune Sensors

By Elene Tsopurashvili (elenet@princeton.edu) and Katelyn C. Cook (katelync@princeton.edu)

September 7, 2020

 

Over evolutionary time, eukaryotic cells have developed a myriad of defense mechanisms against intracellular pathogens, allowing cells to grow and thrive in a world dominated by viruses and other parasitic microbes. These defenses include the ability to sense and respond to invading genomes, whereby protein sensors flag pathogenic DNA/RNA as foreign material and induce a series of anti-viral processes to shut down infection (if you are interested to know more, check out our innate immunity post). For example, nuclear replicating viruses (e.g., influenza viruses, herpesviruses, adenoviruses) are met with sensors in the host cell nucleus, an array of proteins that includes the interferon inducible protein 16 (IFI16) – a major research focus within the Cristea lab.

IFI16 was first described in the 1990s as a gene that increases in expression upon treatment with interferon gamma, a type of cytokine. This discovery marked IFI16 as a key player in the progression of human inflammatory diseases, motivating numerous studies that described its functions in cancer, DNA damage responses, and autoimmune disorders. It was not until 2010 that IFI16 was implicated in innate immune sensing, when Unterholzner et al. reported that IFI16 binds to herpes simplex virus 1 (HSV-1) genomes and induces cytokine production. This discovery was truly groundbreaking for the immunology and virology fields as it marked IFI16 as the first described nuclear sensor of foreign DNA, placing it at a fundamental axis in distinguishing the host cell genome from that of a pathogen. Scientists in our lab and others have worked to characterize the regulation and downstream functions of this sensor during infection. We have been particularly interested in the mechanisms underlying IFI16 recruitment to HSV-1 genomes, viral strategies that co-evolved to inhibit IFI16 functions, and the protein-protein interactions underlying IFI16-induced anti-viral responses.

Our researchers each bring their own expertise and creative flavor to these investigations. Timothy Howard, a fifth-year PhD student, strives to better understand the spatiotemporal dynamics of IFI16’s association with viral genomes near the nuclear periphery. This phenomenon occurs rapidly and with remarkab

timothy howard

le specificity, yet how IFI16 coordinates other host or viral factors to achieve immune signaling remains unknown. Tim’s primary goal is to characterize how the interactions between IFI16, viral genomes, and other downstream proteins change across time to control the IFI16-induced anti-viral response. To address this question, Tim bridges numerous technical fields by integrating mass spectrometry-based proteomics with genomics, microscopy, and molecular virology techniques.

This broad interdisciplinary engagement is something Tim finds to be crucial for his investigations. “The best way to carry out science is by using a variety of techniques because then you can approach each question from multiple angles,” Tim stated. Currently, he is incorporating leading-edge genomics tools for exploring his hypotheses about IFI16’s ability to suppress the HSV-1 genome. “I want to see how IFI16 affects the chromatin landscape of viral genomes. With genomics, I can obtain specific information on DNA that is bound to IFI16.”

To capture protein dynamics, Tim uses mass spectrometry to examine IFI16 protein interactions at the timing of virus entry and links this with molecular virology perturbations to assess their contributions to anti-viral response. “Investigating interactions is helpful,” Tim explained. “Since IFI16 does not have an enzymatic domain, it has to either function as a physical entity, blocking other chemical reactions, or coordinate those reactions by interacting with other proteins that have enzymatic activity.”

While combining these techniques makes for a powerful approach, it also presents significant challenges. For instance, linking genomics with proteomics allows Tim to obtain global profiles of IFI16 interactions across infection time, but it also necessitates the analysis, visualization, and interpretation of massive amounts of data. This can be quite difficult to navigate without significant computational expertise or resources. In addition, not all interactions can be functionally validated, and molecular virology investigations often result in negative findings, requiring the researcher to pursue numerous hypotheses. Despite disappointments that are a part of scientific research, Tim gets inspiration from this process: “There are moments when you have a success and make a discovery. It is not just that you are the first person to have thought of something, you are the first person to have observed it: a natural phenomenon that will be occurring regardless of human observation. The idea of being that person is very exciting.” Tim is actively analyzing his datasets and hopes to submit his findings for peer-review in the coming months.

Like Tim, another graduate student in the Cristea lab, Bokai Song, takes joy in making new discoveries about innate immune pathways. Bokai dedicates his time to investigating another viral DNA sensor, cGAS. Just before he joined the lab in 2015, cGAS was discovered as a potent anti-viral protein that – in contrast to IFI16 – detects foreign DNA in the cytoplasm, a location where DNA is not found unless a cell is infected or damaged. While scientists have devoted much effort towards identifying the mechanisms underlying downstream cGAS signaling, much remains unknown about how cGAS functions are regulated by the cell. Bokai is interested in how cGAS anti-viral responses are dictated by post translational modifications (PTMs), alterations in protein structure that can affect protein interactions, localizations, or enzymatic functions.

bokai song

In his recently published study in Molecular and Cellular Proteomics, Bokai used a targeted MS technique known as parallel reaction monitoring (PRM) to detect and quantify cGAS PTMs during infection. “PRM was very useful for characterization and especially quantification of PTMs since endogenous cGAS is in such low amounts in non-immune cells,” Bokai said, explaining that PRM is well-suited for measuring low-abundance proteins such as cGAS. He discovered multiple cGAS PTMs and showed that they change in abundance during HSV-1 infection. Moreover, after mutating these sites to observe their role in cGAS function, Bokai determined that many of them are required for cGAS-mediated anti-viral sensing and signaling. Despite roadblocks along the way, Bokai’s work altogether demonstrates that cGAS is regulated by PTMs, opening the door to multiple new avenues for understanding how cells control innate immune pathways.

Beyond viral infections, both Tim and Bokai anticipate that their work will lay a foundation for understanding autoimmune diseases. “Everything that medical establishments can do relies on a basic understanding of molecular biology,” Tim stated. Building off his own work and that from other groups, Tim highlighted the links between IFI16 and non-viral inflammatory responses, suggesting that therapeutic developments can build on these themes to achieve similar goals. Similarly, Bokai considers that exploring the way cGAS and other viral sensors are regulated will reveal mechanisms that trigger their over-activation and malfunction in immune disorders. He hopes that in the next two decades, the innate immunity field will have a solid grasp of autoimmune diseases and be better equipped to combat them. Additionally, as both IFI16 and cGAS have been implicated in cancer, Tim and Bokai predict that further discoveries of innate immune sensors will directly impact oncology investigations. Both lab mates expressed a passion for the broad impact of their own findings on studies of human health and disease, and have worked to make their methodologies accessible to other researchers by co-authoring a Methods in Enzymology review of techniques well-suited for investigations of anti-viral sensors.

When Tim and Bokai are not pondering innate immunity at the lab bench, they occupy themselves with different activities. Tim likes to travel, lift weights, connect with other scientists over happy hours, and occasionally see a cheap movie with his wife Katelyn. He is also the current co-chair of the Princeton MolBio community outreach and science communication program. Bokai is a guitar aficionado, something that has become a favorite aspect of Cristea lab parties and a great way to boost morale during long days in the lab. He also applies his scientific skills within the kitchen. “Cooking is like doing experiments and a protocol is like a recipe. There are some steps that are really important for experiments and there are some that are not. With cooking, it’s the same,” Bokai said with a grin.

Stay updated on Tim and Bokai’s research contributions, academic accomplishments, and beyond-the-bench adventures by visiting the tabs on our lab website! We also encourage you to shoot them a quick email if you’re curious to know more.