RNA viruses often induce massive morphological changes upon infecting their host cells, in particular by generating unconventional membrane-bound organelles that become viral replication factories. This project studies formation of this organelle and its relevance to viral replication in a model system of cellular infection by Coxsackievirus (a member of the picornavirus family that also includes polio virus). Earlier findings documented the sensitivity of picornavirus replication to the activity of the cellular small GTPase Arf, which is known to regulate organelle dynamics in the secretory pathway of eukaryotic cells. Hence, it is hypothesized that picornaviruses may selectively harness the nascent organelle building activities of the secretory pathway machinery (including Arf GTPases and their effectors) to build unconventional organelles that are optimal for viral replication. This study will rely on live-cell imaging methodologies to monitor the host-pathogen interaction. Fluorescence tagging of specific proteins will unravel the dynamics of replication organelle formation in infected cells. A new methodology, based on molecular beacons (i.e. nucleic acid probes that can detect viral RNA in living cells) will be developed to probe the dynamic interplay between viral RNA replication and the secretory pathway machinery. The combination of these two techniques is particularly well-suited to the study of the virus-cell interface because they can provide valuable spatio-temporal information without perturbing the dynamic events themselves, thereby allowing investigation of virus host events at the single-cell level. Ultimately, this project will glean information on the formation, organization and dynamics of replication organelles in individual cells. In particular, the roles of known components of the secretory pathway machinery in generating the viral RNA replication organelles will be investigated. A biological screen will be performed to identify as yet uncharacterized components of the secretory pathway whose expression regulates organelle formation and viral RNA replication. Determining the cellular components of these virus-specific organelles will shed light on how specific host activities contribute to the formation of an organelle that facilitates viral RNA replication. Moreover, characterizing the role of cellular components for the formation of unconventional organelles, such as those for viral replication, may provide important information about their respective roles in conventional organelle biogenesis taking place in eukaryotic cells. Finally the strategies developed in the project will provide a novel framework for others to explore the dynamics of host-pathogen interactions.
Broader Impacts Live-cell imaging is a critical component of current research in the life sciences, providing unparalleled insights. However, the majority of biology majors and M.S./Ph.D. candidates do not have the opportunity to see the power of this approach much less gain experience with it. This project will provide opportunity for students to learn and apply to a complex biological phenomenon such as virus-host dynamics a variety of state of the art live-cell imaging methodologies, image processing/analysis techniques and quantitative data analysis and modeling. In addition, a live-cell imaging course has been created to introduce undergraduate and graduate students to the power of imaging methodologies towards tackling a wide range of cell physiology problems. The interdisciplinary nature of the project and its focus on organelle biogenesis and viral RNA dynamics has generated an excellent opportunity to forge collaborations with mathematicians and chemists from neighboring institutions in Newark, New Jersey. This project will generate new perspectives on and novel methods of inquiry into the fundamental biological problem of host-pathogen interactions.
The viral repertoire on our planet is dominated by RNA viruses of which many are pathogenic to life forms and root cause of many human diseases such as Polio, Influenza, Hepatitis, AIDS and the common cold. Upon infecting a "host" cell ( human, plant, animal), many RNA viruses dramatically remodel the host cells intracellular membranes into specialized organelles which provide a membrane based platform on which viral genomes are replicated. The functional properties of the membranes that are required to support replication and whether different RNA viruses rely on the same specific organelle features for replication remain unknown. Often individuals can be infected with multiple different RNA viruses and hence development of a single panviral antiviral targeted against a common replication organelle feature is greatly beneficial since pan-vaccination against the viruses would be near impossible and the available vaccine repertoire is limited compared to the wide variety of pathogenic viruses. In my lab we have made a key contribution to the generation of panviral therapeutics by revealing a critical role for how specific host cell lipids within membrane platforms can facilitate the replication of a variety of different RNA viruses. Dissecting how host lipid -virus interactions drive viral replication has implied a combination of approaches taken from virology and cell biology. My laboratory has emphasized the synthesis of live-cell high–resolution-imaging techniques with spectroscopic (NMR), computational, molecular/biochemical and genetic approaches to understand the virus-host interface. Using this multi-lateral approach, from molecular to macro level, in vitro to in vivo level, we have revealed how different RNA viruses all selectively exploit host cell Phosphatidylinositol-4-kinase (PI4K) family of lipid modifying enzymes, to assemble membrane platforms specialized for replication that are highly enriched in host phosphatidylinositol-4–phosphate (PI4P) lipids. We have revealed that these PI4P lipids, are panviral regulators of replication, required by a wide variety of viruses. We have also provided some novel mechanistic insight into how these lipids maybe critical for these viruses by demonstrating that viral RNA polymerases, the critical RNA synthesizing viral enzymes, have unique binding sites for PI4P lipids that in turn regulate their activity and potentially targeting to the membrane platforms. Discovering PI4P lipids as regulators of viral RNA metabolsim has also opened up for us and others a new avenue of research into a wider role for lipids in regulating RNA metabolic reactions in eukaryotic and prokaryotic cells. Finally our studies have led to productive collaborations with pharmaceutical industries in the development of panviral therapeutics targeting host PI4 kinases, which if successful will enable us to combat multiple different viral infections at any given time.
