Friday, July 26, 2024
7/26/2024
Project Summary/Abstract Flaviviruses (a family of over 90 known viruses, including Zika, dengue and West Nile) are significant human pathogens affecting 3.1 billion people annually, and most of these enveloped viruses do not have any viable vaccines or antivirals capable of combating their spread. The primary objective of current flavivirus antiviral design is to disrupt specific mechanisms in the flavivirus life cycle that are key for virus survival, including virus attachment to the host cell, viral endocytosis, genome uncoating, genome replication, and virus maturation through the Golgi. These potentially druggable mechanisms have been explored in great detail both within our lab and elsewhere. However, little is known about the assembly mechanisms that drive infectious virus particle formation. Because of the high structural homology among all flaviviruses, the identification of assembly mechanisms will provide ubiquitous targets for therapeutic intervention in virus proliferation. In other enveloped virus systems, assembly depends on the interaction of virus core proteins with lipid membranes or membrane bound glycoproteins to produce infectious virus particles. Exploration via single particle Cryo-EM reconstruction has shown that the nucleocapsid core (NC) of immature Zika virus, is found in close proximity with the envelope glycoproteins on the inner side of the virus’s lipid bilayer. Due to this close proximity, I hypothesized that the NC interacts with the envelope glycoproteins during virus assembly. I further hypothesized and that these interactions occur between the capsid protein (CP) and transmembrane helices of the precursor membrane (prM) protein and the envelope (E) protein while the particle is in the immature state. Since no information currently exists on virus assembly relying on CP-prM/E interactions, these interactions have the potential to be exploited as new drug targets capable of inhibiting the proliferation of flaviviruses. To this end, I am investigating two independent strategies to validate the hypothesized NC-prM/E interactions using dengue virus serotype 2 (DENV2) as a model system. The Kuhn lab is particularly adept in mutagenesis studies using DENV2, which is why the decision was made to use this virus as our model system instead of Zika. Firstly, amino acids within the prM/E transmembrane helices that were posited to be key in the assembly process of DENV2 and other flaviviruses have been or will be mutated to investigate their role in promoting particle assembly. Secondly, the ability of the DENV2 prM and E transmembrane helices to interact with CP is being examined through the use of reconstituted prM and E proteins within SMA lipid nanoparticles. Due to the high similarity of all flaviviruses, the techniques, results and mechanisms identified in this study can be applied to other flaviviruses. Combining these essential experimental studies with the proposed training skills and collaborative opportunities for effective scientific communication through writing, speaking and mentoring will prepare me well for a future in biomedical research.
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