Friday, May 23, 2025
5/23/2025
Investigating the conformational changes of the portal protein that drive DNA packaging in a dsDNA virus - PROJECT SUMMARY Assembly of dsDNA viruses, including the tailed bacteriophages, herpesviruses, and adenoviruses, is a highly coordinated process involving a series of protein interactions that lead to the formation of an infectious virion. The central goal of this research is to achieve a detailed mechanistic understanding of the specific protein:protein interactions that govern this assembly process. During assembly, proteins interact temporally, driven by their conformational plasticity. The dodecameric portal protein complex, which is essential for tailed dsDNA virus assembly, exemplifies this. In bacteriophage P22, a model for dsDNA viruses, scaffolding protein drives the oligomerization of portal protein monomers into rings which are subsequently incorporated into procapsids (PCs). Terminase proteins then preferentially bind the PC conformation of portal, and package DNA until the head is full. At this point, portal undergoes a conformational switch to its mature virion (MV) form that triggers the release of the terminase proteins and the binding of proteins that stopper the portal channel resulting in the complete MV. Portal takes on another distinct conformation from its PC and MV forms after the virion injects its DNA into host cells. Despite the portal complex being critical for successful virion assembly, owing to it being the conduit for DNA translocation, its roles during DNA packaging and signaling for the completion of packaging have not been mechanistically defined. The central hypothesis is that conformational changes of the portal complex are required to regulate the processes involved in successful viral maturation. I will test this hypothesis using bacteriophage P22 which provides an excellent dsDNA model assembly system. Overall, its simple genetics and well-established biochemistry offer advantages over more complex dsDNA viruses. The experiments proposed in Aim 1 will elucidate the signal for DNA packaging completion by interrogating portal protein variants’ ability to make infectious phages with the appropriate morphology that package the correct amount of DNA. In Aim 2, I will obtain the cryo-EM structure of the portal complex bound to terminase, which has not been solved at high-resolution. Collectively, these data will represent a significant advancement of our understanding of the dsDNA viral assembly pathway by shedding light on how conformational changes of portal drive DNA packaging. Overall, elucidation of the details involved in P22 assembly can lead to the identification of potential anti-viral drug target sites to inhibit assembly of dsDNA human pathogens.
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