Wednesday, January 22, 2025
1/22/2025
This proposal aims to fill a critical knowledge gap in the assembly of icosahedral viruses; mechanisms and controls by which a viral genome flows into and out of a virus capsid with high precision and fidelity. The tailed dsDNA bacteriophage T4 is our model virus. The proposal will also translate this basic knowledge into a gene therapy for muscular dystrophy, a debilitating degenerative muscular disease that causes early death. We propose an innovative experimental design by advancing a novel “molecular valve” hypothesis. The hypothesis states that a sophisticated molecular valve at the unique portal vertex controls genome flow into and out of a virus capsid through dynamic structural and conformational changes. By integrating genetics, biochemistry, and cryo-electron microscopy, we will generate a series of asymmetric reconstructions of nanomachines in different structural and conformational states. These machines translocate genome into virus capsid creating a pressurized condensate (inward genome flow), arrest genome flow and position for delivery, and allow genome ejection upon encountering new host (outward genome flow). In specific aim 1, we will generate atomic level structures of the DNA packaging machine consisting of the portal vertex-bound packaging motor and its intermediate states during active translocation. A detailed packaging mechanism will be formulated that will have broad implications to phages, eukaryotic and archaeal viruses including herpes and adeno viruses. A strong work-flow has been established for preparation and data collection of packaging complexes using a newly constructed super-charged “acidic capsid” mutant. In specific aim 2, we will elucidate the dynamic mechanism of the neck-connector valve complex bound to portal by generating structures in different assembly states. These structures would illustrate conformational changes in the valve complex that lead to arrest of genome flow post-packaging and position it for delivery following tail docking. We present a novel discovery involving the participation of a host nucleic acid binding protein Hfq in these dynamic transactions. In specific aim 3, we will probe the mechanism of genome ejection by asymmetric cryo-EM reconstructions of the genome ejection machine pre-infection, during-infection, and post-infection. A preliminary cryo-EM reconstruction revealed, for the first time, density for a helical tape measure protein-DNA complex in the innermost core of the ejection tube. In specific aim 4, we will incorporate the basic knowledge gained from specific aims 1-3 to establish a novel, large capacity, T4 gene therapy vehicle to deliver the full- length ~11-kb dystrophin gene into human muscle cells as well as into a muscular dystrophy mouse model. Relying on our 42-years of expertise on T4 phage assembly and genome packaging, our synergistic research team will uncover the basic mechanisms of genome flow in viruses and their translation into potentially transformative phage-based gene therapeutics. These will have broad implications to virology and human disease therapies.
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