Structure, dynamics, and mechanism of genome packaging motors in dsDNA viruses

Project Summary/Abstract The mechanism of DNA packaging for double-stranded DNA viruses will be studied in the Bacillus subtilis bacteriophage φ29, the most efficient in vitro viral packaging system known. Using an integrated genetic, biochemical, single-molecule and structural approach, we will characterize protein conformational change and movement in the transiently assembled packaging motor during DNA encapsidation. The mechanism of packaging in φ29 will serve as a model for animal virus packaging in the analogous herpesvirus and adenovirus systems, and aid in the search for new antiviral therapies. Due to similarities between the φ29 ATPase and other ring translocases, insights gained from the study of φ29 packaging will also provide insight into the basic principles of macromolecular motor function in higher organisms. As part of our long-term and multicomponent efforts to interrogate the mechanism of DNA packaging, here we will: 1 – Characterize the structure, dynamics, and energy landscape of DNA packaging motors. In Aim 1.1, we will use standard cryoEM SPA and nested highly focused reconstructions to test structural predictions of the helical to planar model. In Aim 1.2: we will utilize advanced image processing approaches to characterize the dynamics and energy landscape of packaging motors, and 2 – Complete single- molecule studies of ring motor mechanism. In Aim 2.1 we will use single molecule optical laser tweezers to dissect how key residues participate in critical functions during DNA translocation. Further, we will utilize combined single molecule imaging and force measurements to characterize DNA rotation during DNA packaging (Aim 2.2) and to correlate changes in motor conformation with DNA translocation (Aim 2.3).