Viral Genome Delivery

The goal of our research is to understand how double-stranded DNA viruses deliver their large genomes (~40-250kb) into living cells. Most of the work carried out in my laboratory is based on a simple model system, the Salmonella-phage P22. Using a combination of structural e.g. X-ray crystallography, cryo-electron microscopy (cryo-EM), small angle X-ray scattering (SAXS), biochemical and functional techniques, we investigate the structure, composition, and assembly of P22 genome ejection machinery. In collaboration with Dr. Sherwood Casjens (University of Utah), we have isolated and reconstituted the five polypeptide chains forming P22 tail machinery and determined their assembly and structural composition in vitro. In collaboration we Dr. Jack Johnson (at the Scripps Research Institute) and Dr. Timothy Baker (UCSD), we determined a 7.8A asymmetric cryo-EM reconstruction of P22 mature virion (Fig. 1A) (Tang et al., Structure, 2011). This wonderful structure served as a molecular framework to identify and fit individual components previously solved in my laboratory, namely the dodecameric portal protein (Fig. 1B), gp4 (Fig. 1C) (Olia et al., Nature Struc Mol Biol., 2011) and the tail needle gp26 (Fig. 1D) (Olia et al., Nature Struc Mol Biol., 2007). Overall, the synergy of top-down (e.g. cryo-EM) and bottom-up approaches (e.g. high-resolution crystal structures of individual components) provides an invaluable tool to study the mechanisms of viral genome ejection at the most fundamental molecular level.


The Genome Ejection Machinery of Bacteriophage P22

Figure 1. Bacteriophage P22 DNA injecting machine. (A) 7.8 Å asymmetric cryo-EM reconstruction of phage P22. The virus genome (in green) is surrounded by a shell of coat protein (shaded in blue), which is interrupted at a unique 5-fold vertex by a 2.8 MDa tail apparatus. The portal protein (gp1) (B), tail factor gp4 (C), and tail needle gp26 (D) have been identified and modeled in the cryo-EM density.

A second project in the lab focuses on viral genome packaging, which is the mirror process of genome ejection. The packaging of viral genomes into empty procapsids is powered by a large DNA-packaging motor. In most viruses, this machine is composed of a large and a small terminase subunit (referred to as TerL and TerS, respectively) complexed with a dodecamer of portal protein. We recently described the 1.75 Å crystal structure of the bacteriophage P22 TerS in a nonameric conformation (Fig. 2) (Roy et al., Structure, 2012). The structure presents a central channel ~23 Å in diameter, sufficiently large to accommodate hydrated B-DNA. The last 23 residues of TerS are essential for binding to DNA and assembly to TerL. Upon binding to its own DNA, TerS functions as a specific activator of TerL ATPase activity. The DNA-dependent stimulation of ATPase activity rationalizes the exclusive specificity of genome-packaging motors for viral DNA in the crowd of host DNA, ensuring fidelity of packaging and avoiding wasteful ATP hydrolysis. This posits a model for DNA-dependent activation of genome-packaging motors of general interest in virology.

Small Terminase subunit

Figure 2. Quaternary structure of the nonameric S-terminase subunit of phage P22. Ribbon diagram of TerS in side (A) and top (B) views.

We also characterized the structure and regulation of P22 TerL subunit (gp2) (Roy and Cingolani, J. Biol Chem, 2012). This protein has a bipartite organization, consisting of an N-terminal ATPase core flexibly connected to a C-terminal nuclease domain. The 2.02 Å crystal structure of P22 headful nuclease obtained by in drop proteolysis of full-length TerL reveals a central seven-stranded b-sheet core that harbors two magnesium ions (Fig. 3). Modeling studies with DNA suggest the two ions are poised for two-metal ion-dependent catalysis, but the nuclease DNA-binding surface is sterically hindered by a loop-helix motif, which is incompatible with catalysis. Accordingly, the isolated nuclease is completely inactive in vitro, while it exhibits endonucleolytic activity in the context of the full length (FL) TerL. Deleting the auto-inhibitory L1-a2 motif (or just the loop L1) restores nuclease activity to a level comparable to FL-TerL. Together, these results suggest that the activity of P22 headful nuclease is regulated by an intramolecular cross-talk with the N-terminal ATPase domain. This cross-talk allows for precise and controlled cleavage of DNA that is essential for genome-packaging.P22-Large-Terminase-nuclease domain

Figure 3. The architecture of TerL nuclease active site. Left panel: ribbon diagram of P22 nuclease domain highlighting only active site residues and metal ions. In the right panel is a magnified view of the active site, which includes essential residues involved in catalysis, two magnesium ions (MgA and MgB) colored in purple and several water molecules (small red spheres). The Fo-Fc density map (in cyan) overlaid to the Mg sites is contoured at 7sigmas above background.