Mechanism of Viral Genome Delivery into Cells

Project Summary Nearly 120 years since the discovery of the first virus, our understanding of how viruses deliver genomes into cells overcoming the complexity of biological membranes remains limited. While a vast scientific literature exists on viral surface proteins and their interaction with host receptors, and the immune system, little emphasis has been devoted to studying the delivery of entire viral genomes into cells. For instance, how do bacteriophages eject DNA through the cell envelope of Gram-negative bacteria? Or, in humans, how do herpesviruses deliver ~200 kb genome through the Nuclear Pore Complex (NPC) into the cell nucleus? For a quarter of a century, first as a trainee (1995-2003), and since 2004 as a principal investigator, I have investigated the mechanisms of nucleocytoplasmic transport and viral genome packaging. My work has resulted in close to 85 publications that contributed to elucidating the atomic structure and regulation of crucial factors implicated in nuclear import, and viral genome packaging. In this R35, I propose to combine the study of these two seemingly distinct biological processes by focusing on the mechanisms of viral genome delivery into living cells. Specifically, I will ask two biological questions that seek to compare and contrast how simple bacterial viruses (or bacteriophages) eject their DNA into bacteria with how Herpesviruses deliver their complex genomes into the nucleus of eukaryotic cells. The first question explores how bacteriophages eject ~45 kb genomes through the cell envelope of gram-negative bacteria. Long-thought to be a simple pressure-driven injection, this process uses a virus-encoded nanomachine, which we have begun to study in my laboratory. The second question explores how Herpesviruses deliver their large genome through the Nuclear Pore Complex (NPC) of human cells into the cell nucleus. This is a signal- and energy-mediated process that uses host importins and the GTPase Ran, exploiting the cellular transport machinery to promote entry of an exogenous genome into the nucleus. Overall, understanding how viruses transfer genetic information through biological membranes into cells and organelles is vital for deciphering the molecular mechanisms of virulence as well as the development of novel therapeutic approaches. The common denominator of this R35 lies in our interest in the structure and transport mechanisms of biological macromolecules. Our research approach marries established sciences like protein biochemistry and X-ray crystallography with the power of cryo-electron microscopy (cryo-EM) to visualize biological macromolecules in near-native conditions. We believe that this R35 MIRA funding mechanism will fuel the creative and diligent pursuit of answers to the questions we pose, permitting our research program to achieve significant advancements in structural biology.