James M. Hogle
Our laboratory uses structural approaches to explore how viruses enter cells and replicate. Current research in the laboratory is focused in two areas: 1) a multiscaled approach to characterizing the cell entry pathway of poliovirus and other simple nonenveloped viruses, 2) structural studies of key proteins in the replication of herpes viruses (in collaboration with Don Coen) and poliovirus and it close relatives.
Membrane fusion provides a conceptually simple mechanism for enveloped viruses to deliver their genomes into the cytoplasm of target cells. In contrast, viruses which lack an outer membrane must provide a mechanism that allows a large nucleocapsid, or at the very least the viral genome, to cross a membrane in order to gain access to the interior of the cell. This process remains poorly understood. We are taking a combined structural approach to characterize the cell entry pathway of poliovirus as a simple model for studying nonenveloped virus entry. The combined approach uses cryoelectron microscopy to characterize soluble forms of cell entry intermediates at high resolution, and cryoelectron microscopy and cryoelectron tomography to characterize membrane-associated intermediates at intermediate resolutions, using a receptor-decorated liposome model developed in the lab. In recent studies (in collaboration with Xiaowei Zhuang) we have extended these studies, using live cell optical microscopy of cells infected with poliovirus labeled such that capsids and genomes have been tagged with distinct fluorescent dyes to follow virus particles in the early stage of infection and identify pathways leading to productive genome release. These studies show that poliovirus entry utilizes an unusual endocytic pathway that is dependent on energy, actin, and an as yet unidentified tyrosine kinase, but independent of clathrin, caveoli, and microtubules. These studies have also shown that genome release is surprisingly efficient and occurs from an internal compartment within 100-200nm of the cell surface. In the coming years we hope to combine fluorescence microscopy and electron tomography to further probe the entry pathway, and characterize the location and structure of the cell entry intermediates in the context of the intact cell. The combination of these approaches will allow us to address questions which span over six orders of magnitude in scale (from atoms to cells), and should be generally applicable to a wide range of intracellular machines.
Once viruses enter cells and release their genomes further steps in replication require complex interactions between viral gene products and host cell components for replication, assembly and release. Although many of these processes may eventually prove addressable by the combined approach, we are currently focusing on obtaining high-resolution structures of key players by x-ray crystallography. Recent structures include a structure of a complex of a portion of the capsid protein of alfalfa mosaic virus with a structured region of the 3’ end of its RNA genome (this interaction is required for the initiation of RNA replication in AMV), a structure of the 3CD protein of poliovirus (a multifunctional protein which is a protease and an RNA-binding protein with roles in priming and initiation of RNA replication) and the processivity factors of herpes simplex and cytomegalovirus.
D. Bubeck, D. J. Filman and J. M. Hogle (2005). Cryo-electron microscopy reconstruction of a poliovirus-receptor-membrane complex. Nat Struct Mol Biol 12: 615-8.
T. J. Tuthill, D. Bubeck, D. J. Rowlands and J. M. Hogle (2006). Characterization of early steps in the poliovirus infection process: receptor-decorated liposomes induce conversion of the virus to membrane-anchored entry-intermediate particles. J Virol 80: 172-80.
B. Brandenburg, L. Y. Lee, M. Lakadamyali, M. J. Rust, X. Zhuang and J. M. Hogle (2007). Imaging poliovirus entry in live cells. PLoS Biol 5: e183.
B. A. Appleton, J. Brooks, A. Loregian, D. J. Filman, D. M. Coen and J. M. Hogle (2006). Crystal structure of the cytomegalovirus DNA polymerase subunit UL44 in complex with the C terminus from the catalytic subunit. Differences in structure and function relative to unliganded UL44. J Biol Chem 281: 5224-32.
L. L. Marcotte, A. B. Wass, D. W. Gohara, H. B. Pathak, J. J. Arnold, D. J. Filman, C. E. Cameron and J. M. Hogle (2007). Crystal structure of poliovirus 3CD protein: virally encoded protease and precursor to the RNA-dependent RNA polymerase. J Virol 81: 3583-96.
Baltz, J. L., Filman, D. J., Ciustea, M., Silverman, J. E., Lautenschlager, C. L., Coen, D. M., Ricciardi, R. P., and Hogle, J. M. (2009) The crystal structure of PF-8, the DNA polymerase accessory subunit from Kaposi's sarcoma-associated herpesvirus, J Virol 83, 12215-12228.
Sam, M. D., Evans, B. T., Coen, D. M., and Hogle, J. M. (2009) Biochemical, biophysical, and mutational analyses of subunit interactions of the human cytomegalovirus nuclear egress complex, J Virol 83, 2996-3006.
Levy, H. C., Bostina, M., Filman, D. J., and Hogle, J. M. (2010) Catching a virus in the act of RNA-release: a novel poliovirus uncoating intermediate characterized by cryoelectron microscopy, J Virol 84, 4426-41.