Identification of new molecular targets for blocking viral infections, including polio virus, herpes simplex virus, and cytomegalovirus; small molecule inhibitors of polio virus; technology innovation for coupling fluorescent microscopy and electron microscopy of samples embedded in vitreous ice.

Dr. Hogle has been studying picornaviruses, such as the well-studied poliovirus, for more than two decades, and has created an impressive array of technologies that have significant commercial appeal. The picornavirus family, which includes rhinovirus and hepatitis A, is linked to a variety of human illnesses. Dr. Hogle has developed cell-free screening methods for small molecule compounds that specifically bind to the outer capsid proteins of picornaviruses, resulting in the isolation of specific viral binders. High-resolution structural studies on viral intermediates that are generated during infection, coupled with proteomic analysis of in vitro reconstituted liposomes containing viral proteins, may identify new host molecular targets that are required for viral entry and viral replication. Consequently, these approaches are laying the foundation for future screening efforts to identify new antiviral agents.

Dr. Hogle’s molecular and structural studies on viral processivity factors and viral proteins that enable viral nuclear egress are also poised to uncover key vulnerabilities in the viral machinery that are susceptible to therapeutic intervention.

In parallel with these commercially relevant biochemical studies outlined above, Dr. Hogle has had a longstanding interest in  technology development, and his current focus on developing a cryo-optical microscope to facilitate coupling fluorescent microscopy and electron microscopy of samples embedded in vitreous ice has a clear practical benefit.
 

Dr. Hogle is extending his work on poliovirus cellular entry, trying to understand the cellular pathways that mediate entry of the virus into the cell and translocation of the virus genome across a membrane to gain access to the cytoplasm. These studies include electron cryomicroscopy and cryotomography to identify and structurally characterize soluble forms of viralcell entry intermediates, intermediates linked to membranes, using a liposome-based model, and intermediates inside infected cells. Isolation of virus-enriched liposomes using techniques such as density gradient centrifugation and fluorescence activated sorting will facilitate proteomic analysis of the vesicles, as well as structural experiments of intravesicular virus particles. These data should add new insights into the virus-host interactions that lead to productive infections.

As part of these efforts, Dr. Hogle is also building cryo-optical microscope for characterizing biological samples at cryo-temperatures. This new apparatus would wed fluorescent microscopy with electron cryomicroscopy, and avoid the use of contrast agents, fixation, and sectioning, allowing characterization of intracellular cell entry intermediates and other intracellular macromoleclar complexes in a fully hydrated state.
Dr. Hogle is also interested in molecular and structural aspects of viral processivity factors, including those for herpes simplex virus, cytomegalovirus, and the Kaposi’s sarcoma associated human herpes virus 8, which act as linkers within the viral replication complexes, helping to increase the local concentration of viral DNA polymerase in the proximity of DNA, and thereby increase the processivity and fidelity of DNA replication. One method used in this project involves the cross-linking of the processivity factor to DNA, since these proteins do not recognize specific nucleic acid motifs.

On a related subject, Dr. Hogle is exploring the nuclear egress of cytomegalovirus nuclear capsids, which involve the concerted action of multiple viral proteins, such as the cytomegalovirus-encoded UL50 and UL53. For the structural phase of this project, the lab is employing nuclear magnetic resonance (in collaboration with Gerhard Wagner) as well as x-ray crystallography.

Dr. Hogle is an expert on high-resolution structural characterization of viruses and viral proteins.  In 1985 Dr. Hogle’s group solved the structure of poliovirus using x-ray crystallographic methods, and since then has continued to use a combination of structural and biochemical approaches to characterize key events intermediates in the virus lifecycle. Poliovirus is a member of the picornavirus family and contains a single strand RNA as its genetic material. For many years after the introduction of the polio vaccine, important questions pertaining to poliovirus infection have persisted. One of the current major focuses of the laboratory is the characterization of the cell entry pathway of poliovirus as a model for understanding how nonenveloped viruses deliver their genomes into the cells. Dr. Hogle has published numerous articles on the intricate sequence of events related to poliovirus entry into host cells, employing techniques such as x-ray crystallograophy, single particle cryoelectron microscopy, and cryoelectron tomography to characterize the structures of key cell-entry intermediates as soluble particles in vitro, in complex with membranes using a liposome-based model developed in the Hogle Lab, and inside infected cells. Dr. Hogle’s studies have primarily addressed two key facets of poliovirus infection, the mechanism responsible for viral genome translocation into the cytoplasm, and the pathway that mediates targeting of the viral genome to specific subcellular compartments where viral replication is carried out.

In a recent study, the Hogle laboratory, working with Xiaowei Zhuang from the Department of Chemistry and Chemical Biology, used real time, live cell, single molecule fluorescence microscopy to visualize poliovirus infection. The strategy was to fluorescently label both the poliovirus capsid and RNA genome, and follow cell entry by real-time fluorescent microscopy. The experiments revealed that infection was an energy-requiring process. Moreover, following association with the cell surface, the virus gains entry into the cell by a endocytic mechanism distinct from classical clathrin- or caveolin-mediated endocytosis, requiring the activity of a tyrosine kinase as well as actin. Dr. Hogle’s novel imaging method demonstrated that the poliovirus genetic material is liberated intracellularly from vesicles that reside on near the cell surface.

Biological Mechanisms and Pathways

• Genome translocation •
• Membrane fusion •
• Nuclear egress •
• Processivity factor •
• Viral endocytosis •
• Viral replication •
• Viral structure •
• Virus infection
•  

Disease Mechanisms

• Cytomegalovirus •
• Herpes simplex virus •
• Poliovirus •
• Virus •
• Virus entry
•  

Infectious Disease

• Cytomegalovirus •
• Cytomegalovirus •
• Viral structure •
• Virus entry
•  

Research Tools and Instrumentation

• Cryoelectron microscopy •
• Cryoelectron tomography •
• Liposome •
• Technology development •
• Viral entry imaging •
• Virus-membrane complex reconstitution •
• X-ray crystallography
•  

Therapeutic Discovery Tools and Assays

• Technology development
•  

Therapeutics

• Antiviral small molecules
•