Molecular mechanisms of cell entry, evolution and innate immune recognition of enveloped RNA viruses
Our overarching goal is to gain a mechanistic understanding at the molecular level of how important pathogens interact with their host cells during infection. We seek to understand the following major questions: (1) How do enveloped viruses assemble and recognize host cells? (2) How do enveloped viruses deliver their genome into the cytoplasm? (3) What are the evolutionary origins of the genes that drive virus entry? (4) How are innate immune responses to microbial nucleic acids generated, amplified and regulated? To answer these questions, we are employing a diverse set of complementary biophysical, biochemical and cell biological approaches. By integrating approaches across scales (in space and time), we envision moving towards a structure-based understanding of biological processes at the cellular level. Each of our projects has important potential applications in global health.
1. Mechanisms of virus entry into cells
Recent work in the laboratory has focused on the roles of envelope glycoproteins in the assembly and cell-entry mechanisms of viruses from the flavivirus, bunyavirus and pestivirus families. These viruses use their envelope protein to bind a receptor and enter cells. Viral entry occurs when the reduced pH of an endosome triggers a conformational rearrangement in the envelope protein, which induces fusion of the viral and host-cell membranes. Building on our structural studies of dengue and West Nile viruses, we aim to discover the full complement of structral classes of viral envelope proteins. We also aim to apply our expertise in structural biology to trace the evolution of the different classes of env viruses back to their molecular origins in the host. This will provide insights on cellular fusion processes. Our third major goal is to obtain a molecular-level understanding of cell entry by dengue virus and related viruses. To achieve this, we are tracking single virus particles in real time as they fuse to synthetic target membrane, and as they infect live cells. We have developed and optimized sophisticated fluo labeling strategies, a TIRF microscopy in vitro fusion assay, and a live-cell confocal microscopy fusion assay.
2. Molecular mechanism of microbial nucleic acid sensing by the innate immune system
Most organisms rely on an innate immune system as their first line of defense against infection. Within the innate immune system, the Toll-like receptors (TLRs), a family of evolutionarily ancient receptors found on the surface of many cell types, are critical for pathogen recognition outside the cell. About 12 TLRs recognize structures specific to pathogens, such as bacterial cell wall components, bacterial filament proteins, or certain types of nucleic acid. This recognition event initiates a signal inside the cell, which induces the rapid secretion of antimicrobial and inflammatory proteins. Remarkably, given the structural diversity of the structures that they recognize, all TLRs rely on a “leucine-rich repeat” (LRR) domain to recognize pathogen-associated structures.
The overall goal of our research program on innate immune sensors is to understand how they recognize conserved molecular patterns in pathogens, and how this recognition is translated into an innate immune response. Our structural approach will provide unique insights into these important processes. Our structures will likely define novel principles of molecular recognition. By revealing the conformational changes associated with ligand binding, the structures will provide insight on how pathogen recognition is translated into a signal in the cell that elicits an immune response. Our work will also guide efforts to design synthetic agonists or antagonists with immunomodulatory properties. Such compounds would have a wide range of medical applications, particularly as vaccine adjuvants or anti-inflammatory therapeutics.
3. Technical Approach
We employ a diverse set of complementary biophysical approaches including X-ray crystallography, electron microscopy, solution biophysics, fluorescence microscopy and cell biological approaches to understand the mechanisms that underlie virus cell-entry, assembly and innate immune sensing in molecular-level detail.