SINGLE-MOLECULE IMAGING OF THE REPLICATION OF VIRAL PATHOGENS

In the most general terms, we aim to understand the function of biological macromolecules in terms of conformational dynamics. In particular, we are interested in the molecules that support the life cycles of human viral pathogens. We take a highly inter-disciplinary approach, which centers on single-molecule fluorescence microscopy and spectroscopy, and incorporates kinetic and thermodynamic analysis of single-molecule data, virology and molecular biology, and computational modeling. Students and postdocs of all backgrounds are welcome to contact James to discuss potentially joining the lab.

MACROMOLECULAR DYNAMICS THAT UNDERLY ENVELOPED VIRUS ENTRY

All enveloped viruses enter target cells by fusing the viral membrane with a cell membrane, which releases the viral genetic information into the cell. This process is catalyzed by envelope glycoproteins found on the virion surface. Although the details of the entry pathways and the structures of the glycoproteins among different viruses can vary significantly, viruses have evolved common mechanisms of surmounting the energetic barrier to fusing two membranes. The energy needed to cross this barrier on a physiologically relevant timescale is contained entirely within the metastable conformation of the glycoprotein. This energy is released through transition from the metastable pre-fusion conformation to a highly stable post-fusion conformation, which occurs through multiple intermediate steps. We aim to characterize this dynamic process in molecular detail, with a focus on common mechanistic strategies utilized by phylogenetically distinct viruses.

IMMUNE EVASION BY CONFORMATIONAL MASKING

Prior to membrane fusion, viral envelope glycoproteins interact with receptors or other moieties embedded in cellular membranes. These early interactions are crucial to forward progress in the entry pathway. In addition, being uniquely exposed on the virion surface, envelope glycoproteins are primary targets of antibodies. Therefore, envelope glycoproteins must maintain their responsiveness to cellular signals in order to promote entry, while at the same time concealing functional centers, such as receptor-binding sites, from neutralization by antibodies. Different viruses have evolved some distinct strategies for maintaining this balance. We are especially interested in a strategy involving the masking of functional centers through conformational changes in the glycoprotein, which is central to the immune-evasion tactics of HIV, among others. We are also interested in how neutralizing antibodies, and potentially therapeutic agents, can overcome this protective ability.

GENOME DYNAMICS DURING RETROVIRAL ASSEMBLY

Proteins are often regarded as the central machines that support the replication of viruses. While this is true in many cases, the RNA genome of retroviruses also plays a crucial role during virion assembly and viral gene expression. The genomic 5' LTR is a key regulatory element that determines whether the genome dimerizes and is recruited into assembling virions, or is used by the cellular translation machinery to express the viral genes. We are interested in using single-molecule fluorescence methods to understand how the conformational dynamics of the genome determine its fate during retroviral replication. That is, how does the genome select between packaging by the retroviral Gag protein, or translation by the ribosome? And conversely, what influence, if any, do these factors have on the conformation of the genome?