Virus structure and cell entry

Virus particles are carriers of genetic material from one cell to another. The key structural distinction is between viruses that have lipid-bilayer membranes and those that lack them --"enveloped" and "non-enveloped", respectively.  These differences reflect different mechanisms of cell entry and different pathways of assembly and maturation.  Enveloped viruses enter by membrane fusion, either from an internal compartment following an endocytic step, or at the cell surface.  Non-enveloped viruses require some form of membrane "perforation". For these viruses to penetrate a cell, a large macromolecular complex (either a subviral particle or just the viral genome) must cross a cellular membrane to access the cytosol, and some mode of local membrane disruption must accompany the translocation.

Rotavirus structure and entry

A principal objective of our research on virus structure is a molecular description of the earliest events leading to infection of a cell: attachment, uptake, and penetration into the cytosol.  Rotavirus, a major cause of infectious infant diarrhea, is a particularly suitable non-enveloped virus for deriving such a "molecular movie".   We have used electron cryomicroscopy (with Nikolaus Grigorieff, Brandeis) to obtain an atomic model for the complete rotavirus particle, taking advantage of a number of high-resolution structures (from x-ray crystallography) of individual structural proteins and protein fragments and of the intact inner capsid particle.  The structural analysis has enabled us to design experiments using live-cell imaging (with Tomas Kirchhausen, Children's Hospital) and electron cryo-tomography (with Daniela Nicastro, Brandeis) to follow molecular events during virus uptake and penetration into cells in culture. 

Viral membrane fusion

Membrane fusion is thermodynamically favorable, but it generally presents a high kinetic barrier. Fusion proteins lower this barrier and thus are catalysts for the merger of two bilayers -- "suicide" catalysts in the case of viral fusion proteins.  Sensitive measurements of fusion kinetics, like careful analysis of enzyme kinetics, can yield information about mechanism, by examining effects of directed mutations on rates, cooperativity, on so forth. We have devised a single-virus-particle fusion assay to study fusion in this way. For influenza virus, we have shown that fusion requires engagement with the target bilayer of fusion peptides from 3 or 4 neighboring HA trimers and that a relatively long-lived extended intermediate is a fundamental aspect of the fusion mechanism and a potential target of small-molecule inhibitors.  We are extending this kind of analysis to flaviviruses -- in particular, to West Nile and dengue viruses.