Title: Molecular rearrangements of recombinant and authentic rotavirus VP5*
Description: Rotavirus is a non-enveloped virus that kills over 600,000 children annually. It consists of three layers of protein enclosing a double-stranded RNA genome. The outer capsid proteins, VP4 and VP7, deliver a large subviral particle across a membrane to initiate infection. Understanding the function of the outer capsid proteins provides insight into rotavirus and, more generally, non-enveloped virus cell entry. Because VP4 and VP7 are also the viral neutralization antigens, understanding their antigenic properties will aid the rational design of immunogens. VP4 performs a series of molecular gymnastics during viral entry. Prior to trypsin cleavage, it is flexible. Trypsin cleaves VP4 into VP5* and VP8* triggering its rearrangement into rigid spikes with approximate two-fold symmetry of their protruding parts. After an unknown second triggering event, cleaved VP4 undergoes another rearrangement, in which two VP5* subunits fold back on themselves and join a third subunit to form a tightly associated trimer.

To examine the role of VP5* in cell entry and probe its antigenic properties, I developed an efficient means to produce a globular domain of the protein containing the putative membrane interaction region and all known neutralizing epitopes. The biochemical characteristics, high-resolution structure, and antigenicity of the purified VP5* antigen domain were characterized. The crystal structures were determined in two biologically relevant conformations that elucidate details of alternative molecular interactions of identical residues facilitating formation of multiple oligomeric states. To link high-resolution structures to rearrangements of trace quantities of virion-derived protein, trimeric VP5* produced from virion-derived material was characterized. A panel of conformationally specific monoclonal antibodies allowing the state of VP4 and its derivatives during cell entry to be probed was generated. Then, conditions inducing trimer formation from virion-derived VP5* were identified. These experiments demonstrated the necessity of trypsin priming of virions prior to rearrangement of the spike into folded-back trimers. These studies provided new information about the mechanisms of rotavirus entry, produced useful reagents to track conformational rearrangements of VP4 during cell entry, and generated a potentially useful immunogen for future vaccine development.