Title: The Role of Outer Capsid Glycoprotein VP7 in Assembly, Neutralization, and Maturation of the Rotavirus Triple Layered Particle
Description: Rotavirus is an important cause of gastroenteritis in infants and children and a severe public health problem in countries unable to provide proper supportive medical care to children with severe, dehydrating diarrhea. The virion is composed of three concentric layers of structural proteins, with the outer layer necessary for cellular entry and the target for protective antibodies. The outer capsid glycoprotein, VP7, is a calcium dependent trimer. It is required for virus entry, for regulating viral transcription, and for virion assembly in the endoplasmic reticulum (ER). The following thesis describes our efforts to understand the structure and function of VP7 in rotavirus replication and host defense. Crystallization of the VP7 trimer in complex with a neutralizing antibody fragment allowed us to visualize how calcium mediates trimer formation and to reclassify linear epitopes into two conformational regions. Our structure implies that all protective antibodies targeting VP7 will inhibit infection by crosslinking trimer subunits. We confirmed this model by testing other VP7 monoclonal antibodies for their potential to neutralize virus as intact divalent IgGs or as monovalent Fabs.

We also designed a VP7 disulfide inter-subunit crosslinked mutant that has properties similar to those of antibody inhibited virus. The crystal structure does not explain how VP7 bound to the virus particle, but a high-resolution electron cryomicroscopy (cryoEM) reconstruction shows how the N-terminal arms of a VP7 trimer, not ordered in the crystals, clamp onto the underlying, trimeric VP6. Truncation mutations of the N-terminal arm confirm the importance of this segment for incorporation into virions and infectious particle assembly; truncations at the C-terminus reveal a role for the C-terminal arm in rotavirus cell entry. To study the final maturation steps in the ER lumen, we have sought to isolate intermediate assembly particles enriched with tunicamycin, a glycosylation inhibitor. The current protocol does not enrich particles to an extent adequate for biochemical or structural analyses but we have established a starting point to study the ER assembly mechanism. Important questions remain regarding VP7 and rotavirus replication. CryoEM promises to make on-going contributions in answering mechanistic questions involving non-enveloped virus entry and assembly.