Didier Poncet, INRAE-I2BC

Didier Poncet, Ana Arteni, Malika Ouldali, Patrice Vende, Stephane Roche, Annie Charpilienne, Jean Lepault, Cecile Laroche, Mariela Duarte.

Structural Biology

We have established by cryo-electron microscopy the structure of bovine rotavirus TLP produced in the absence (aTLP) or presence (tTLP) of porcine trypsin. Superposition of the 3D structures of tTLP and aTLP reveals the presence of an additional electron density around the icosahedral threefold axis in the type III channels of tTLP, slightly below the VP7 layer. This density cannot be attributed to any viral protein, whereas the crystallographic structure of porcine trypsin can be fitted into it. The molecular model obtained from the 3D structure of tTLP shows that trypsin is attached to TLP through interaction with glutamine 201 of one protomers from three distinct VP7 trimers. However, mutation Q201A does not abolish trypsin's interaction with TLP completely. The position of the trypsin catalytic site toward the VP7 layer but away from any viral proteins is consistent with trypsin activation only after solubilization of the outer capsid layer. A biochemical test (zymogram) shows that tryptic activity is indeed associated with the TLPs of rotavirus strains RRV, OSU, and SA11, even though trypsin is not visible on their electron density maps. Tryptic activity is also detected with a recombinant rotavirus carrying a VP7 protein from a field isolate of human rotavirus, showing that this association is not the result of rotavirus adaptation to cell culture. Our results show that ordered and functional trypsin associates with rotavirus particles in a symmetrical position, which is a unique observation for a non-enveloped virus. The role of the rotavirus-associated trypsin remains to be established.