A novel glycoform of the rotavirus outer capsid protein VP7 is secreted from polarized cells and activates innate immune cells

Rotaviruses (RVs) are unique among non-enveloped animal viruses encoding a structural and a nonstructural glycoprotein in their genome. The outer capsid of the virion is formed by 260 trimers of VP7, which is glycosylated at an asparagine residue(s) in the endoplasmic reticulum of infected cells, where it is retained until a late stage of virion morphogenesis. We investigated the fate of VP7 produced during synchronous infection of polarized Caco-2 cells and observed that a previously undetected form with a molecular weight greater than virion-associated VP7 was secreted into the medium. This secreted form of VP7 (sVP7) was not associated with virions and was insensitive to endoglycosidase H treatment consistent with the dominance of complex type N-glycans revealed in structural details using LC-MS/MS. We also detected core 1 and 2-type O-glycans on sVP7 contributing to the higher-than-predicted molecular weight. Purification of sVP7 from the medium of virus-infected cells revealed that it was trimeric and retained conformationally sensitive epitopes recognized by neutralizing monoclonal antibodies. Like the secreted nonstructural protein 4(NSP4), sVP7 exhibited agonism of TLR2 and TLR4 and caused activation of innate immune cells in human blood. Our results identify a novel form of the rotavirus capsid glycoprotein previously undetected and suggest that VP7 may possess extracellular functions beyond its role in the virion as a modulator of innate and adaptive antiviral immunity.IMPORTANCERotavirus (RV) exhibits a mode of assembly unique among non-enveloped animal viruses whereby virion morphogenesis occurs within an endomembrane compartment. The outer shell of rotavirus is assembled within the lumen of the ER where a glycoprotein VP7 is transferred to immature particles that bud from cytoplasmic inclusions. Lytic release of virions from non-polarized cells has obscured the fate of rotavirus glycoproteins from cells with a differentiated phenotype. This study reveals that in polarized cells, a significant proportion of VP7 is secreted, independently of virions, and acquires novel glycan signatures through extensive modification of N- and O-linked glycosylation within the medial Golgi complex. Secreted VP7 exhibited agonism of Toll-like receptors and activated several distinct subsets of innate immune cells while retaining conformationally dependent neutralizing epitopes associated with a trimeric structure indicating a potential role for the secreted capsid glycoprotein in modulating both innate and adaptive immune responses to rotavirus infection.