The Protease Domain in HEV pORF1 Mediates the Replicase’s Localization to Multivesicular Bodies and Its Exosomal Release

Abstract

BackgroundA peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization. MethodsSubcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western Blot analyses or RT-qPCR to analyze separated particles in more detail. ResultsWe found pORF1 to be accumulating within the endosomal system, most dominantly to MVBs. Expression of the polyprotein’s seven subdomains revealed that the PCP (papain-like cysteine-protease) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3. ConclusionsTaken together we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, as replication and release could be coupled at the endosomal interface. Additionally, this may mediate capsid-independent spread or may facilitate the spread of viral infection, as genomes entering the cell during de novo infection readily encounter exosomally-transferred pORF1.