New insights into the ORF2 capsid protein, a key player of the hepatitis E virus lifecycle

Hepatitis E Virus (HEV) genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Recently, we developed an efficient HEV cell culture system and demonstrated that this virus produces three different forms of its capsid protein: (i) the ORF2i form (infectious/intracellular) which is the form associated with the infectious particles, (ii) the ORF2g (glycosylated ORF2) and ORF2c (cleaved ORF2) forms that are massively secreted glycoproteins not associated with infectious particles, but are the major antigens present in HEV-infected patient sera. The ORF2 protein sequence contains three highly conserved potential N-glycosylation sites (N1, N2 and N3). Although ORF2 protein is the most characterized viral protein, its glycosylation status and the biological relevance of this post-translational modification is still unclear. In the present study, we constructed and extensively characterized a series of ORF2 mutants in which the three N-glycosylation sites were mutated individually or in combination. We demonstrated that the ORF2g/c protein is N-glycosylated on N1 and N3 sites but not on the N2 site. We showed that N-glycosylation of ORF2 protein does not play any role in replication and assembly of infectious HEV particles. We found that glycosylated ORF2g/c forms are very stable proteins which are targeted by patient antibodies. During our study, we also demonstrated that the ORF2i protein is translocated into the nucleus of infected cells. In conclusion, our study led to new insights into the molecular mechanisms of ORF2 expression.