Biochemical Characterization of Hepatitis E Virus ORF3 Protein

Background and Aim: Hepatitis E virus (HEV) infection is the most common cause of acute hepatitis worldwide. This faecal-orally transmitted virus is responsible for epidemics and sporadic cases of hepatitis E in emerging countries but has now been recognized as a zoonotic infection in developed countries through consumption of contaminated food. HEV is a small icosahedral, non-enveloped virus with a positive-strand RNA genome of 7.2 kb encoding three open reading frames (ORFs). ORF1 encodes the replicase, ORF2 the viral capsid and ORF3 a small, hitherto poorly characterized protein. To date, ORF3 protein is believed to play an important role in particle secretion. The aim of this study was to further characterize the ORF3 protein by biochemical approaches. Methods: Various plasmids allowing the expression of wild-type and mutant ORF3 protein were transfected into mammalian cells. Cell free expression systems were employed to translate recombinant ORF3 protein in vitro. Protein lysates were analysed by SDS-PAGE and western blot (WB) analyses. An immunoprecipitation protocol using FLAG® superscript M2 magnetic beads was developed to purify ORF3-FLAG protein for mass spectrometry (MS) analysis. Results: We observed that ORF3 protein displays different and higher apparent molecular weight (MW) when expressed in mammalian cells as compared to cell free expression system. Furthermore, the observed higher MW was not due to potential phosphorylation at Ser 70. In addition, co-expression of the ORF2 capsid protein did not influence the apparent MW of ORF3 protein. We successfully optimized an immunoprecipitation protocol to efficiently and specifically recover a C-terminally FLAG-tagged ORF3 protein expressed in mammalian cells. Despite efforts to increase the amount of protein recovered for MS analysis, the latter did not reveal any substantial modification in ORF3 protein. Finally, a more targeted approach did not reveal any glycosylation of ORF3 protein. Conclusion: Our findings reveal that HEV ORF3 protein has a higher MW than expected and suggest that it is likely post-translationally modified. Therefore, it opens new perspectives in the understanding of this viral protein as well as of the HEV life cycle