An Arginine-Rich Motif in the ORF2 Capsid Protein Regulates the Hepatitis E Virus Lifecycle and Interactions with the Host Cell

Abstract

Producing multifunctional proteins is one of the major strategies developed by viruses to condense their genetic information. Here, we investigated the molecular determinants of the multifunctionality of hepatitis E virus (HEV) ORF2 capsid protein. We previously identified 3 isoforms of ORF2 which are partitioned in different subcellular compartments to perform distinct functions. Notably, the infectious ORF2 (ORF2i) protein is the structural component of the virion, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. We identified a 5 amino acid Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region as a central regulator of the subcellular localizations and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation, promoting regulation of host antiviral responses. This motif also regulates the dual topology and functionality of ORF2 signal peptide, leading to the production of either cytosolic infectious ORF2i or reticular non-infectious glycosylated ORF2 forms. Furthermore, the ARM likely serves as a cleavage site of the glycosylated ORF2 protein. Finally, it promotes ORF2 membrane association that is likely essential for particle assembly. In conclusion, our observations highlight ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle.

The Endocytic Recycling Compartment Serves as a Viral Factory for Hepatitis E Virus

“…Indeed, by combining the gt3 p6 strain [15] and the highly transfectable PLC3 cells, we identified 3 forms of the ORF2 capsid protein that perform distinct functions in the HEV lifecycle and display different sequences, post-translational modifications and subcellular localization [6,14]. The ORF2i form is the component of infectious particles and derives from the assembly of intracellular ORF2i (ORF2intra) protein [8]. The ORF2i and ORF2intra proteins are not glycosylated and display the same sequence starting at Leu14 corresponding to the middle of the signal peptide (Fig.…”

“…These glycosylated forms are produced by translocation of ORF2 proteins into the secretory pathway where they are N-glycosylated on two N-x-S/T sequons [14], O-glycosylated and sialylated, and quickly secreted [6,7]. They are cleaved by cellular proteases notably by the proprotein convertase furin [8]. Here, we capitalized on these distinctive features to generate and characterize four anti-ORF2 monoclonal antibodies, including three antibodies (P1H1, P2H1 and P2H2) directed against the ORF2i form and one antibody (P3H2) directed against the different ORF2 isoforms (Fig.…”

“…4C) and are deleterious for viral particle assembly [8]. Of note, HEV RNA replication is not altered in PLC3/HEV-∆ORF3 and PLC3/HEV-5R/5A cells [8].…”

“…The plasmid pBlueScript SK(+) carrying the DNA of the full length genome of adapted gt3 Kernow C-1 p6 strain, (GenBank accession number JQ679013, kindly provided by S.U Emerson) was used [15]. The ORF3-null mutant (HEV-p6-∆ORF3) and the 5R/5A mutant (HEV-p6-5R/5A) of HEV-p6 were generated as described in [16] and [8], respectively. Capped genomic HEV RNAs were prepared with the mMESSAGE mMACHINE kit (Ambion) and delivered to PLC3 or Huh-7.5 cells by electroporation using a Gene Pulser Xcell TM apparatus (Bio-Rad) [6].…”

The Hepatitis E Virus Open Reading Frame 2 Protein: Beyond Viral Capsid

“…It appears that the HEV capsid interacts with β-TRCP to block the ubiquitin-proteasome-mediated degradation of IκB which subsequently inhibits NF-κB activation (Surjit et al, 2012). Moreover, a recent preprint report suggested that the argininerich motifs of the N-terminal of ORF2 could serve as a nuclear location signal to direct the nuclear translocation of ORF2 therefore inhibit the downregulation of NF-κB-related signaling as well (Hervouet et al, 2021).…”