An Arginine-Rich Motif in the ORF2 capsid protein regulates the hepatitis E virus lifecycle and interactions with the host cell

Abstract

Hepatitis E virus (HEV) infection is the most common cause of acute viral hepatitis worldwide. Hepatitis E is usually asymptomatic and self-limiting but it can become chronic in immunocompromised patients and is associated with increased fulminant hepatic failure and mortality rates in pregnant women. HEV genome encodes three proteins including the ORF2 protein that is the viral capsid protein. Interestingly, HEV produces 3 isoforms of the ORF2 capsid protein which are partitioned in different subcellular compartments and perform distinct functions in the HEV lifecycle. Notably, the infectious ORF2 (ORF2i) protein is the structural component of virions, whereas the genome-free secreted and glycosylated ORF2 proteins likely act as a humoral immune decoy. Here, by using a series of ORF2 capsid protein mutants expressed in the infectious genotype 3 p6 HEV strain as well as chimeras between ORF2 and the CD4 glycoprotein, we demonstrated how an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region controls the fate and functions of ORF2 isoforms. We showed that the ARM controls ORF2 nuclear translocation likely to promote 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. It serves as maturation site of glycosylated ORF2 by furin, and promotes ORF2-host cell membrane interactions. The identification of ORF2 ARM as a unique central regulator of the HEV lifecycle uncovers how viruses settle strategies to condense their genetic information and hijack cellular processes.

The endocytic recycling compartment serves as a viral factory for hepatitis E virus

“…2 a). Of note, P3H2 and 1E6 antibodies recognize the different ORF2 isoforms notably the ORF2g/c forms, which are proteins going through the secretory pathway [ 7 , 10 , 19 ], leading to the diffuse ORF2 staining observed in some cells. Of note, all antibodies also recognized ORF2 proteins from HEV-gt1 (Supplementary data, Fig.…”

“…To further understand which viral determinant is important for the formation of HEV-induced subcellular structures, we transfected PLC3 cells with an ORF3-null mutant (HEV- ORF3) [ 15 ] (Fig. 5 c) and an ORF2 assembly mutant (HEV-5R/5A) [ 10 ] (Fig. 5 d).…”

“…The plasmid pBlueScript SK(+) carrying the DNA of the full-length genome of gt3 Kernow C-1 p6 strain (GenBank accession number JQ679013, kindly provided by S.U Emerson) was used [ 20 ]. The ORF3-null mutant (HEV-p6- ORF3) and the 5R/5A mutant (HEV-p6-5R/5A) of HEV-p6 were generated as described in [ 15 ] and [ 10 ], respectively. The plasmid pBlueScript SK(+)/HEV-p6 expressing a V5-tagged ORF1 (V1 insertion) has been described previously [ 21 ].…”

“…ORF2g and ORF2c (herein referred to as ORF2g/c) likely act as humoral decoys that inhibit antibody-mediated neutralization [ 8 ]. Recently, we demonstrated that a 5 amino acid arginine-rich motif (ARM) located in the ORF2 N-terminal region is a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle [ 10 ]. Indeed, the ARM controls ORF2 nuclear translocation, promoting regulation of host antiviral responses.…”

“…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 [ 10 ].…”