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, 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. It serves as maturation site of glycosylated ORF2 by cellular proteases, 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. Author summary Hepatitis E virus (HEV) is the major cause of acute viral hepatitis worldwide. Although infection with HEV is usually self-resolving, it can cause up to 30% mortality in pregnant women in the third trimester. There is no specific treatment nor universal vaccine to fight against HEV. In our study, we focused on the HEV ORF2 capsid protein which is produced as different forms that perform distinct functions in the HEV lifecycle. The infectious ORF2i form is the structural component of virions, while the other forms likely act as an immune decoy. Herein, we deciphered the molecular determinants of ORF2 multifunctionality. We identified an Arginine-Rich Motif (ARM) located in the ORF2 N-terminal region that controls the subcellular localization, the fate and functions of ORF2 forms. It also promotes ORF2-host cell interactions. Our observations highlight the ORF2 ARM as a unique central regulator of ORF2 addressing that finely controls the HEV lifecycle.

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