Palmitoylation mediates membrane association of hepatitis E virus ORF3 protein and is required for infectious particle secretion

Abstract

Hepatitis E virus (HEV) is a positive-strand RNA virus encoding 3 open reading frames (ORF). HEV ORF3 protein is a small, hitherto poorly characterized protein involved in viral particle secretion and possibly other functions. Here, we show that HEV ORF3 protein forms membrane-associated oligomers. Immunoblot analyses of ORF3 protein expressed in cell-free vs. cellular systems suggested a posttranslational modification. Further analyses revealed that HEV ORF3 protein is palmitoylated at cysteine residues in its N-terminal region, as corroborated by 3H-palmitate labeling, the investigation of cysteine-to-alanine substitution mutants and treatment with the palmitoylation inhibitor 2-bromopalmitate (2-BP). Abrogation of palmitoylation by site-directed mutagenesis or 2-BP treatment altered the subcellular localization of ORF3 protein, reduced the stability of the protein and strongly impaired the secretion of infectious particles. Moreover, selective membrane permeabilization coupled with immunofluorescence microscopy revealed that HEV ORF3 protein is entirely exposed to the cytosolic side of the membrane, allowing to propose a model for its membrane topology and interactions required in the viral life cycle. In conclusion, palmitoylation determines the subcellular localization, membrane topology and function of HEV ORF3 protein in the HEV life cycle.

Hepatitis E virus replication in human intestinal cells

“…The antiviral effect of ribavirin on HEV has been linked to mutagenesis of the HEV genome. Moreover, ORF3 protein is involved in HEV secretion due to its interaction with lipid membrane and intracellular endosomal sorting complexes required for transport (ESCRT) machinery 19 20. Therefore, we sequenced the ORF3 gene in HEV particles isolated from the apical and basal compartments of infected enterocytes during ribavirin treatment.…”

“…This could be due to differences in local intracellular ribavirin concentration produced by distinct uptake/efflux ratios at the apical and basal membranes. The ORF3 protein is the major HEV protein involved in virus egress by interacting with the cell lipid membrane and ESCRT machinery 19 20. The effect of ribavirin on basal virus release was not due to it causing specific mutagenesis in the ORF3 protein.…”

Optimized Hepatitis E Virus (HEV) Culture and Its Application to Measurements of HEV Infectivity

“…Insertions in the HVR can confer advantages for replication [15,16]. ORF2 is translated from two initiation codons giving two forms of ORF2, the capsid protein, and a soluble glycosylated protein [17,18], while ORF3 is a small phosphorylated and palmitoylated protein involved in HEV morphogenesis and release [19][20][21][22][23]. The HEV particles that leave infected cells are in a quasi-enveloped form (eHEV) [24].…”

Hepatitis E Virus Infection: Circulation, Molecular Epidemiology, and Impact on Global Health

“…It has been shown to bind to microtubules and be involved in particle assembly and egress by interaction with the tumor susceptibility gene 101 protein (TSG101), a key protein involved in the endosomal sorting complexes of the ESCRT (endosomal sorting complexes required to transport) transport pathway and involved in the budding of the viruses [ 47 , 48 , 49 , 50 ]. Furthermore, it may play a role in infectious particle secretion via its palmitoylation and membrane association [ 51 ]. Additionally, there are also reports of its role in intracellular transduction pathways, the potential to reduce host immune responses, and protection of virus-infected cells [ 52 , 53 , 54 ].…”