Activation of ERAD Pathway by Human Hepatitis B Virus Modulates Viral and Subviral Particle Production

Catalin Lazar,Alina Macovei,Stefana Petrescu,Norica Branza-Nichita,Wang-Shick Ryu

doi:10.1371/journal.pone.0034169

Catalin Lazar, Alina Macovei + Show 3 more

Open Access

https://doi.org/10.1371/journal.pone.0034169

Copy DOI

Journal: PloS one	Publication Date: Mar 26, 2012
Citations: 104	License type: CC BY 4.0

Affiliation: Romanian Academy, Institute of Biochemistry

Abstract

Hepatitis B virus (HBV) belongs to the Hepadnaviridae family of enveloped DNA viruses. It was previously shown that HBV can induce endoplasmic reticulum (ER) stress and activate the IRE1-XBP1 pathway of the unfolded protein response (UPR), through the expression of the viral regulatory protein X (HBx). However, it remained obscure whether or not this activation had any functional consequences on the target genes of the UPR pathway. Of these targets, the ER degradation-enhancing, mannosidase-like proteins (EDEMs) are thought to play an important role in relieving the ER stress during UPR, by recognizing terminally misfolded glycoproteins and delivering them to the ER-associated degradation (ERAD). In this study, we investigated the role of EDEMs in the HBV life-cycle. We found that synthesis of EDEMs (EDEM1 and its homologues, EDEM2 and EDEM3) is significantly up-regulated in cells with persistent or transient HBV replication. Co-expression of the wild-type HBV envelope proteins with EDEM1 resulted in their massive degradation, a process reversed by EDEM1 silencing. Surprisingly, the autophagy/lysosomes, rather than the proteasome were involved in disposal of the HBV envelope proteins. Importantly, inhibition of the endogenous EDEM1 expression in HBV replicating cells significantly increased secretion of both, enveloped virus and subviral particles. This is the first report showing that HBV activates the ERAD pathway, which, in turn, reduces the amount of envelope proteins, possibly as a mechanism to control the level of virus particles in infected cells and facilitate the establishment of chronic infections.

Highlights

Hepatitis B virus (HBV) is a noncytopathic, hepatotropic virus which belongs to the Hepadnaviridae family
HBV activates expression of EDEM proteins To investigate the relationship between HBV replication and EDEM proteins expression, the level of EDEM1-3 transcripts was quantified in HepG2.2.2.15 cells, which support active HBV replication, assembly and secretion of infectious virions, and the parental HepG2 cell line
To further determine whether the increased level of mRNA was accompanied by an accumulation of the corresponding proteins, EDEM1-3 biosynthesis was analysed by Western blotting of both cell lines lysates, following digestion with PNGase F

Summary

Introduction

Hepatitis B virus (HBV) is a noncytopathic, hepatotropic virus which belongs to the Hepadnaviridae family. The viral DNA genome is packaged inside the nucleocapsid, surrounded by a lipid bilayer derived from the host cell, which contains three transmembrane proteins translated from alternative start codons of the same open reading frame (ORF). These surface proteins are designated as large (L), middle (M) and small (S) and share a 226 amino acid- long S domain, at the C-terminal region [3]. Misfolded proteins are retro-translocation into the cytosol, followed by polyubiquitylation and proteasomal degradation [6] This tightly regulated ERassociated degradation (ERAD) pathway is initiated by the oligomerization and autophosphorylation of the ER stress-sensor IRE1, which, once activated, removes an intron from the X-box binding protein 1 (XBP1) mRNA [7]

Methods

Results

Conclusion