Abstract
BackgroundHepatitis E virus (HEV) is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2) is known to induce endoplasmic reticulum stress in ORF2 expressing cells.Methodology/Principal FindingsIn this study we found that HEV ORF2 activates the expression of the pro-apoptotic gene C/EBP homologous protein (CHOP). ORF2 stimulates the CHOP promoter mainly through AARE (amino acid response elements) and to a minor extent the ERSE (endoplasmic reticulum stress response elements). Activating transcription factor 4 (ATF4) protein binds and activates the AARE regulatory sites of the CHOP promoter. ORF2 expression also leads to increased phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) that in turn initiates the translation of ATF4 mRNA. The pro-apoptotic gene CHOP is an important trigger to initiate endoplasmic reticulum stress induced apoptosis. However, the activation of CHOP by ORF2 in this study did not induce apoptosis, nor did BCL2-associated X protein (Bax) translocate to mitochondria. Microarray analysis revealed an ORF2 specific increased expression of chaperones Hsp72, Hsp70B', and co-chaperone Hsp40. Co-immunoprecipitation (Co-IP) and in silico molecular docking analysis suggests that HEV ORF2 interacts with Hsp72. In addition, Hsp72 shows nuclear accumulation in ORF2 expressing cells.Conclusions/SignificanceThese data provide new insight into simultaneously occurring counter-acting effects of HEV ORF2 that may be part of a strategy to prevent host suicide before completion of the viral replication cycle.
Highlights
Hepatitis E virus (HEV), the causative agent of viral hepatitis, is a non-enveloped positive-stranded RNA virus with an icosahedral capsid of about 27 to 34 nm in diameter [1,2,3]
open reading frame 2 (ORF2) activates the C/EBP homologous protein (CHOP) promoter Processing of hepatitis E virus proteins leads to the accumulation in the endoplasmic reticulum (ER) of the viral capsid protein ORF2
We have used luciferase reporter construct driven by the full-length 954 bp CHOP promoter and either pcDNA3.1 or pcDNA-HEV ORF2 was transiently transfected into hepatoma derived Huh7 and H1299 human lung cancer cells
Summary
Hepatitis E virus (HEV), the causative agent of viral hepatitis, is a non-enveloped positive-stranded RNA virus with an icosahedral capsid of about 27 to 34 nm in diameter [1,2,3]. The viral genome has three open reading frames called ORF1, ORF2, and ORF3. The nonstructural proteins required for virus replication and protein processing are encoded by ORF1, while ORF2 encodes the viral capsid protein, and ORF3 a small protein that regulates the cellular environment [4]. The N-terminal part of ORF2 is reported to contain an endoplasmic reticulum (ER) translocation signal [6], and its C-terminal region has an RNA binding site. Hepatitis E virus (HEV) is a non-enveloped plus-strand RNA virus that causes acute hepatitis. The capsid protein open reading frame 2 (ORF2) is known to induce endoplasmic reticulum stress in ORF2 expressing cells
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