Abstract
The hepatitis E virus (HEV) is a small RNA virus and the cause of acute viral hepatitis E. The open reading frame 3 protein (pORF3) of HEV appears to be a pleiotropic regulatory protein that helps in the establishment, propagation and progression of viral infection. However, the global cellular effects of this protein remain to be explored. In the absence of traditional in vitro viral infection systems or efficient replicon systems, we made an adenovirus based ORF3 protein expression system to study its effects on host cell gene expression. We infected Huh7 hepatoma cells with recombinant adenoviruses expressing pORF3 and performed microarray-based gene expression analyses. Several genes down regulated in pORF3-expressing cells were found to be under regulation of the liver-enriched hepatocyte nuclear factor 4 (HNF4), which regulates hepatocyte-specific gene expression. While HNF4 localizes to the nucleus, its phosphorylation results in impaired nuclear localization of HNF4. Here we report that pORF3 increases HNF4 phosphorylation through the ERK and Akt kinases, which results in impaired nuclear translocation of HNF4 and subsequently the down modulation of HNF4-responsive genes in pORF3-expressing cells. We propose that modulation of several hepatocyte specific genes by pORF3 will create an environment favorable for viral replication and pathogenesis.
Highlights
Hepatitis E virus (HEV) is the causative agent for hepatitis E, a waterborne disease that occurs sporadically and as focused outbreaks [1,2]. It is endemic in large parts of Asia, Africa and Latin America from where large and small outbreaks and sporadic disease have been reported [3]; about 2 billion people are estimated to live in areas where HEV infection is endemic [4]
We recently showed that pORF3 delays the movement of activated epidermal growth factor (EGF) receptors (EGFRs) and hepatocyte growth factor (HGF) receptors (c-Met) into the degradative compartment, which is likely to result in extended growth factor signaling from intracellular sites [7,13]
Recombinant adenoviruses expressing the orf3-egfp or egfp were constructed as described in Materials and Methods
Summary
Hepatitis E virus (HEV) is the causative agent for hepatitis E, a waterborne disease that occurs sporadically and as focused outbreaks [1,2]. The orf of HEV encodes a protein of 123 amino acids; a recent report proposed pORF3 to be translated from a bicistronic subgenomic RNA and to be 9 aa shorter [6]. Both the ORF3 proteins (123aa and 114aa) behave in terms of their localization and functional effects in subgenomic expression systems [7]. We recently showed that pORF3 delays the movement of activated epidermal growth factor (EGF) receptors (EGFRs) and hepatocyte growth factor (HGF) receptors (c-Met) into the degradative compartment, which is likely to result in extended growth factor signaling from intracellular sites [7,13] This effect on EGFR movement was found to retard the nuclear transport of phosphorylated signal transducer and activator of transcription 3
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