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The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4.

Chandra V, Holla P, Ghosh D, Chakrabarti D, Padigaru M, Jameel S - PLoS ONE (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

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.

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The ORF3 protein modulates HNF4 phosphorylation.(A) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein, and cells were harvested 48 hr post-infection. Cell lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and then western blotted with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pERK and anti-ERK to show the effect of pORF3 on ERK activation. (B) Huh7 cells were infected with ORF3 expressing (+) and control (−) recombinant adenoviruses. At 36 hr post infection, the cells were treated with either 50 µM LY294002 or an equal volume of DMSO for 12 hr. Cells were harvested 48 hr post-infection and lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and followed by western blotting with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pAkt and anti-Akt to show the effect of pORF3 on Akt activation. (C) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein. At 36 hr post-transfection, the cells transfected with ΔD1-ORF3-EGFP expressing plasmid were treated with 50 µM LY294002 and others were treated with an equal volume of DMSO for 12 hr. Nuclear lysates were prepared and western blotted with anti-HNF4. STAT3 served as a loading control. (D) Huh7 cells were transfected and treated as described in (C). RT-PCR analysis was performed as mentioned earlier. Histone H4 served as a loading control for RNA.
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pone-0022412-g005: The ORF3 protein modulates HNF4 phosphorylation.(A) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein, and cells were harvested 48 hr post-infection. Cell lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and then western blotted with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pERK and anti-ERK to show the effect of pORF3 on ERK activation. (B) Huh7 cells were infected with ORF3 expressing (+) and control (−) recombinant adenoviruses. At 36 hr post infection, the cells were treated with either 50 µM LY294002 or an equal volume of DMSO for 12 hr. Cells were harvested 48 hr post-infection and lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and followed by western blotting with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pAkt and anti-Akt to show the effect of pORF3 on Akt activation. (C) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein. At 36 hr post-transfection, the cells transfected with ΔD1-ORF3-EGFP expressing plasmid were treated with 50 µM LY294002 and others were treated with an equal volume of DMSO for 12 hr. Nuclear lysates were prepared and western blotted with anti-HNF4. STAT3 served as a loading control. (D) Huh7 cells were transfected and treated as described in (C). RT-PCR analysis was performed as mentioned earlier. Histone H4 served as a loading control for RNA.

Mentions: The ORF3 protein is likely to modulate the subcellular localization of HNF4 in two possible ways. It could interact with HNF4 and trap it in the cytoplasm or endosomal compartments where pORF3 is mainly present. Alternatively, pORF3 could interfere with the regulatory mechanism responsible for the nuclear localization of HNF4. A recent report suggested that phosphorylation of a highly conserved serine (Ser78) in HNF4 resulted in its impaired nuclear localization [21]. Further, HNF4 activity was shown to depend upon ERK and Akt kinases [22], [23]. Our previous studies have shown that ERK and Akt are activated in ORF3-expressing cells as well [10], [12]. To test for these alternative pathways, Huh7 cells were transfected with plasmids expressing ORF3-EGFP or EGFP (control). The cell lysates were immunoprecipitated with anti-HNF4 followed by western blotting with either anti-phosphoSerine (for phospho-HNF4) or anti-EGFP (for pORF3) antibodies. We did not find co-immunoprecipitation of pORF3 and HNF4 (data not shown) but found significant increase in phosphorylated HNF4 levels in ORF3-expressing cells (Fig. 5A). It was not possible to demonstrate these effects of the ORF3 protein on HNF4 phosphorylation with the full-length replicon due to its poor replication efficiency and a delay of 3-4 weeks in producing measurable virions in culture. We have earlier shown the hydrophobic domain 1 of pORF3 to be responsible for ERK activation. Here we found reduced HNF4 phosphorylation in cells expressing the domain 1-deleted (ΔD1) ORF3 protein compared to the wild type protein (Fig. 5A). Blocking of Akt activation with its pharmacological inhibitor LY294002 also resulted in attenuation of pORF3-mediated HNF4 phosphorylation (Fig. 5B). The use of a dominant negative mutant of the catalytic subunit of phosphoinositide 3-kinase (p110DN) did not show any measurable effects on pORF3-mediated HNF4 phosphorylation (not shown). To confirm that the pORF3-mediated effect on HNF4 nuclear localization was due to its phosphorylation, we blocked both the kinases by treating ΔD1-ORF3 expressing cells with LY294002, and checked the nuclear levels and transcription factor activity of HNF4. In these cells, the nuclear levels of HNF4 were comparable to control cells (Fig. 5C), and the transcriptional repression of HNF4-responsive genes was also relieved (Fig. 5D). Together these results confirm that HEV pORF3 increases HNF4 phosphorylation through the activation of ERK and Akt pathways. This is responsible for reduced nuclear localization and transcription factor activity of HNF4.


The hepatitis E virus ORF3 protein regulates the expression of liver-specific genes by modulating localization of hepatocyte nuclear factor 4.

Chandra V, Holla P, Ghosh D, Chakrabarti D, Padigaru M, Jameel S - PLoS ONE (2011)

The ORF3 protein modulates HNF4 phosphorylation.(A) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein, and cells were harvested 48 hr post-infection. Cell lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and then western blotted with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pERK and anti-ERK to show the effect of pORF3 on ERK activation. (B) Huh7 cells were infected with ORF3 expressing (+) and control (−) recombinant adenoviruses. At 36 hr post infection, the cells were treated with either 50 µM LY294002 or an equal volume of DMSO for 12 hr. Cells were harvested 48 hr post-infection and lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and followed by western blotting with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pAkt and anti-Akt to show the effect of pORF3 on Akt activation. (C) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein. At 36 hr post-transfection, the cells transfected with ΔD1-ORF3-EGFP expressing plasmid were treated with 50 µM LY294002 and others were treated with an equal volume of DMSO for 12 hr. Nuclear lysates were prepared and western blotted with anti-HNF4. STAT3 served as a loading control. (D) Huh7 cells were transfected and treated as described in (C). RT-PCR analysis was performed as mentioned earlier. Histone H4 served as a loading control for RNA.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3140526&req=5

pone-0022412-g005: The ORF3 protein modulates HNF4 phosphorylation.(A) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein, and cells were harvested 48 hr post-infection. Cell lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and then western blotted with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pERK and anti-ERK to show the effect of pORF3 on ERK activation. (B) Huh7 cells were infected with ORF3 expressing (+) and control (−) recombinant adenoviruses. At 36 hr post infection, the cells were treated with either 50 µM LY294002 or an equal volume of DMSO for 12 hr. Cells were harvested 48 hr post-infection and lysates containing equal amounts of total protein were immunoprecipitated with anti-HNF4 and followed by western blotting with anti-pSerine antibody. Normalized lysates were western blotted with anti-HNF4 and anti-GFP antibodies for loading and expression controls, respectively. Normalized lysates were also western blotted with anti-pAkt and anti-Akt to show the effect of pORF3 on Akt activation. (C) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein. At 36 hr post-transfection, the cells transfected with ΔD1-ORF3-EGFP expressing plasmid were treated with 50 µM LY294002 and others were treated with an equal volume of DMSO for 12 hr. Nuclear lysates were prepared and western blotted with anti-HNF4. STAT3 served as a loading control. (D) Huh7 cells were transfected and treated as described in (C). RT-PCR analysis was performed as mentioned earlier. Histone H4 served as a loading control for RNA.
Mentions: The ORF3 protein is likely to modulate the subcellular localization of HNF4 in two possible ways. It could interact with HNF4 and trap it in the cytoplasm or endosomal compartments where pORF3 is mainly present. Alternatively, pORF3 could interfere with the regulatory mechanism responsible for the nuclear localization of HNF4. A recent report suggested that phosphorylation of a highly conserved serine (Ser78) in HNF4 resulted in its impaired nuclear localization [21]. Further, HNF4 activity was shown to depend upon ERK and Akt kinases [22], [23]. Our previous studies have shown that ERK and Akt are activated in ORF3-expressing cells as well [10], [12]. To test for these alternative pathways, Huh7 cells were transfected with plasmids expressing ORF3-EGFP or EGFP (control). The cell lysates were immunoprecipitated with anti-HNF4 followed by western blotting with either anti-phosphoSerine (for phospho-HNF4) or anti-EGFP (for pORF3) antibodies. We did not find co-immunoprecipitation of pORF3 and HNF4 (data not shown) but found significant increase in phosphorylated HNF4 levels in ORF3-expressing cells (Fig. 5A). It was not possible to demonstrate these effects of the ORF3 protein on HNF4 phosphorylation with the full-length replicon due to its poor replication efficiency and a delay of 3-4 weeks in producing measurable virions in culture. We have earlier shown the hydrophobic domain 1 of pORF3 to be responsible for ERK activation. Here we found reduced HNF4 phosphorylation in cells expressing the domain 1-deleted (ΔD1) ORF3 protein compared to the wild type protein (Fig. 5A). Blocking of Akt activation with its pharmacological inhibitor LY294002 also resulted in attenuation of pORF3-mediated HNF4 phosphorylation (Fig. 5B). The use of a dominant negative mutant of the catalytic subunit of phosphoinositide 3-kinase (p110DN) did not show any measurable effects on pORF3-mediated HNF4 phosphorylation (not shown). To confirm that the pORF3-mediated effect on HNF4 nuclear localization was due to its phosphorylation, we blocked both the kinases by treating ΔD1-ORF3 expressing cells with LY294002, and checked the nuclear levels and transcription factor activity of HNF4. In these cells, the nuclear levels of HNF4 were comparable to control cells (Fig. 5C), and the transcriptional repression of HNF4-responsive genes was also relieved (Fig. 5D). Together these results confirm that HEV pORF3 increases HNF4 phosphorylation through the activation of ERK and Akt pathways. This is responsible for reduced nuclear localization and transcription factor activity of HNF4.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Virology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, India.

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.

Show MeSH
Related in: MedlinePlus