The ORF3 protein of hepatitis E virus delays degradation of activated growth factor receptors by interacting with CIN85 and blocking formation of the Cbl-CIN85 complex

Abstract

Hepatitis E virus (HEV) causes an acute self-limiting disease that is endemic in developing countries. Previous studies suggested that the ORF3 protein (pORF3) of HEV is required for infection in vivo and is likely to modulate the host response. Our previous work showed that pORF3 localizes to early and recycling endosomes and causes a delay in the postinternalization trafficking of epidermal growth factor receptor (EGFR) to late endosomes/lysosomes. Here we report that pORF3 also delays the trafficking and degradation of activated hepatocyte growth factor receptor (c-Met) and delineate the mechanistic details of these effects. A mutant ORF3 protein, which does not localize to endosomes, also showed similar effects on growth factor receptor trafficking, making this effect independent of the endosomal localization of pORF3. The ORF3 protein was found to interact with CIN85, a multidomain adaptor protein implicated in the Cbl-mediated downregulation of receptor tyrosine kinases. This interaction competed with the formation of the growth factor receptor-Cbl-CIN85 complex, resulting in the reduced ubiquitination of CIN85 and trafficking of the growth factor receptor complex toward late endosomes/lysosomes. We propose that through its effects on growth factor receptor trafficking, pORF3 prolongs endomembrane growth factor signaling and promotes cell survival to contribute positively to viral replication and pathogenesis.

FIG. 1.

The ORF3 protein delays degradation of the activated hepatocyte growth factor receptor, c-Met. (A) Flow cytometric analysis of surface or total c-Met in Huh7 cells transfected with either pEGFP-N1 (gray area) or pEGFP-ORF3 (black line). Single-parameter histograms are shown for EGFP-positive cells. (B) Huh7 cells were transfected as described above and 36 h later were serum starved for 12 h. The cells were then pulsed with 50 ng/ml HGF for the indicated times. Cell lysates containing equal amounts of total protein were Western blotted with anti-phospho-Met antibodies. The graph on the right shows decay curves, taking the highest intensity (at 30 min post-HGF treatment) to be 100%. The gel and decay curves are representative of three separate experiments.

FIG. 2.

The ORF3 protein also affects total growth factor receptor levels in cells. Huh7 cells were transfected with plasmid pORF3-EGFP (+) or pEGFP-N1 (−). After 36 h, six sets of cells were serum starved, and two sets were kept in complete medium. Three sets of serum-starved cells were pulsed with 100 ng/ml EGF, and the other three sets were pulsed with 50 ng/ml HGF for the indicated times. Cell lysates were prepared, and equal amounts of total protein were Western blotted with anti-EGFR (left) or anti-c-Met (right) antibodies. Normalized lysates were also Western blotted with anti-ERK1 and anti-EGFP antibodies for loading and expression controls, respectively.

FIG. 3.

The endosomal localization of pORF3 is not responsible for its effect on EGFR degradation. (A) Huh7 cells were cotransfected with pRab5-RFP and either pORF3-EGFP or expression vectors for mutant ORF3-EGFP fusion proteins. The fixed cells were then imaged directly for fluorescent proteins by confocal microscopy. The pORF3 and Rab5 signals were pseudocolored green and red, respectively. The merged pictures of single confocal planes are shown. In all cases, representative images that were observed for at least 70% of coexpressing cells are shown. (B) Huh7 cells were transfected to express EGFP or the wild-type or mutant ORF3-EGFP fusion protein in two sets. After 36 h, the cells were serum starved for 12 h. One set was pulsed with 100 ng/ml EGF for 30 min, and another set was pulsed with 50 ng/ml HGF for 45 min. Cell lysates containing equal amounts of total protein were immunoprecipitated (IP) with anti-EGFR (left) or anti-c-Met (right) and then Western blotted (WB) with anti-pTyr and either anti-EGFR (left) or anti-c-Met (right) antibodies. Normalized lysates were also Western blotted with anti-ERK1 and anti-green fluorescent protein (GFP) antibodies for loading and expression controls, respectively.

FIG. 4.

The ORF3 protein interacts with CIN85. (A) Huh7 cells were transfected to express EGFP (control) or the ORF3-EGFP or ΔD1-ORF3-EGFP fusion protein. After 48 h, cell lysates were prepared and immunoprecipitated with either anti-EGFP or anti-CIN85 antibodies, followed by Western blotting with anti-CIN85 or anti-EGFP antibodies, respectively. Normalized lysates were also Western blotted with anti-EGFP and anti-CIN85 antibodies, which served as expression controls for the expressed proteins. (B) Huh7 cells were cotransfected with pcDNA3-FLAG-CIN85 and either pEGFP-N1 (control) or pORF3-EGFP. After 48 h, cell lysates were prepared, and equal amounts of total protein were immunoprecipitated with anti-GFP, followed by Western blotting with anti-FLAG antibodies. Cell lysates were also Western blotted with anti-FLAG and anti-GFP antibodies to serve as expression controls. (C) Huh7 cells were transfected with the pORF3-EGFP expression vector (1), or S10-3 cells were transfected with in vitro-synthesized capped replicon transcript (2), as described in Materials and Methods. The cells were fixed and stained for CIN85 using a rabbit anti-CIN85 antibody and for pORF3 with a monoclonal anti-ORF3 antibody (2). The stained cells were imaged by confocal microscopy. The pORF3 and CIN85 signals were pseudocolored green and red, respectively. The merged pictures of single confocal planes are shown. Details are also shown for the boxed regions. In all cases, representative images that were observed for at least 70% of coexpressing cells are shown.

FIG. 5.

Overexpression of CIN85 reverses the effect of pORF3 on growth factor endocytosis and cell survival. Control and ORF3-expressing stable cell lines were transfected with different amounts of the pcDNA3-FLAG-CIN85 expression vector as indicated and a vector control to keep the total amount of plasmid constant. After 36 h, cells were serum starved overnight and then pulsed with either 100 ng/ml EGF for 30 min or 50 ng/ml HGF for 45 min. Cell lysates were prepared, and equal amounts of total protein were immunoprecipitated by using anti-EGFR (left) or anti-Met (right) and then Western blotted with anti-pTyr and anti-EGFR or anti-Met antibodies, as shown. Normalized lysates were also Western blotted with anti-FLAG and anti-ERK1 antibodies for CIN85 expression and loading controls, respectively. (B) pCN and ORF3/4 cells were transiently transfected with the pcDNA3-FLAG-CIN85 expression vector and control empty vector. After 48 h, the cells were washed, and an MTT assay was carried out as described in Materials and Methods. The readings were converted into percent survival, taking the control as 100%.

FIG. 6.

The ORF3 protein reduces formation of the growth factor receptor-Cbl-CIN85 complex. (A) Huh7 cells were transfected with plasmid pORF3-EGFP (+) or pEGFP-N1 (−). After 36 h, two sets of cells were serum starved, and one set was kept in complete medium. One set of serum-starved cells was pulsed with 100 ng/ml EGF for 30 min, and the other was pulsed with 50 ng/ml HGF for 45 min. Cell lysates were prepared, and equal amounts of total protein were immunoprecipitated with anti-CIN85 antibodies followed by Western blotting with anti-pCbl, anti-EGFP, or anti-CIN85 antibodies. Normalized lysates were also Western blotted with anti-ERK1 and anti-EGFP antibodies as loading and expression controls, respectively. (B) The anti-CIN85 immunoprecipitates from A were Western blotted with anti-EGFR antibodies (for EGF-stimulated lysates) or anti-c-Met antibodies (for HGF-stimulated lysates). The immunoprecipitates from non-serum-starved cells (steady state) were Western blotted with both antibodies. Immunoprecipitates were also Western blotted with anti-CIN85 antibodies.

FIG. 7.

The ORF3 protein reduces ubiquitination of CIN85. Huh7 cells were transfected with plasmid pORF3-EGFP (+) or pEGFPN1 (−) and treated as described in the legend of Fig. 5. Cell lysates containing equal amounts of total protein were immunoprecipitated with anti-CIN85 antibodies and then Western blotted with anti-ubiquitin (Ub) or anti-CIN85 antibodies. Normalized lysates were also Western blotted with anti-ERK1 and anti-GFP antibodies for loading and expression controls, respectively. Huh7 cells were transfected with plasmid pORF3-EGFP (+) or pEGFP-N1 (−). After 36 h, cells were serum starved and were pulsed with either 100 ng/ml EGF for 30 min or 50 ng/ml HGF for 45 min. Cell lysates were prepared, and equal amounts of total protein were immunoprecipitated with anti-EGFR (left) or anti-c-Met (right) antibodies followed by Western blotting with anti-ubiquitin and either anti-EGFR or anti-c-Met antibodies. Normalized lysates were also Western blotted with anti-ERK1 and anti-GFP antibodies for loading and expression controls, respectively.

FIG. 8.

Model for ORF3 protein-mediated regulation of growth factor receptor endocytosis. Following its ligation by growth factor (GF), the growth factor receptor (GFR) is phosphorylated at Tyr residues in its cytoplasmic domain. The pTyr residues bind Cbl, which in turn binds the multidomain adaptor protein CIN85 through proline-arginine-rich sequences in Cbl and the SH3 domains in CIN85. The CIN85 protein separately binds endophilin, which recruits the GFR-Cbl-CIN85 complex to clathrin-coated vesicles. The RING finger domain of Cbl recruits ubiquitin-conjugating enzymes and ubiquitinates GFR as well as CIN85, which regulates GFR trafficking toward late endosomes/lysosomes. The ORF3 protein binds CIN85, directly competes with the binding of Cbl to CIN85, and leads to a reduced ubiquitination of CIN85. This delays the trafficking of GFR to the degradative compartments.