Functional study of two flexible regions of the hepatitis E virus ORF1 replicase
Fig 5
Effect of pharmacological inhibitors on ORF1 processing, and determination of the molecular weights of ORF1-derived products.
(a) H7-T7-IZ cells were transfected with a pTM plasmid expressing the untagged (WT) or V5-HA-tagged wildtype ORF1 protein (V5 blue arrowhead, HA red arrowhead). (b, c, d) At 18h post-transfection, cells were treated with the proteasome inhibitor MG132 (10µM, + /-), the HSP90 inhibitor NVP-HSP990 (1µM, -/+), or both (+/+) for 9h. Cells treated with solvent (DMSO, -/-) and non-transfected cells (Mock) were used as controls. Total cell lysates were next collected in the presence of protease inhibitors and analyzed by WB. Immunoblots were probed either with antibodies directed against the V5 epitope (b, left), the HA epitope (b, right), the MetY domain (c, top), the macrodomain X (c, middle), or the HSP70 (c, bottom). The latter served as control for HSP70 expression, which is upregulated in response to chemical stress. The full-length ORF1 protein (ORF1), the HEV-derived SMAD activator (HDSA) and the Met domain (Met) are indicated on the left. Molecular weight markers are indicated in kilodaltons on the right. The asterisk on the WB anti-MetY indicates a non-specific band. (d) The MolecularWeightEstimator ImageJ macro [35] was used to determine the molecular weights of ORF1-derived products. A representative picture of WB anti-V5 is shown along with the colored protein markers used (Page Ruler and ProSieve Quadcolor). Regions of interest (ROI) were selected for both the marker bands of known size and the ORF1-derived bands of unknown size (ORF1 and blue, green, orange and brown arrowheads). The macro indexes their positions and calculates molecular weights based on a calibration curve fitted to the marker bands. Calculated sizes of full-length ORF1 and derived products are boxed. This analysis estimated the full-length ORF1 at ∼235 kDa, the blue arrowhead at ∼182 kDa, the green arrowhead/HDSA at ∼139 kDa, the orange arrowhead at ∼97 kDa, and the brown arrowhead at ∼86 kDa.