Fig 5.

VKORC1v2 in intracellular retention of vIL-6. (A) The influence of VKORC1v2 on intracellular retention of vIL-6 was tested by overexpression of vBD as a fusion with hIL-6 (secreted) or hIL-6-KDEL (ER retained) in vIL-6-cotransfected HEK293T cells. While ER-expressed vBD led to a reduction in the very low levels of secreted vIL-6, consistent with its binding the viral cytokine, vBD in the context of secreted hIL-6 was insufficient to induce, in a dominant fashion, secretion of vIL-6. These data indicate that a mechanism independent of vIL-6–VKORC1v2 association is responsible for ER retention of vIL-6. (B) The ability of hIL-6-fused vBD to disrupt vIL-6–VKORC1v2 binding was verified in a coprecipitation assay utilizing lysates from transfected HEK293T cells. Chitin bead-sedimented VKORC1v2-CBD was able to coprecipitate vIL-6 in the presence of hIL-6-KDEL (control) but not its vBD-containing counterpart. (C) Testing by RT-PCR (top panels) of lentivirus-cloned shRNAs directed to VKORC1 variants 1 and/or 2 transcripts (see Materials and Methods) for their abilities to deplete the respective mRNAs in BCBL-1 cells. Selected, active shRNAs were then tested in VKORC1v1- and VKORC1v2-vector transfected cells to verify by immunoblotting (IB; bottom panels) their activities in respect of VKORC1 protein depletion. (D) shRNA-mediated VKORC1v2 depletion had little effect relative to controls (VKORC1v1 and NS shRNA transduction) on the levels of secreted, medium-derived versus intracellular vIL-6. Secreted vIL-6 was immunoprecipitated from medium, and the proportion loaded onto the gel and immunoblotted represents 50 times the fraction of corresponding cell lysate analyzed. (E) Western analysis of density gradient-purified ER membrane preparations by immunoblotting revealed that there was no influence of VKORC1v2 depletion on ER localization of vIL-6. Calreticulin (Crt; ER marker) and EEA1 (endosomal marker) were detected to check the quality of the ER preparations.