title: Human herpesvirus 8 viral interleukin-6 interacts with splice variant 2 of vitamin K epoxide reductase complex subunit 1.
abstract: Viral interleukin-6 (vIL-6) specified by human herpesvirus 8 is, unlike its cellular counterpart, secreted very inefficiently and can signal via vIL-6(2):gp130(2) signaling complexes from the endoplasmic reticulum (ER) compartment. Intracellular, autocrine activities of vIL-6 are important for proproliferative and prosurvival activities of the viral cytokine in latently infected primary effusion lymphoma (PEL) cells. However, the molecular determinants of vIL-6 ER localization and function are unclear. Using yeast two-hybrid analysis, we identified the database-documented but uncharacterized splice variant of vitamin K epoxide reductase complex subunit 1 (VKORC1), termed VKORC1 variant 2 (VKORC1v2), as a potential interaction partner of vIL-6. In transfected cells, epitope-tagged VKORC1v2 was found to localize to the ER, to adopt a single-transmembrane (TM) topology placing the C tail in the ER lumen, and to bind vIL-6 via these sequences. Deletion mutagenesis and coprecipitation assays mapped the vIL-6-binding domain (vBD) of VKORC1v2 to TM-proximal residues 31 to 39. However, while sufficient to confer vIL-6 binding to a heterologous protein, vBD was unable to induce vIL-6 secretion when fused to (secreted) hIL-6, suggesting a VKORC1v2-independent mechanism of vIL-6 ER retention. In functional assays, overexpression of ER-directed vBD led to suppression of PEL cell proliferation and viability, effects also mediated by VKORC1v2 depletion and, as reported previously, by vIL-6 suppression. The growth-inhibitory and proapoptotic effects of VKORC1v2 depletion could be rescued by transduced wild-type VKORC1v2 but not by a vIL-6-refractory vBD-altered variant, indicating the functional relevance of the vIL-6-VKORC1v2 interaction. Notably, gp130 signaling was unaffected by VKORC1v2 or vBD overexpression or by VKORC1v2 depletion, suggesting an alternative pathway of vIL-6 activity via VKORC1v2. Combined, our data identify a novel and functionally significant interaction partner of vIL-6 that could potentially be targeted for therapeutic benefit.
authors: Sidney Kimmel Comprehensive Cancer Center, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
authors: Daming
authors: D
authors: Chen
authors: Emily
authors: E
authors: Cousins
authors: Gordon
authors: G
authors: Sandford
authors: John
authors: J
authors: Nicholas
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D019288:Herpesvirus 8, Human / Q000378:metabolism*; D006801:Humans; D015850:Interleukin-6 / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D006899:Mixed Function Oxygenases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011485:Protein Binding; D012326:RNA Splicing; D020798:Two-Hybrid System Techniques; D064417:Vitamin K Epoxide Reductases
chemical_list: D015850:Interleukin-6; D006899:Mixed Function Oxygenases; C531327:VKORC1L1 protein, human; D064417:Vitamin K Epoxide Reductases