Determinants of Secretion and Intracellular Localization of Human Herpesvirus 8 Interleukin-6

Abstract

Human herpesvirus 8 (HHV-8) interleukin-6 (vIL-6) is distinct from human and other cellular IL-6 proteins in that it does not require the nonsignaling ␣-receptor subunit for the formation of gp130-based signal transducing complexes and also is largely retained intracellularly rather than being secreted. We and others have reported that vIL-6 is retained and is active in the endoplasmic reticulum (ER) compartment, and data from our laboratory have demonstrated that intracellular vIL-6 is functional in the autocrine promotion of proliferation and survival of HHV-8 latently infected primary effusion lymphoma cells. It has also been reported that vIL-6 secretion in gp130-deficient cells can be enhanced by introduced gp130, thereby implicating the signal transducer in vIL-6 trafficking to the cell surface. We examine here the requirements for intracellular retention and localization of vIL-6. Using vIL-6-hIL-6 chimeric and point-mutated vIL-6 proteins, we identified regions and residues of vIL-6 influencing vIL-6 secretion. However, there was no correlation between vIL-6 secretion and gp130 interaction. We found that vIL-6, but not hIL-6, could associate stably with ER-resident chaperone protein calnexin. Glycosylation-dependent interaction of vIL-6 with calnexin correlated with proper protein folding, but there was no direct relationship between vIL-6-calnexin interaction and intracellular retention. While calnexin depletion had little influence on absolute amounts of secreted vIL-6, it led to markedly reduced levels of intracellular cytokine. This was reversed by gp130 transduction, which had no detectable effect on vIL-6 secretion, but redistributed vIL-6 into ER-distinct locations in calnexin-depleted cells, specifically. Our data reveal that calnexin plays a role in ER localization of vIL-6 and that gp130 promotes ER exit, but not secretion, of the viral cytokine.

Modulation of Kaposi's Sarcoma-Associated Herpesvirus Interleukin-6 Function by Hypoxia-Upregulated Protein 1

“…1B). Over 40 unique calnexin peptides and 9 unique gp130 peptides were identified in the vIL-6 sample, consistent with previous findings (28). The vIL-6 sample also had 9 unique peptides identified for the protein hypoxia-upregulated protein 1 (HYOU1; ORP150), while no HYOU1 peptides were identified in the empty vector control.…”

“…This causes STAT3 to dimerize and relocate to the nucleus, where it upregulates IL-6-responsive proinflammatory genes. Others have shown that vIL-6 induces STAT3 Y705 phosphorylation through activation of gp130 (21,28). To determine whether HYOU1's interaction with vIL-6 is involved in the induction of this signaling cascade, HEK293 cells were transfected with the empty vector or the FLAG-tagged vIL-6 plasmid and then transfected with an NTC or HYOU1-targeting siRNA 24 h later.…”

“…In this compartment, vIL-6 binds gp130 in a tetrameric complex to induce intracellular signaling (12). The cellular ER protein calnexin has been shown to interact with vIL-6 to stabilize vIL-6 folding and maintain its intracellular distribution (28). The ER transmembrane protein vitamin K epoxide reductase complex subunit 1 variant 2 (VKORC1v2) was recently identified as an additional intracellular binding partner of vIL-6 (29,30).…”

Human Herpesvirus 8 Viral Interleukin-6 Interacts with Splice Variant 2 of Vitamin K Epoxide Reductase Complex Subunit 1

“…However, the duration of vIL-6 interaction with calnexin is shorter than that of intracellular retention of vIL-6, suggesting that the vIL-6 -calnexin association is not the principal determinant of the slow secretion kinetics of vIL-6 (3,12). In addition to calnexin involvement, gp130 has been reported to promote vIL-6 secretion when introduced into Ba/F3 cells, which do not express endogenous gp130 (12).…”

“…The mechanism(s) responsible for ER and intracellular retention of vIL-6 has not been resolved, although interaction of vIL-6 with the ER chaperone calnexin appears to contribute, directly or indirectly, to ER localization and stability of the viral cytokine (3). However, the duration of vIL-6 interaction with calnexin is shorter than that of intracellular retention of vIL-6, suggesting that the vIL-6 -calnexin association is not the principal determinant of the slow secretion kinetics of vIL-6 (3,12).…”