Genetic Analyses of Contributions of Viral Interleukin-6 Interactions and Signaling to Human Herpesvirus 8 Productive Replication

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) is a cytokine that is poorly secreted and largely localized to the endoplasmic reticulum (ER). It has been implicated, along with other HHV-8 pro-inflammatory and/or angiogenic viral proteins, in HHV-8-associated Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), in addition to an MCD-related disorder involving systemic elevation of pro-inflammatory cytokines, including vIL-6 and human IL-6 (hIL-6). In these diseases, lytic (productive) replication, in addition to viral latency, is believed to play a critical role. Pro-replication activity of vIL-6 has been identified experimentally in PEL and endothelial cells, but the relative contributions of different vIL-6 interactions have not been established. Productive interactions of vIL-6 with the IL-6 signal transducer, gp130, can occur within the ER, but vIL-6 also interacts in the ER with a non-signaling receptor called vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2), calnexin, and VKORC1v2- and calnexin-associated proteins UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) and glucosidase II (GlucII). Here, we report the systematic characterization of interaction-altered vIL-6 variants and the lytic phenotypes of recombinant viruses expressing selected variants. Our data identify the critical importance of vIL-6 and its ER-localized activity via gp130 to productive replication in iSLK (inducible epithelial) cells, absence of detectable involvement of vIL-6 interactions with VKORC1v2, GlucII or UGGT1, and the insufficiency and lack of direct contributory effects of extracellular signaling by vIL-6 or hIL-6. These findings, obtained through genetics-based approaches, complement and extend previous analyses of vIL-6 activity.IMPORTANCE Human herpesvirus 8 (HHV-8)-encoded viral interleukin-6 (vIL-6) was the first viral IL-6 homologue to be identified. Experimental and clinical evidence suggests that vIL-6 is important for the onset and/or progression of HHV-8-associated endothelial- and B-cell pathologies, including AIDS-associated Kaposi's sarcoma and multicentric Castleman's disease. The protein is unusual in its poor secretion from cells and its intracellular activity; it interacts, directly or indirectly, with a number of proteins beyond the IL-6 signal transducer, gp130, and can mediate activities through these interactions in the endoplasmic reticulum. Here, we characterize with respect to protein interactions and signal transducing activity a panel of vIL-6 variants and utilize HHV-8 mutant viruses expressing selected variants in phenotypic analyses. Our findings establish the importance of vIL-6 in HHV-8 productive replication and the contributions of individual vIL-6-protein interactions to HHV-8 lytic biology. This work furthers understanding of the biological significance of vIL-6 and its unique intracellular interactions.