PMID- 32669340 OWN - NLM STAT- MEDLINE DCOM- 20201221 LR - 20210315 IS - 1098-5514 (Electronic) IS - 0022-538X (Print) IS - 0022-538X (Linking) VI - 94 IP - 19 DP - 2020 Sep 15 TI - Genetic Analyses of Contributions of Viral Interleukin-6 Interactions and Signaling to Human Herpesvirus 8 Productive Replication. LID - 10.1128/JVI.00909-20 [doi] LID - e00909-20 AB - Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) is a cytokine that is poorly secreted and localized largely to the endoplasmic reticulum (ER). It has been implicated, along with other HHV-8 proinflammatory 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 proinflammatory 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. Proreplication 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 nonsignaling 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 inducible SLK (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-cell 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 report the characterization with respect to protein interactions and signal-transducing activity of a panel of vIL-6 variants and utilization of 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. CI - Copyright (c) 2020 American Society for Microbiology. FAU - Li, Qian AU - Li Q AD - Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Xiang, Qiwang AU - Xiang Q AD - Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Chen, Daming AU - Chen D AD - Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA. FAU - Nicholas, John AU - Nicholas J AUID- ORCID: 0000-0002-4058-9652 AD - Sidney Kimmel Comprehensive Cancer Center at Johns Hopkins, Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA nichojo@jhmi.edu. LA - eng GR - R01 CA076445/CA/NCI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural DEP - 20200915 PL - United States TA - J Virol JT - Journal of virology JID - 0113724 RN - 0 (Interleukin-6) RN - 0 (Viral Proteins) RN - 133483-10-0 (Cytokine Receptor gp130) RN - 139873-08-8 (Calnexin) RN - EC 1.17.4.4 (VKORC1 protein, human) RN - EC 1.17.4.4 (Vitamin K Epoxide Reductases) RN - EC 2.4.1.- (Glucosyltransferases) RN - EC 2.4.1.- (UGGT1 protein, human) RN - EC 3.2.1.- (4-nitrophenyl-alpha-glucosidase) RN - EC 3.2.1.20 (alpha-Glucosidases) RN - Multi-centric Castleman's Disease SB - IM MH - Amino Acid Substitution MH - Calnexin/metabolism MH - Castleman Disease/virology MH - Cytokine Receptor gp130/metabolism MH - Endoplasmic Reticulum/metabolism MH - Endothelial Cells/metabolism MH - Glucosyltransferases/metabolism MH - Herpesvirus 8, Human/*genetics/*physiology MH - Humans MH - Interleukin-6/*genetics/*metabolism MH - Lymphoma, Primary Effusion/virology MH - Sarcoma, Kaposi/metabolism MH - Sequence Analysis, Protein MH - Signal Transduction/*physiology MH - Viral Proteins/metabolism MH - Virus Latency MH - Vitamin K Epoxide Reductases/metabolism MH - alpha-Glucosidases/metabolism PMC - PMC7495386 OTO - NOTNLM OT - UDP-glucose:glycoprotein-glucosyltransferase 1 OT - endoplasmic reticulum OT - glucosidase II OT - gp130, signal transduction OT - human herpesvirus 8 OT - lytic replication OT - viral interleukin-6 OT - vitamin K epoxide reductase complex subunit 1 variant 2 EDAT- 2020/07/17 06:00 MHDA- 2020/12/22 06:00 PMCR- 2021/03/15 CRDT- 2020/07/17 06:00 PHST- 2020/05/09 00:00 [received] PHST- 2020/07/08 00:00 [accepted] PHST- 2020/07/17 06:00 [pubmed] PHST- 2020/12/22 06:00 [medline] PHST- 2020/07/17 06:00 [entrez] PHST- 2021/03/15 00:00 [pmc-release] AID - JVI.00909-20 [pii] AID - 00909-20 [pii] AID - 10.1128/JVI.00909-20 [doi] PST - epublish SO - J Virol. 2020 Sep 15;94(19):e00909-20. doi: 10.1128/JVI.00909-20. Print 2020 Sep 15.