Kaposi's Sarcoma Herpesvirus K15 Protein Contributes to Virus-Induced Angiogenesis by Recruiting PLCγ1 and Activating NFAT1-dependent RCAN1 Expression

Abstract

Kaposi's Sarcoma (KS), caused by Kaposi's Sarcoma Herpesvirus (KSHV), is a highly vascularised angiogenic tumor of endothelial cells, characterized by latently KSHV-infected spindle cells and a pronounced inflammatory infiltrate. Several KSHV proteins, including LANA-1 (ORF73), vCyclin (ORF72), vGPCR (ORF74), vIL6 (ORF-K2), vCCL-1 (ORF-K6), vCCL-2 (ORF-K4) and K1 have been shown to exert effects that can lead to the proliferation and atypical differentiation of endothelial cells and/or the secretion of cytokines with angiogenic and inflammatory properties (VEGF, bFGF, IL6, IL8, GROα, and TNFβ). To investigate a role of the KSHV K15 protein in KSHV-mediated angiogenesis, we carried out a genome wide gene expression analysis on primary endothelial cells infected with KSHV wildtype (KSHVwt) and a KSHV K15 deletion mutant (KSHVΔK15). We found RCAN1/DSCR1 (Regulator of Calcineurin 1/Down Syndrome critical region 1), a cellular gene involved in angiogenesis, to be differentially expressed in KSHVwt- vs KSHVΔK15-infected cells. During physiological angiogenesis, expression of RCAN1 in endothelial cells is regulated by VEGF (vascular endothelial growth factor) through a pathway involving the activation of PLCγ1, Calcineurin and NFAT1. We found that K15 directly recruits PLCγ1, and thereby activates Calcineurin/NFAT1-dependent RCAN1 expression which results in the formation of angiogenic tubes. Primary endothelial cells infected with KSHVwt form angiogenic tubes upon activation of the lytic replication cycle. This effect is abrogated when K15 is deleted (KSHVΔK15) or silenced by an siRNA targeting the K15 expression. Our study establishes K15 as one of the KSHV proteins that contribute to KSHV-induced angiogenesis.

Activation of NF-κB by the Kaposi's Sarcoma-Associated Herpesvirus K15 Protein Involves Recruitment of the NF-κB-Inducing Kinase, IκB Kinases, and Phosphorylation of p65

“…The K15 Y 481 F mutant was impaired in the induction of NF-B/DNA complex formation (Fig. 1C), which, in addition to previously reported experiments (16)(17)(18), provides further evidence for the importance of the Y 481 EEV motif for K15-mediated downstream signaling.…”

“…Previous studies showed that the K15 protein of KSHV induces several signal transduction pathways, including the activation of NF-B (16)(17)(18). Induction of NF-B via the classical or the alternative pathway is accompanied by the translocation of specific NF-B dimers from the cytoplasm to the nucleus.…”

“…The open reading frame (ORF) K15 of KSHV is located between the terminal repeat region and the right end of the long unique coding region of the genome. Several divergent alleles of K15 have been identified, among them the predominant K15-P type and the minor K15-M type, which share only 33% of their amino acids (13)(14)(15)(16) (Fig. 1A).…”

“…K15-P activates the mitogen-activated protein (MAP) kinases c-jun-N-terminal kinase 1 (JNK 1) and extracellular signal-regulated kinase (Erk2), and the transcription factors NF-B and AP-1 (14,17,18). Moreover, K15, by recruiting and activating PLC␥1, contributes to KSHV-mediated angiogenesis (16). The phosphorylation of Y 481 in the C-terminal cytoplasmic domain of K15-P by Src kinases (17) is required for most of the K15-P-induced gene expression (17).…”

“…The phosphorylation of Y 481 in the C-terminal cytoplasmic domain of K15-P by Src kinases (17) is required for most of the K15-P-induced gene expression (17). Mutation of tyrosine 481 to phenylalanine (K15-P Y 481 F) leads to impaired downstream signaling by K15-P (16)(17)(18).…”