Defining the Link between the Kaposi's Sarcoma-associated Herpesvirus E3 Ubiquitin Ligase, K5, and the Notch Signaling Pathway

Kaposi's Sarcoma-associated Herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, some forms of Multicentric Castleman's disease, and Primary Effusion Lymphoma. KSHV undergoes biphasic replication, with both a latent and a lytic phase of replication. When KSHV-associated malignancies are examined, the majority of cells harbor virus that is undergoing latent replication, but approximately 3% of cells have viral genomes that are undergoing spontaneous lytic replication. If lytic replication is inhibited in KSHV-associated malignancies, patients have reduced KSHV viral loads. Therefore identifying the factors that control the switch from latent to lytic replication is an important step towards developing therapeutics to treat the diseases caused by KSHV.;K5 is a virally encoded E3 ubiquitin ligase that modulates cell-surface signaling molecules that are involved in immune detection (MHCI, CD54 and CD86), viral entry (DC-SIGN and DC-SIGNR), viral egress (tetherin) and cellular metabolism (receptor tyrosine kinases). K5 is classified as an immediate early lytic protein but has the unusual property that its expression can be induced independent of lytic replication through over-expression of the Notch1 Intracellular Domain (NICD1). Over-expression of NICD1 leads to viral reactivation in a PEL cell line. Given that K5 appears to groom the cells for reactivation, we hypothesized that K5 was manipulating the Notch signaling pathway to control entry into the lytic replication cycle. To this end we investigated the effects of K5 on expression of Notch ligands and receptors in B cells (BJABs) and monocytes (THP-1 cells). Our results showed that K5 was modulating both the RNA and protein levels of some Notch receptors and ligands and was acting differently in the two cell lines tested. This is the first report of K5 expression modulating the RNA levels of specific genes.;Although multiple receptors and ligands were modulated by K5 expression in one or both cell lines, we decided to focus on the Notch1 receptor in THP-1 cells and the Jagged1 ligand in BJABs. K5 caused a reduction in Notch1 RNA expression in THP-1 cells but not BJABs. Reduction of the Notch1 protein in THP-1 cells expressing K5 required the E3 ubiquitin ligase activity, the endocytic motif, and the putative SH3 binding domain of K5. This is the first lime that the putative SH3 binding domain has been shown to be required for modulation of a target by K5. Interestingly, expression of K5 led to the inefficient processing of the receptor in the Golgi, which may be due to K5-mediated, ER-associated degradation of Notch1, but this still remains to be confirmed.;Reduction of Jagged1 protein by K5 in BJABs required the E3 ubiquitin ligase activity of K5 as well as the endocytic motif. Using chimeras in which the extracellular domain of CD4 was fused to the transmembrane and C-terminus of Jagged1 or Jagged2, we showed that K5 could down regulate both CD4-Jagged1 and CD4-Jagged2 from the cell surface in BJABs. K5 caused increased endocytosis of CD4-Jagged1 through ubiquitination of lysine residues in the C-terminus of Jagged1. Importantly, this modulation of CD4-Jagged1 was also observed in BJABs where wild-type KSHV was reactivated, and this was dependent upon expression of K5.;Taken together, this work demonstrates that K5 modulates the Notch signaling pathway by affecting both the RNA and protein levels of certain Notch receptors and ligands. Future studies will focus on how these interactions influence the viral life cycle. We hypothesize that this modulation of the Notch signaling pathway by K5 is involved in viral reactivation and initiation of lytic replication.