KSHV LANA MODULATES THE STABILITY AND ACTIVITY OF THE E3 UBIQUITIN LIGASE RLIM

The Kaposi’s sarcoma associated herpesvirus (KSHV) encoded LANA protein functions in latently infected cells as an essential participant in KSHV genome replication and as a driver of dysregulated cell growth. In a previous study, we have identified LANA interacting proteins using a protein array screen. Here, we explored the effect of LANA on the stability and activity of RLIM (RING-finger LIM-domain-interacting protein, encoded by the RNF12 gene) a novel LANA interacting protein identified in that protein screen. RLIM is an E3 ubiquitin ligase that leads to the ubiquitination and degradation of several transcription regulators, such as LMO2, LMO4, LHX2, LHX3, LDB1 and the telomeric protein TRF1. Expression of LANA leads to down-regulation of RLIM protein levels. This LANA-mediated RLIM degradation is prevented in the presence of the proteasome inhibitor, MG132. Therefore, the interaction between LANA and RLIM could be detected in co-immunoprecipitation assay only in the presence of MG132 to prevent RLIM degradation. A RING finger mutant RLIM (HH 590, 593 EE) is resistant to LANA mediated degradation, suggesting that LANA promotes RLIM auto-ubiquitination. We found that RLIM down regulation in LANA expressing cells, lead to the accumulation of RLIM substrates such as TRF1, and LHX3. On the other hand, the protein levels of other RLIM substrates, such as LDB1 and LMO2, were diminished in the presence of LANA. Furthermore, Luciferase reporter assay and protein expression revealed a complex effect of LANA on transcription regulated by LHX3/LDB1. In conclusion, our study demonstrates down regulation of the E3 ubiquitin ligase RLIM by LANA and the specific implication of this interaction on transcription factors levels and activity in the cell.