Alpha-human herpesviruses cause cutaneous lesions and establish neuronal latency post primary cycle of infection. In order to evade host immune line of defense, effector proteins encoded by these viruses disrupt nuclear bodies (PML-NBs). In case of Varicella zoster virus, causative agent of Chicken Pox, an immediate early protein – ORF61 is encoded. It carries a conserved RING domain that drives its ubiquitin E3 ligase activity. It also carries SUMO-Interacting Motifs (SIMs) that enable its interaction with SUMOylated proteins in the PML-NBs. However, it has not been exclusively reported as a SUMO-Targeted Ubiquitin Ligase (STUbL) and its implications in onset of pathogenesis remain unclear.
Employing a three-pronged approach involving computational, biophysical and in-vitro biochemical assays, we report the structure of ORF61-RING~E2~Ub complex with the interaction map of residues of ORF61 RING involved at the interface and their significance. We screened for potential E2 enzymes that could pair with ORF61 and only the promiscuous family (E2D) interacts efficiently. We confirmed ORF61 to function as a STUbL with tetra-SUMO2 as the substrate even though the substrate preference may differ. We have estimated binding affinities for the individual SIM regions through titrations. Utilizing in vitro assays, their importance in the STUbL activity of ORF61 was also assessed.
Overall, our study highlights one of the strategies used by viruses for immune evasion in this specific case of herpesvirus. The study has revealed ORF61 mimics host STUbL to interact with and potentially ubiquitinate SUMO/ SUMOylated substrates within PML-NBs to cause their dispersal.