Investigating the activity of Varicella Zoster Virus (VZV) SUMO-targeted Ubiquitin Ligase ORF61

Varicella Zoster Virus (VZV) is a dsDNA virus that infects dermal cells and causes characteristic cutaneous lesions. The virus undergoes neurotropism and later causes secondary cycles of infection. In the host nucleus, Promyelocytic Leukaemia Nuclear Bodies (PML-NBs) spontaneously form around the VZV genome to repress viral gene expression. VZV encodes for a ubiquitin E3 ligase ORF61 to disperse PML-NBs and alleviate repression. ORF61 functions as a ubiquitin E3 ligase with a conserved RING domain at the N-terminal end. It carries three SUMO-interacting motifs (SIMs) that mediate interactions with SUMOylated proteins within PML bodies. The mechanism by which ORF61 disperses PML-NBs is poorly understood. To understand how ORF61 interacts with SUMOylated proteins, we investigated its interaction with SUMO and studied its SUMO-Targeted Ubiquitin Ligase (STUbL) activity. Our studies reveal that ORF61 co-opts the E2D family for ubiquitination activity. A specific network of interactions between the E2 enzyme, ORF61, and Ub facilitates polyubiquitination. ORF61 can synthesize branched polyubiquitin chains of K11, K48, and K63 linkages. The C-terminal SIM in ORF61 is a high-affinity binder of SUMO chains. Utilizing the SIM, ORF61 targets specific lysines on SUMO chains for ubiquitination. These studies provide crucial insights into the functional mechanism of viral STUbL ORF61.