A Viral Ubiquitin Ligase Has Substrate Preferential SUMO Targeted Ubiquitin Ligase Activity that Counteracts Intrinsic Antiviral Defence
Figure 3
ICP0 interacts with SUMO in a SIM-dependent manner.
(A) Locations of predicted SIM-Like Sequences (SLS, black vertical bars), the RING finger (grey box), nuclear localization sequence (nls, black horizontal bar), and USP7 binding domain (white box) within ICP0. Number arrows refer to amino acid coordinates. (B) Alignment of SLS in HSV-1 and HSV-2 ICP0. Grey boxes highlight the hydrophobic core within individual SLSs. SLS-4 is additionally aligned with SIMs of hDaxx and HCMV IE2. Square boxes adjacent to SLS-4 highlight phosphorylated serine residues. (C) Y2H analysis showing ICP0 interaction with SUMO-2/3, but not SUMO-1, in a C-terminal di-glycine independent manner. Ubc9 and USP7 are positive controls for SUMO and ICP0 interaction respectively. Mated diploids were plated out onto media lacking leucine and tryptophan (-L/-W, indicating the presence of both plasmids) or leucine, tryptophan, and histidine (-L/-W/-H). Positive interactions are indicated by growth on medium lacking histidine. GAL4 activation domain (AD) or binding domain (BD) fusion orientations are highlighted. Vec indicates the respective empty vector control. (D) ICP0 interacts with SUMO-2/3 in a RING finger-independent manner and requires residues between amino acids 241 to 388. (E) Table highlighting residue mutations made within SLS-4, -5 and -7. (F) Mutation of SLS-4 inhibits ICP0’s ability to interact with SUMO-2/3 in Y2H assay. (G) GST pull down assay demonstrating that the C-terminal third of ICP0 (residues 594-775) interacts with SUMO-1.