Abstract
Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication. In particular, phosphorylation and SUMOylation can distinctly regulate the activity of the human cytomegalovirus (HCMV) transactivator immediate early 2 (IE2). However, the molecular mechanism of this process is unknown. Using various structural, biochemical, and cell-based approaches, here we uncovered that IE2 exploits a cross-talk between phosphorylation and SUMOylation. A scan for small ubiquitin-like modifier (SUMO)-interacting motifs (SIMs) revealed two SIMs in IE2, and a real-time SUMOylation assay indicated that the N-terminal SIM (IE2-SIM1) enhances IE2 SUMOylation up to 4-fold. Kinetic analysis and structural studies disclosed that IE2 is a SUMO cis-E3 ligase. We also found that two putative casein kinase 2 (CK2) sites adjacent to IE2-SIM1 are phosphorylated in vitro and in cells. The phosphorylation drastically increased IE2-SUMO affinity, IE2 SUMOylation, and cis-E3 activity of IE2. Additional salt bridges between the phosphoserines and SUMO accounted for the increased IE2-SUMO affinity. Phosphorylation also enhanced the SUMO-dependent transactivation activity and auto-repression activity of IE2. Together, our findings highlight a novel mechanism whereby SUMOylation and phosphorylation of the viral cis-E3 ligase and transactivator protein IE2 work in tandem to enable transcriptional regulation of viral gene.
Highlights
Many viral factors manipulate the host post-translational modification (PTM) machinery for efficient viral replication
The immediate early 2 (IE2)–SIM1 peptide was titrated into a sample of 15N-isotope–labeled SUMO1, and a series of 15N-edited HSQC experiments monitored its effect on 15NSUMO1
The pattern of chemical shift perturbations (CSP) in SUMO1 upon binding IE2–SIM3 is similar to IE2–SIM1, indicating that both the SUMO-interacting motif (SIM) bind at the same interface
Summary
IE2–SIM3, but not IE2– SIM2, bound to SUMO1 (Fig. S1A). IE2– SIM3, but not IE2–SIM2, interacted with SUMO2 (Fig. S1D). NMR titrations were repeated using a synthetic peptide of IE2–SIM1, where the two serines Ser-203 and Ser-205 are phosphorylated (IE2-ppSIM1, Table 1), to examine the effect of Ser-203/205 phosphorylation on its interaction with SUMO1/2. The pattern of CSPs observed in SUMO1 upon titration with IE2–ppSIM1 is similar to that observed during interaction with unphosphorylated IE2–SIM1 (Fig. S5A), indicating that the interface of binding is identical. Fitting of NMR chemical shifts against ligand/protein concentration yielded the dissociation constant to be 7.1 (Ϯ0.4) M, which is 8-fold lower than unphosphorylated IE2–SIM1 (Fig. S5B and Table 1). When IE2– ppSIM1 was titrated to SUMO2, the interface of interaction was similar to IE2–SIM1 (Fig. S6A).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
