A genome-wide screen of Epstein-Barr virus proteins that modulate host SUMOylation identifies a SUMO E3 ligase conserved in herpesviruses

Abstract

Many cellular processes pertinent for viral infection are regulated by the addition of small ubiquitin-like modifiers (SUMO) to key regulatory proteins, making SUMOylation an important mechanism by which viruses can commandeer cellular pathways. Epstein-Barr virus (EBV) is a master at manipulating of cellular processes, which enables life-long infection but can also lead to the induction of a variety of EBV-associated cancers. To identify new mechanisms by which EBV proteins alter cells, we screened a library of 51 EBV proteins for global effects on cellular SUMO1 and SUMO2 modifications (SUMOylation), identifying several proteins not previously known to manipulate this pathway. One EBV protein (BRLF1) globally induced the loss of SUMOylated proteins, in a proteasome-dependent manner, as well as the loss of promeylocytic leukemia nuclear bodies. However, unlike its homologue (Rta) in Kaposi’s sarcoma associated herpesvirus, it did not appear to have ubiquitin ligase activity. In addition we identified the EBV SM protein as globally upregulating SUMOylation and showed that this activity was conserved in its homologues in herpes simplex virus 1 (HSV1 UL54/ICP27) and cytomegalovirus (CMV UL69). All three viral homologues were shown to bind SUMO and Ubc9 and to have E3 SUMO ligase activity in a purified system. These are the first SUMO E3 ligases discovered for EBV, HSV1 and CMV. Interestingly the homologues had different specificities for SUMO1 and SUMO2, with SM and UL69 preferentially binding SUMO1 and inducing SUMO1 modifications, and UL54 preferentially binding SUMO2 and inducing SUMO2 modifications. The results provide new insights into the function of this family of conserved herpesvirus proteins, and the conservation of this SUMO E3 ligase activity across diverse herpesviruses suggests the importance of this activity for herpesvirus infections.

Author summary

The functions of many cellular proteins important for anti-viral responses and oncogenesis are controlled by modifications by small ubiquitin-like modifiers (SUMOs). Here we present the first screen of Epstein-Barr virus (EBV) proteins for those that can globally alter SUMO modifications of cellular proteins. We identify four distinct EBV proteins that increase SUMO modifications and one that decreases them. One of the SUMO upregulating proteins (SM) is conserved in other herpesviruses and we show that this activity is conserved in homologues from herpes simplex virus 1 (HSV1) and cytomegalovirus (CMV). We also show that these three homologues have SUMO E3 ligase activity in in vitro assays and that they bind SUMO and Ubc9, consistent with the expectation of SUMO E3 ligases. The results provide new insights into the functions and mechanisms of action of this family of herpesvirus proteins. Our study identifies the first SUMO E3 ligases for EBV, HSV1 and CMV and provides a new mechanism by which EBV can manipulate cellular processes, through global effects on cellular SUMOylation.