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

<div><p>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.</p></div

BRLF1 binds SUMO and induces loss of PML proteins and nuclear bodies.

<p>A. BRFL1-FLAG was purified from 293T cell lysate on anti-FLAG resin. Control resin loaded with the same 293T lysate lacking BRLF1 was generated as a control (EV). Resins were then incubated with <i>E</i>.<i>coli</i> extract containing equal amounts of GST, GST-SUMO1 or GST-SUMO2, washed and eluted in SDS buffer (FLAG pulldowns). The Coomassie gel on the left shows the BRLF1-FLAG and GST proteins used in the assay (arrowheads indicating full length proteins). The GST Western blot on the right shows the recovery of GST and GST-SUMOs. B. CNE2Z cells were transfected with plasmids expressing FLAG-tagged BRLF1, then, 24 hours later, fixed and stained for FLAG and PML. The number of PML NBs were counted in 50 FLAG-positive and 50 FLAG-negative cells on the same slide in two independent experiments, and average values are shown on the bar graph. The effect of ICP0 expression on the number of PML NBs in the CNE2Z cells is also shown on the graph as a positive control. p values (** = 0.001<<i>P</i> < 0.01; *** = <i>P</i> < 0.001) are shown. C. CNE2Z cells were transfected with plasmids expressing FLAG-tagged BRLF1, ICP0 or empty FLAG vector. 48 hours later, cells were lysed and immunoblotted for PML, ICP0, FLAG (BRLF1) and actin.</p

Effect of selected EBV proteins on endogenous SUMO1 and SUMO2 modifications.

<p>293T cells were transfected with plasmids expressing the indicated viral protein or empty vector control. 36 hours later, cell lysates were generated and equal amounts were immunoblotted for SUMO1, SUMO2, FLAG and actin.</p

SM, UL54 and UL69 interact with SUMO1, SUMO2 and Ubc9 in human cells.

<p>A and B. HeLa cells containing integrated His6-SUMO1 (A) or His6-SUMO2 (B) were transfected with plasmids expressing FLAG-SM, FLAG-UL54, FLAG-UL69 or empty vector (pCMV). 36 hours later, His6-SUMO was recovered from cell lysates on metal chelating resin under nondenaturing conditions and immunoblotted for His and FLAG (left panels). 5% of the input lysate was also immunoblotted for His and FLAG (right panels). C. 293T cells were transfected with plasmids expressing FLAG-SM, FLAG-UL54, FLAG-UL69 or empty vector (pCMV). 36 hours later, immunoprecipitations were performed for endogenous Ubc9, followed by immunoblots with Ubc9 and FLAG antibody (left panels). Immunoblots were also performed on 5% of the input lysates (right panels). For all experiments, longer exposures were used for pulldowns/IPs than for inputs in order to provide optimum exposures to show differences between recoveries of different viral proteins in pulldowns and even levels of the viral proteins in inputs.</p

Induction of p53 SUMOylation by SM, UL54 and UL69 in cells.

<p>293T cells were co-transfected with plasmids expressing His6-SUMO1 (A) or His6-SUMO2 (B) and plasmids expressing LMP1, ICP0, FLAG- SM, FLAG-UL54 or FLAG-UL69 or empty vector. His6-tagged proteins were recovered on metal chelating resin under denaturing conditions and immunoblotted for p53. Samples of the lysates (Input) were also immunoblotted for p53 and FLAG. Positions of SUMO1- or SUMO2-modified p53 are indicated.</p

Screens of EBV proteins for global effects on cellular SUMO1 and SUMO2 modifications.

<p>A. 293T cells were co-transfected with plasmids expressing His6-SUMO1 (top panels) or His6-SUMO2 (bottom panels) and the indicated viral protein or empty vector control. B. HeLa cells stably expressing His6-SUMO1 (left panels) or His6-SUMO2 (right panels) were transfected with plasmids expressing the indicated viral protein or empty vector control. C. CNE2Z cells were co-transfected with plasmids expressing His6-SUMO1 (left panels) or His6-SUMO2 (right panels) and the indicated viral protein or empty vector control. In all cases, His6-tagged proteins were recovered from cell lysates on metal chelating resin under denaturing conditions (Pull-down panels) and immunoblotted for SUMO1 or SUMO2 as indicated. Samples of the lysates (Inputs) were also immunoblotted for actin and FLAG, to detect the FLAG-tagged EBV library proteins. Note that LMP1 and ICP0 did not contain FLAG-tags and hence are not seen in the FLAG blots.</p

<i>In vitro</i> SUMO E3 ligase assays.

<p><i>In vitro</i> SUMOylation assays were performed using Abcam SUMOylation Assay Kit with SUMO1 (left panels) or SUMO2 (right panels) and <i>E</i>.<i>coli</i> purified full length p53 as a substrate. Various amounts of His6-SM-3FLAG (A), His6-UL54-3FLAG (B) and His6-UL69-3FLAG (C), generated as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007176#ppat.1007176.g007" target="_blank">Fig 7A</a>, were added to the indicated reactions where 1x is estimated to be 100 ng of full length protein. Negative controls lacking SAE are also shown. All lanes contained equal amounts of p53 substrate, SUMO and Ubc9. After 1.5 hrs reactions, Western blots were performed with antibodies against p53, SUMO1 (left panels), SUMO2 (right panels) and FLAG (to detect SM, UL54, UL69). The position of SUMO-modified p53 is indicated by the arrowheads.</p

Screen of EBV Lytic proteins for global effects on cellular SUMOylation^*.

<p>Screen of EBV Lytic proteins for global effects on cellular SUMOylation^{<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007176#t001fn001" target="_blank">*</a>}.</p

Effect of SM, UL54 and UL69 on global SUMO1 and SUMO2 modifications.

<p>293T (A) and CNE2Z (C) cells were co-transfected with plasmids expressing His6-SUMO1 (left panels) or His6-SUMO2 (right panels) and either ICP0, LMP1, FLAG-SM, FLAG-UL54, FLAG-UL69 or empty vector control. HeLa cells (B) stably expressing His6-SUMO1 (left panels) or His6-SUMO2 (right panels) were transfected with plasmids expressing ICP0, LMP1, FLAG-SM, FLAG-UL54, FLAG-UL69 or empty vector control. His6-tagged proteins were recovered as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007176#ppat.1007176.g001" target="_blank">Fig 1</a> and immunoblotted for SUMO1 or SUMO2 as indicated. Samples of the input lysates were also immunoblotted for actin and FLAG.</p