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. 2018 Nov 12;92(23):e01452-18.
doi: 10.1128/JVI.01452-18. Print 2018 Dec 1.

Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by Herpes Simplex Virus 1 ICP0

Wangheng Hou  1   2 , Ruth Cruz-Cosme  1 , Fayuan Wen  1 , Jin-Hyun Ahn  3 , Inez Reeves  4 , Min-Hua Luo  5 , Qiyi Tang  6
Affiliations

Expression of Human Cytomegalovirus IE1 Leads to Accumulation of Mono-SUMOylated PML That Is Protected from Degradation by Herpes Simplex Virus 1 ICP0

Wangheng Hou et al. J Virol. .

Abstract

To countermeasure the host cellular intrinsic defense, cytomegalovirus (CMV) and herpes simplex viruses (HSV) have evolved the ability to disperse nuclear domain 10 (ND10, aka PML body). However, mechanisms underlying their action on ND10 differ. HSV infection produces ICP0, which degrades the ND10-forming protein PML. Human CMV (HCMV) infection expresses IE1 that deSUMOylates PML to result in dispersion of ND10. It has been demonstrated that HSV ICP0 degraded only the SUMOylated PML, so we hypothesized that HCMV IE1 can protect PML from degradation by ICP0. HCMV IE1-expressing cell lines (U-251 MG-IE1 and HELF-IE1) were used for infection of HSV-1 or transfection of ICP0-expressing plasmid. Multilabeling by immunocytochemistry assay and protein examination by Western blot assay were performed to determine the resultant fate of PML caused by ICP0 in the presence or absence of HCMV IE1. Here, we report that deSUMOylation of human PML (hPML) by HCMV IE1 was incomplete, as mono-SUMOylated PML remained in the IE1-expressing cells, which is consistent with the report by E. M. Schilling, M. Scherer, N. Reuter, J. Schweininger, et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). As expected, we found that IE1 protected PML from degradation by ICP0 or HSV-1 infection. An in vitro study found that IE1 with mutation of L174P failed to deSUMOylate PML and did not protect PML from degradation by ICP0; hence, we conclude that the deSUMOylation of PML is important for IE1 to protect PML from degradation by ICP0. In addition, we revealed that murine CMV failed to deSUMOylate and to protect the HSV-mediated degradation of hPML, and that HCMV failed to deSUMOylate and protect the HSV-mediated degradation of mouse PML. However, IE1-expressing cells did not enhance wild-type HSV-1 replication but significantly increased ICP0-defective HSV-1 replication at a low multiplicity of infection. Therefore, our results uncovered a host-virus functional interaction at the posttranslational level.IMPORTANCE Our finding that HCMV IE1 protected hPML from degradation by HSV ICP0 is important, because the PML body (aka ND10) is believed to be the first line of host intrinsic defense against herpesviral infection. How the infected viruses overcome the nuclear defensive structure (PML body) has not been fully understood. Herpesviral proteins, ICP0 of HSV and IE1 of CMV, have been identified to interact with PML. Here, we report that HCMV IE1 incompletely deSUMOylated PML, resulting in the mono-SUMOylated PML, which is consistent with the report of Schilling et al. (J Virol 91:e02049-16, 2017, https://doi.org/10.1128/JVI.02049-16). The mono-SUMOylated PML was subjected to degradation by HSV ICP0. However, it was protected by IE1 from degradation by ICP0 or HSV-1 infection. In contrast, IE1 with L174P mutation lost the function of deSUMOylating PML and failed to protect the degradation of the mono-SUMOylated PML. Whether the mono-SUMOylated PML has any defensive function against viral infection will be further investigated.

Keywords: HSV-1; SUMOylation; cytomegalovirus (CMV); herpes simplex virus (HSV); immediate-early protein 1 (IE1); infected cellular protein (ICP0); nuclear domain 10 (ND10); promyelocytic leukemia protein (PML).

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Figures

FIG 1
FIG 1
HCMV IE1 deSUMOylated hPML and protected hPML from degradation by ICP0. (A, right) U-251 MG or U-251 MG-IE1 cells were mock infected or infected with WT HSV-1 (strain 17) for 24 h at an MOI of 1. The whole-cell lysates were collected for Western blotting to detect the proteins PML, IE1, ICP0, and tubulin. (Left) U-251 MG cells were mock infected (lane 1), HSV-1 infected plus MG132 treated (lane 2), or HSV-1 infected (lane 3) for 12 h. The whole-cell lysates were used for Western blotting to detect the proteins. MW, molecular weight. (B) U-251 MG or U-251 MG-IE1 cells were infected with WT HSV-1 (strain 17) for the indicated times at an MOI of 1. The whole-cell lysates were collected for Western blotting to detect the proteins PML, IE1, ICP0, and tubulin. (C) U-251 MG or U-251 MG-IE1 cells grown on coverslips were infected with WT HSV-1 (strain 17) for 16 h, and the cells were then fixed and permeabilized for IFA to detect PML, ICP0, and DAPI. The arrows show the PML in ICP0-positive cells. Bar, 5 μm.
FIG 2
FIG 2
IFA to show that HCMV IE1 protected hPML from degradation by ICP0. (Upper) U-251 MG-IE1 cells grown on coverslips were transfected with pYFPICP0 for 24 h, and the cells were then fixed and permeabilized for IFA to detect PML, IE1, ICP0, and DAPI. The arrows show the PML in ICP0-positive cells. (Lower) The same experiments were performed in U-251 MG cells. Bar, 10 μm.
FIG 3
FIG 3
Effects of CMV infection on ICP0’s degradation of PML. (A) Human lung fibroblast cells (MRC-5) were mock infected (left) or infected with HCMV (middle) or MCMV at an MOI of 1 for 24 h. The cells then were superinfected with WT HSV-1 (strain 17) for different times, as indicated, at an MOI of 1. The whole-cell lysates were collected for Western blotting to detect the proteins PML, IE1, ICP0, and tubulin. (B) The same experiments were performed in SC1 mouse fibroblast cells.
FIG 4
FIG 4
IFA to show that HCMV IE1 cannot protect mPML, or that MCMV IE1 cannot protect hPML, from degradation by ICP0. MRC-5 cells were cotransfected with plasmids expressing YFP-ICP0 and GFP-mIE1 (MCMV IE1) (upper) or NIH 3T3 cells were cotransfected with plasmids expressing YFP-ICP0 and GFP-hPML (lower) for 24 h. IFA was performed to visualize PML in purple, ICP0 in red, GFP in green, and DAPI in blue. Bar, 10 μm.
FIG 5
FIG 5
In vitro study to determine whether HCMV IE1 protected hPML from degradation by ICP0. (A) Diagram of the mutants of HCMV IE1. (B) Purified HCMV IE1 or its mutants were incubated with HeLa cell nuclear extracts for 30 min at 37°C. Purified ICP0 was then added or not added to the reaction mix, which was incubated for another 30 min. Western blot assay was then performed to detect PML, ICP0, IE1, and lamin A.
FIG 6
FIG 6
HSV-1 replication in IE1-expressing or IE1-negative cell lines. (A and B) Growth curve assays. U-251 MG (or HELF) or U-251 MG-IE1 (or HELF-IE1) cells were infected with WT HSV-1 (strain 17) (A) or FXE strain (ICP0 RING finger helix mutation) (B) at an MOI of 0.01. The cells and supernatant samples were collected at the time points indicated. PFU assay was performed to count the viral particle numbers. Viral growth curves were determined. A Student's t test was applied to perform the comparison between two groups. Significance was set at a P value of <0.001 (*). (C and D) Western blot assays. The cells were infected with HSV-1 at an MOI of 0.01 for different times as shown. The whole-cell lysates were used for Western blot assays to examine the proteins as indicated. The ICP0 band was first compared to tubulin for normalization, and then the normalized ICP0 levels were compared between the non-IE1-expressing cells and the IE1-expressing cells. The ratios are shown under each group. Significance was set at a P value of <0.001 (*). Statistical analysis was carried out using pairwise two-tailed t test to compare the two groups (IE1-expressing cells versus non-IE1 cells) and the differences were significant, as shown by an asterisk (P < 0.001).
FIG 7
FIG 7
Model of interaction between HCMV IE1, hPML, and ICP0. HSV infection produces an immediate-early protein, ICP0, that degrades poly-SUMO-PML but not mono-SUMO-PML. HCMV immediate-early protein, hIE1, deSUMOylates hPML to generate mono-SUMO-PML, which cannot be degraded by ICP0. Therefore, hIE1 protects hPML from degradation by ICP0.
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