Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain

Abstract

Viral hijacking of cellular processes relies on the ability to mimic the structure or function of cellular proteins. Many viruses encode ubiquitin ligases to facilitate infection, although the mechanisms by which they select their substrates are often unknown. The Herpes Simplex Virus type-1-encoded E3 ubiquitin ligase, ICP0, promotes infection through degradation of cellular proteins, including the DNA damage response E3 ligases RNF8 and RNF168. Here we describe a mechanism by which this viral E3 hijacks a cellular phosphorylation-based targeting strategy to degrade RNF8. By mimicking a cellular phosphosite, ICP0 binds RNF8 via the RNF8 forkhead associated (FHA) domain. Phosphorylation of ICP0 T67 by CK1 recruits RNF8 for degradation and thereby promotes viral transcription, replication, and progeny production. We demonstrate that this mechanism may constitute a broader viral strategy to target other cellular factors, highlighting the importance of this region of the ICP0 protein in countering intrinsic antiviral defenses.

Figure 1. The RNF8 FHA domain is required for targeting by ICP0

(A) Functional regions of RNF8 and fragments generated. (B) Flag-RNF8 fragments and mutants were co-transfected into HeLa cells with eGFP-ICP0ΔRING and processed for immunofluorescence 24 h post-transfection. (C) Cells expressing ICP0 and Flag-RNF8 (50 cells for each condition imaged in panel B) were scored for co-localization. (D) Flag-RNF8 or the R42A mutant were transfected into HeLa cells and infected with WT HSV-1 (17syn+) 24 h post-infection. Cells were harvested 8 h post-infection (see also Supplemental Figure S1).

Figure 2. ICP0 requirements for RNF8 interaction

(A) ICP0 was transfected into 293T cells and harvested 24h post-transfection. Cells were lysed in GST lysis buffer and, where indicated, lysates were treated with CIP or CIP plus sodium Na₃VO₄, and then incubated with GST-RNF8-FHA or GST-RNF8-FHA-R42A and precipitated using glutathione-Sepharose beads. (B) Top panel, functional domains of ICP0 and location of the RNF8 consensus motif. Bottom panel, eGFP-ICP0 or the T67A mutant were transfected into 293T cells, harvested 24h post-transfection, and lysates were used in GST pulldowns with GST-RNF8-FHA or GST-RNF8-FHA-R42A. (C) eGFP-ICP0 WT or T67A were co-transfected with Flag-RNF8 into HeLa cells, fixed 24 h post-transfection, and localization assessed by immunofluorescence. (D) eGFP-ICP0 or the T67A mutant was co-transfected into HeLa cells with eGFP-ICP0 in 3× excess over Flag-RNF8. Cells were harvested 24 h post-transfection. (E) eGFP-ICP0 or the T67A mutant were co-transfected into HeLa cells with eGFP-ICP0 in 3× excess over HA-RNF168 and analyzed by immunoblotting. (F) HeLa cells were co-transfected with RNF8 and/or RNF168 in excess of ICP0, subject to 10 Gy IR 24 h post-transfection, and fixed and processed for immunofluorescence 1 h post-IR. (G) Cells expressing ICP0 (30 cells for each condition imaged in (F) were scored for 53BP1 co-localization with γH2AX. Cells in which 53BP1 was localized to non-IRIF foci were excluded from this analysis. In a parallel analysis, it was confirmed that cells expressing ICP0 or mutants thereof had also been transfected with RNF8 and/or RNF168 (see also Supplemental Figures S2, S3, and S4).

Figure 3. Phosphorylation of ICP0 at T67

(A) The RNF8 FHA domain or R42A mutant were purified from E. coli and peptides comprising the region around ICP0 T67 were synthesized. Binding of the peptides to the FHA domains was measured using isothermal calorimetry. 1.2mM peptides were titrated into solutions containing 100mM RNF8 FHA domain. pT67 peptide bound the RNF8 domain. (B) Nonphosphorylated peptide did not bind the RNF8 FHA domain. (C) R42A mutation in the FHA domain prevented binding to pT67 peptide. (D) Antibodies were raised in rabbits against a synthetic peptide containing ICP0 pT67 and affinity purified. Peptide (1 μg) was spotted onto nitrocellulose and analyzed by immunoblotting using sera purified over nonphosphorylated peptide (α-T67 total) or double purified over nonphosphorylated and pT67 peptide (α-pT67). (E) eGFP-ICP0 WT and T67A were transfected into HeLa cells and processed for immunofluorescence using α-pT67 antibodies. (F) eGFP-ICP0 WT or T67A were transfected into HeLa cells and lysates were prepared. Before analysis, one lysate expressing WT eGFP-ICP0 was subjected to dephosphorylation by l phosphatase for 30 min at 37°C (see also Supplemental Figure S5).

Figure 4. Mechanism of ICP0 T67 phosphorylation

(A) Recombinant GST-ICP0-241 or the T67A mutant were purified from E. coli, phosphorylated in vitro by recombinant CK1δ̣ or CK2α̃β, and analyzed by SDS-PAGE and immunoblotting. (B) GST-ICP0-241 or the indicated mutants were phosphorylated in vitro by recombinant rat CK1d and used to precipitate Flag-RNF8 from lysates via glutathione-Sepharose. (C) The region of ICP0 containing T67 (red, bold) and the upstream serines (red) is indicated. Recombinant GST-241 or the indicated mutants were phosphorylated in vitro by CK1. Reactions were quenched at 5 min intervals between 0 and 20 min, and phosphorylation of T67 was measured by reactivity to α-pT67 antibodies. (D) Plasmids expressing the ICP0-nls241, the S64A, or T67A mutants were transfected into HeLa cells and analyzed by SDS-PAGE and immunoblotting with α-pT67 antibodies. (E) Plasmids expressing the indicated ICP0 sequences were transfected into 293T cells, lysed in GST lysis buffer and used in GST pulldown assays with recombinant GST-RNF8-FHA or the R42A mutant. (F) Plasmids expressing the indicated ICP0 proteins were co-transfected into HeLa cells with Flag-RNF8 and localization was assessed by immunofluorescence. (G) Cells expressing ICP0 and Flag-RNF8 proteins (50 cells for each condition represented in F) were counted and scored for the ability of RNF8 to localize with ICP0. Cells in which any ICP0 foci co-localized with RNF8 were scored as positive for RNF8/ICP0 co-localization (see also Supplemental Figure S6).

Figure 5. Effect of the T67A mutation on virus infection

(A) WT, ΔICP0, or T67A mutants of HSV-1 (strain KOS) were used to infect HeLa cells at MOI=3 and harvested at the indicated time points. (B) The indicated viruses were infected at MOI=0.01 in HepaRG cells expressing a scrambled shRNA (shNeg) or shRNA targeting RNF8 (shRNF8) and harvested at the indicated time points. Transcripts were quantified via qPCR with primers targeted to the immediate-early ICP27 gene and are represented as the relative transcript level at each time point. Error bars represent standard deviation of triplicate samples. (C) 25% of the cell pellets from the samples in (B) was used to isolate genomic DNA. ICP27 gene copies, indicative of the relative number of viral genomes, were quantified and analyzed by qPCR as described in (B). Error bars represent standard deviation of triplicate samples (see also Supplemental Figure S7).

Figure 6. ICP0 pT67 can bind other cellular proteins

(A) Biotinylated peptides were synthesized that were nonphosphorylated or phosphorylated at T67, S64, or S64/T67. (B) Peptides were used to precipitate cellular proteins from 293T lysates using Streptavidin beads and analyzed by silver staining after SDS-PAGE. (C) Co-purifying FHA domain proteins were identified by mass spectrometry. FHA domain proteins purifying uniquely in the presence of pS64/pT67 peptide are listed, with the corresponding number of peptides identified. (D) Peptide pulldowns were analyzed by immunoblotting using antibodies to proteins identified by mass spectrometry in (C). (E) TAP-ICP0 or GFP-ICP0 was co-transfected with HA-Chk2 into 293T cells and TAP-ICP0 was precipitated using streptavidin-Sepharose. (F) 293T cells were co-transfected with HA-Chk2 and eGFP-ICP0 WT or the T67A mutant, and eGFP-ICP0 proteins were immunoprecipitated from the resulting lysates using α-GFP antibodies and protein A/G-agarose beads. (G) ICP0 pT67 peptide (0.35 mM) was titrated into a solution containing purified Chk2 FHA (27 μM) and binding affinity measured by ITC. The peptide bound with comparable affinity to RNF8 FHA domain. (H) The unphosphorylated peptide did not bind. (I) The F70A mutation did not significantly affect the binding to Chk2 FHA (see also Supplemental Table S1).

Figure 7. ICP0 mimicry of cellular phosphorylation marks targets cellular FHA domain proteins, promoting viral infection

Parallels between RNF8-Mdc1 interaction during the DNA damage response and RNF8-ICP0 interaction during HSV-1 infection are shown. The motifs on ICP0 and Mdc1 use distinct mechanisms to catalyze phosphorylation, but both employ the induced phosphosite to bind RNF8. Also depicted are consequences for the virus and the possibility that ICP0 (or other viral proteins) can use this mimicking approach to target other cellular proteins.