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Comparative proteomics identifies Schlafen 5 (SLFN5) as a herpes simplex virus restriction factor that suppresses viral transcription

Abstract

Intrinsic antiviral host factors confer cellular defence by limiting virus replication and are often counteracted by viral countermeasures. We reasoned that host factors that inhibit viral gene expression could be identified by determining proteins bound to viral DNA (vDNA) in the absence of key viral antagonists. Herpes simplex virus 1 (HSV-1) expresses E3 ubiquitin-protein ligase ICP0 (ICP0), which functions as an E3 ubiquitin ligase required to promote infection. Cellular substrates of ICP0 have been discovered as host barriers to infection but the mechanisms for inhibition of viral gene expression are not fully understood. To identify restriction factors antagonized by ICP0, we compared proteomes associated with vDNA during HSV-1 infection with wild-type virus and a mutant lacking functional ICP0 (ΔICP0). We identified the cellular protein Schlafen family member 5 (SLFN5) as an ICP0 target that binds vDNA during HSV-1 ΔICP0 infection. We demonstrated that ICP0 mediates ubiquitination of SLFN5, which leads to its proteasomal degradation. In the absence of ICP0, SLFN5 binds vDNA to repress HSV-1 transcription by limiting accessibility of RNA polymerase II to viral promoters. These results highlight how comparative proteomics of proteins associated with viral genomes can identify host restriction factors and reveal that viral countermeasures can overcome SLFN antiviral activity.

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Fig. 1: ICP0 targets SLFN5 for degradation.
Fig. 2: ICP0 interacts with SLFN5.
Fig. 3: SLFN5 colocalizes with HSV-1 DNA.
Fig. 4: ICP0 counteracts SLFN5-mediated suppression of HSV-1 replication.
Fig. 5: SLFN5 associates with vDNA to suppress HSV-1 gene transcription.

Data availability

The MS proteomics data have been deposited with the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE75 partner repository with the dataset no. PXD018773. Source data are provided with this paper.

Code availability

The scripts used to analyse the iPOND proteomics data are available from the corresponding author upon reasonable request or can be accessed via GitHub (https://github.com/JosephDybas/HSV_iPOND).

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Acknowledgements

We thank members of the Weitzman laboratory for insightful discussions and input. We thank G. H. Cohen, R. D. Everett, D. M. Knipe, S. L. Sawyer and P. A. Schaffer for generous gifts of reagents. C.B. was supported by a Medical Research Council grant no. MC_UU_12014/5. Work in the Weitzman laboratory was supported in part by grants from the National Institutes of Health (grant nos. AI115104 and NS082240 to M.D.W.) and funds from the Children’s Hospital of Philadelphia. A.M.P. was supported by the National Cancer Institute T32 Training Grant in Tumor Virology no. T32-CA115299 and Individual National Research Service Award no. F32-AI138432. J.M.D. was supported by the Individual National Research Service Award no. F32-AI147587.

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E.T.K., J.M.D., E.D.R. and M.D.W. conceived and designed the study. E.D.R., K.K. and B.A.G. performed the iPOND MS and subsequent analysis. J.M.D. provided the computational analysis. E.T.K., E.D.R., A.M.P., A.O. and C.B. performed the cell imaging experiments. A.M.P. performed the RNA stability assays. E.T.K. and L.N.A. performed the virological, biochemical and molecular biology experiments. E.T.K. and M.D.W. wrote the manuscript with input from all authors.

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Correspondence to Matthew D. Weitzman.

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Extended data

Extended Data Fig. 1 Comparative analysis of protein abundance between PCA-based clustering of HSV iPOND data and whole cell proteome data identifies potential substrates of ICP0.

a, Venn diagram of PML, ATRX, IFI16, and DNA-PKcs PCA-based clusters identifies proteins that are clustered with each known substrate. b, Volcano plots showing log2 fold change (x-axis) and associated statistical significance (y-axis) for proteins identified by iPOND proteomics, quantifying proteins associated with HSV genomes. Known ICP0 substrates (green) and clustered proteins (blue and red) are indicated. iPOND data comparing HSV WT and ΔICP0 infection shows known substrates and clustered proteins enriched on the ΔICP0 genome. P-values were calculated using the two-tailed unpaired Student’s t-test. n = 3 biologically independent experiments. c, Heatmap showing iPOND abundances as z-scores for mock, WT HSV-1, and ΔICP0 during infection. This list is proteins that are more than 2-fold enriched (dashed gray box in b) on the ΔICP0 virus genome compared to WT HSV-1 genome. These are clustered using a hierarchical clustering algorithm that analyzes the abundance for each condition. Therefore, the proteins with the same trends for mock-HSV-ΔICP0 are close together on the heatmap. d, Whole cell proteome abundance data over a time course of WT HSV-1 infection show decreases in known ICP0 substrates PML, IFI16, and DNA-PKcs, as well as SLFN5. Additional proteins (gray line) showing similar trends in the heat map (c) are not decreased during HSV infection.

Extended Data Fig. 2 Comparison of SLFN5 degradation in different cells.

a, HEK293T cells were transfected with plasmids encoding SLFN5-V5 and GFP-ICP0 (WT or ΔRING), as indicated. At 24 h after transfection, total cell lysates were prepared, and immunoblot analysis was carried out with indicated antibodies. GAPDH was used as a loading control. b, HEK293T cells were transfected with plasmids encoding GFP-SLFN5 and ICP0 (WT or ΔRING), and the cells were monitored for the GFP signals and the fluorescence intensity was measured by ImageJ software. Scale bar, 300 µm. c, HEK293 cells were infected with WT HSV-1 or ΔRING virus at an MOI of 3. Cell lysates were prepared at the indicated time points and subjected to immunoblot analysis. d, HeLa cells were infected with WT HSV-1 or ΔRING virus at an MOI of 3. Cell lysates were prepared at the indicated time points and subjected to immunoblot analysis. e, HeLa cells were transduced with recombinant adenoviral vectors encoding ICP0 together with Ad.C-rtTA, an adenovirus expressing the doxycycline regulated transactivator. At 24 h after transduction in the absence or presence of doxycycline (1 µg/ml), immunoblot analysis was performed with indicated antibodies. f, g, Similar experimental setup as in (d and e); U2OS cells were used. h, SLFN5 degradation is independent of SUMOylation, and is detected in cells depleted of the SUMO E2 conjugating enzyme UBC9. i, In vitro ubiquitination assay with bacterially purified SLFN5 Δ730-891 truncation mutant which lacks the ICP0 interaction region demonstrates it is not ubiquitinated. Immunoblot data shows representative data from n = 3 biologically independent experiments. Source data

Extended Data Fig. 3 ICP0 interacts SLFN5 via the carboxy terminal region of SLFN5.

a, Subcellular localization of SLFN5 mutants was analyzed. HEK293T cells were transfected with plasmids encoding GFP-SLFN5 (full-length or various deletion mutants). At 24 hours after transfection, the cells monitored for the GFP signals. Scale bar, 50 µm. b, HEK293T cells were co-transfected with plasmids encoding various version of GFP-SLFN5 and ICP0 ΔRING or cytoplasmic ICP0 mutant (cICP0, also called D8) ΔRING, as indicated. The cells were subject to co-IP with anti-GFP Ab, followed by immunoblotting. Immunoblot and immunofluorescence images show representative data from n = 3 biologically independent experiments. c, SLFN5 interaction with ICP0 maps to a part of the intrinsicly disordered region of SLFN5. The SLFN5 and SLFN11 protein sequences were analyzed for disorder tendency using IUPred2A. The domain structure of SLFN is shown above. The green bar indicates an interaction region of ICP0 or a homolgous region of SLFN11 with SLFN5. Red lines indicate unique disorder region of SLFN5. Source data

Extended Data Fig. 4 Colocalization of SLFN5 with replication compartments of HSV-1.

a, HFF cells were infected with WT HSV-1 at an MOI of 10 and immunofluorescence was examined at 2 hpi. ICP4 marks viral pre-replication foci from incoming viral genomes. b, HFF cells were infected with WT HSV or ΔICP0 mutant virus at an MOI of 3 and immunofluorescence was examined at 8 hpi. ICP8 marks viral replication compartments and DAPI marks cellular DNA. Scale bar, 10 µm. Fluorescence plot profile analyzed by ImageJ showed colocalization of SLFN5 and viral proteins (at dashed line). Immunofluorescence images show representative data from n = 3 biologically independent experiments.

Extended Data Fig. 5 SLFN5 suppresses HSV-1 replication.

a, Efficiency of SLFN5 knockdown in HFF and HeLa cells. Immunoblot analysis indicating SLFN5 and β-actin levels following transduction with lentiviruses encoding shRNAs that are nontargeting control (Ctrl) or specific for SLFN5 (#1 to #5). b, Viral DNA replication yields from shRNA-transduced HFF cells infected with ΔRING virus at an MOI of 0.1 and harvested at 24 h after infection. Quantification of viral DNA was carried out by qPCR. n = 5 biologically independent experiments. c, HeLa cells transduced with shRNA were mock infected or infected with HSV-1 (WT or ΔRING) at different MOIs (0.5 to 3) as indicated. Cell lysates were prepared at 9 h after infection and immunoblot nalysis was performed with antibodies to ICP0, VP21, and β-actin. d, Phase micrograph of cell morphology under subconfluent or confluent culture conditions. Scale bar, 100 µm. e, Growth of shRNA transduced cells over time. n = 3 biologically independent experiments. f, 2,000 cells were seeded in a 96-well plate, and cell proliferation was measured over 6 days by colorimetric analysis using the water-soluble tetrazolium salt, WST-8 (Cell Counting Kit-8, Dojindo) at the indicated time point. n = 3 biologically independent experiments. g, CRISPR/Cas9-mediated SLFN5 gene editing and permanent depletion from HeLa cells. Guide RNA transfected cells were selected and cloned for a month. Immunoblot analysis of SLFN5 in control cells or SLFN5 knockout cells. Viral DNA replication yields from cells infected with ΔRING at an MOI of 0.1 were analyzed by qPCR. HeLa cells were infected with ΔRING at an MOI of 0.1 for 24 h. Viral DNA was measured by qPCR. n = 3 biologically independent experiments. Data are the mean ± SD. Comparisons between groups were performed using the two-tailed unpaired Student’s t-test. **, p < 0.005, ***, p < 0.0005, n.s., not significant. Immunoblots show representative data from n = 3 biologically independent experiments. Source data

Extended Data Fig. 6 SLFN5 Δ730-891 mutant does not inhibit HSV-1 replication.

a, HeLa cells were stably transduced with lentivirus containing tetracycline-inducible SLFN5-HA genes (WT and Δ730-891). SLFN5-HA was induced with doxycycline (0.5 μg/ml) for 48 h. SLFN5 expression was confirmed by immunoblot analysis. b, c, Cells were infected with HSV-1 (b) and ΔRING virus (c) at an MOI of 0.1. At 24 hpi, viral DNA was measured by qPCR. n = 3 biologically independent experiments. Data are the mean ± SD. Comparisons between groups were performed using the two-tailed unpaired Student’s t-test. **, p < 0.005. n.s., not significant. Source data

Extended Data Fig. 7 Degradation of SLFN5 by other DNA viruses.

a, HFF cells were mock infected or infected with HSV-2 at an MOI of 3, and MG132 (5 µM) was added at 2 h after virus inoculation as indicated. Total cell lysates were prepared at indicated time points and examined by immunoblot analysis with indicated antibodies. ICP8 serves as an infection control. b, HFF cells were mock infected or infected with HCMV (TB40/E) at an MOI of 3 and total cell lysates were prepared at indicated time points and examined by immunoblot analysis with indicated antibodies. UL44 serves as an infection control. Circle marks cross-reacting viral protein. c, A549 cells were mock infected or infected with adenovirus (Ad5) at an MOI of 20 and MG132 (5 µM) was added at 2 h after virus inoculation, as indicated. Cell lysates were prepared at the indicated time points and examined by immunoblot analysis with indicated antibodies. RAD50 served as a degradation control and DBP as a control for infection. Immunoblots show representative data from n = 3 biologically independent experiments. Source data

Extended Data Fig. 8 Restriction of other DNA viruses by SLFN5.

HeLa cells were mock infected or infected with other DNA viruses as indicated. a, Immunoblot analysis demonstrating increased HSV-2 protein levels in SLFN5-depleted cells. b, HCMV proteins are slightly increased in SLFN5-depleted cells at immediate-early time point (24 hpi), but decreased at late times (72 hpi) as compared to control cells. The ISG15 protein was increased in the absence of SLFN5 and further increased upon HCMV infection. c, SLFN5 does not affect adenovirus protein levels. d, SLFN5 knockdown results in higher level of ISG15 expression in HFF and HeLa cells. Immunoblots show representative data from n = 3 biologically independent experiments. Source data

Extended Data Fig. 9 Effects of SLFN5 on HCMV infection.

a, b, HCMV mRNA (UL123, an immediate-early gene and UL94, a late gene) expression from shRNA-transduced HFF cells infected with HCMV at an MOI of 3 and harvested at 12 h (a) and 72 h (b) after infection. Quantification of viral transcripts was carried out by RT-qPCR. Viral DNA replication yields from shRNA-transduced HFF cells infected with ΔRING virus at an MOI of 0.1 and harvested at 24 h after infection. Quantification of viral DNA was carried out by qPCR. c, Progeny production of HCMV was monitored by infectious center assays using anti-IE1 antibody. n = 3 biologically independent experiments. Data are the mean ± SD. Comparisons between groups were performed using the two-tailed unpaired Student’s t-test. *, p < 0.05, **, p < 0.005. Source data

Extended Data Fig. 10 Interaction of SLFN5 with HSV-1 DNA.

a. ChIP assay with anti-HA antibody was performed in SLFN5-HA-expressed HeLa cells. ΔRING virus was infected at an MOI of 3 for indicated time points in the absence or presence of PAA. ChIP DNA was assayed by qPCR using primers specific for US11 TSS. n = 3 biologically independent experiments. b. ΔRING virus was infected at an MOI of 3 for 3 h. ChIP DNA was assayed by qPCR using primers specific for the HSV-1 DNA or human DNA loci. n = 3 biologically independent experiments. Data are the mean ± SD. Comparisons between groups were performed using the two-tailed unpaired Student’s t-test. *, p < 0.05, **, p < 0.005. Source data

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Kim, E.T., Dybas, J.M., Kulej, K. et al. Comparative proteomics identifies Schlafen 5 (SLFN5) as a herpes simplex virus restriction factor that suppresses viral transcription. Nat Microbiol 6, 234–245 (2021). https://doi.org/10.1038/s41564-020-00826-3

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