Fig 1.
HSV-1 γ134.5 downregulates immune genes linked to RNA sensing.
(A) Effects of γ134.5 on global host gene expression. MEF cells were mock infected, infected with wild type HSV-1 or Δγ134.5 (5 pfu/cell). At 8 h postinfection, RNAs were extracted for RNA deep sequencing. Data from triplicate samples, processed as described in the Materials and Methods, are presented as Scatterplot. X axis denotes log2 fold change in HSV-1 to mock ratios. Y axis indicates log2 fold change in Δγ134.5 to mock ratios. Dots are single genes. A few representative genes upregulated more by Δγ134.5 than by wild type HSV-1 are highlighted. (B) GSEA hallmark analysis on genome wide gene expression. Pathways enriched in gene sets upregulated by Δγ134.5 relative to wild type HSV-1 are ranked based on the normalized enrichment score (NES). False discovery rate (FDR) q value < 0.25 is defined as significantly enriched. Nominal p values are also indicated for top ranked pathways. (C) Heatmap visualization of RNA transcripts linked to the IFN response. The map shows 46 genes including IFN-stimulated genes, intracellular DNA sensors and RNA sensors. G1, G2 and G3 are distinct experimental replicates. The data represents the Log2FC (Fold Changes). (D) Effect of γ134.5 on antiviral gene expression. MEF cells infected as in (A) were subjected to quantitative PCR analysis to test the expression of Ifit1, Ccl5, Ifi204, Ddx58, Dhx58, Ddx60, and Ifih1, Mx2, Oas1b, Oas2, Oas3, and Oasl1. The results were expressed as fold activation relative to 18S ribosomal RNA, with standard deviations among triplicate samples. The data were statistically analyzed by one-way ANOVA (**, P < 0.01).
Fig 2.
The γ134.5 protein dampens antiviral responses mediated by RIG-I in MEF cells.
(A) Effects of γ134.5 on antiviral gene expression. Rig-I+/+ or Rig-I-/- MEF cells were infected with wild type HSV-1 or Δγ134.5 (5 pfu/cell). At 8 h after infection, RNA transcript levels of IFN-β, Ifit1, Ifit2, and Ccl5 were assessed by quantitative PCR analysis. The results were expressed as fold activation relative to 18S ribosomal RNA, with standard deviations among triplicate samples. The data were statistically analyzed by one-way ANOVA (**, P < 0.01). (B) Effects of γ134.5 on IRF3 phosphorylation. Rig-I+/+ or Rig-I-/- MEF cells were mock infected or infected with the indicated viruses (5 pfu/cell). At 8 h postinfection, cell lysates were processed for western blot analysis with antibodies against p-IRF3, IRF3, ICP27, γ134.5, RIG-I, and β-actin. The experimental data are representative of results from three independent experiments.
Fig 3.
HSV-1 γ134.5 reduces RIG-I dependent antiviral responses in human lung fibroblasts (HEL).
(A) Validation of RIG-I knockdown in HEL cells. Cell lysates from HELs expressing control shRNA (shCtrl) or RIG-I target shRNA (shRIG-I) were subjected to western blot analysis with anti-RIG-I and β-actin antibodies. (B) Effects of γ134.5 on antiviral gene expression in control or RIG-I knockdown HEL cells. Cells infected with wild type HSV-1 or Δγ134.5 (5 pfu/cell) for 8 h were analyzed for transcript levels of IFN-β, Ifit1, Ifit2, and Ccl5 by quantitative PCR analysis. The data were statistically analyzed by one-way ANOVA (**, P < 0.01) with SD (n = 3). (C) Effects of γ134.5 on IRF3 phosphorylation in shCtrl-transfected HEL or RIG-I knockdown HEL. Cells were infected as described in panel B and processed for Western blot analysis with antibodies against p-IRF3, IRF3, ICP27, γ134.5 and β-actin. The experimental data are representative of results from three independent experiments.
Fig 4.
The γ134.5 protein interacts with RIG-I.
(A and B) The γ134.5 protein interacts with endogenous RIG-I in infected cells. MEFs were infected with wild-type HSV-1 or Δγ134.5 (10 pfu/cell). At 8 h postinfection, the cells were processed for immunoprecipitation (IP) with anti-RIG-I antibody or normal mouse IgG (A), or with anti-γ134.5 antibody or normal rabbit IgG (B). Whole-cell lysates (WCL) and precipitated proteins were probed with antibodies against RIG-I, γ134.5, ICP27 and β-actin. (C and D) HSV-1 γ134.5 interacts with RIG-I in the absence of other viral proteins. HEK-293T cells were transfected with Myc-RIG-I together empty vector (Vec), Flag-γ134.5 or Flag-mCherry for 36 h. Cell lysates were subjected to immunoprecipitation (IP) with anti-Myc antibody (C) or anti-Flag antibody (D). Precipitated proteins and WCLs were probed with antibodies against Flag, Myc, and β-actin. The data are representative of results from three independent experiments.
Fig 5.
The γ134.5 protein does not inhibit the K63-linked ubiquitination of RIG-I.
(A) Effect of γ134.5 on ubiquitination. HEK-293T cells were transfected with plasmids encoding Myc-RIG-I and HA-Ub (K63 only) together with empty vector (Vec), Flag-mCherry, Flag-γ134.5 or pCAGGS-NS1 (positive control). At 24 h after transfection, the cells were treated with SeV (100 HA/ml) for additional 12 h and then harvested for ubiquitination analysis as described in the Materials and Methods. Whole-cell lysates (WCLs) were subjected to immunoprecipitation (IP) with anti-Myc antibody. Precipitated proteins and WCLs were probed with antibodies against HA, Flag, Myc, and β-actin. (B) Effect of different doses of γ134.5 on RIG-I ubiquitination. HEK-293T cells were transfected with increasing amounts of Flag-γ134.5 and assayed as described as in (A). (C) The γ134.5 protein does not disrupt the interaction of RIG-I and TRIM25. HEK-293T cells were transfected with plasmids encoding Myc-RIG-I and V5-TRIM25 together with empty vector (Vec) or different doses of Flag-γ134.5 for 36 h. WCLs were subjected to IP with anti-Myc antibody. Precipitated proteins and WCLs were probed with antibodies against Flag, Myc, V5, and β-actin. The experimental data are representative of results from three independent experiments.
Fig 6.
The γ134.5 protein blocks RIG-I translocation to the mitochondria.
(A) Ectopic expression of γ134.5 inhibits RIG-I mitochondrial translocation in response to SeV infection. HEK-293T cells were transfected with empty vector (Vec) or increasing amounts of Flag-γ134.5. At 24 h posttransfection, the cells were infected with SeV (100 HA/ml) for additional 24 h and harvested for cytoplasmic and mitochondrial fractionation. Samples were subjected to Western blot analysis with antibody against RIG-I, LDHA, COX IV and Flag. (B) The γ134.5 protein inhibits IFN-β promoter activation by SeV. HEK-293T cells were co-transfected with pIFN-β-luc (50 ng) and pRL-TK (10 ng) along with either vector or Flag-γ134.5 (range from 200 to 800 ng). At 24 h posttransfection, the cells were treated with SeV (100 HA/ml) for 24 h and harvested for luciferase assays. Results are expressed as means ± standard deviations (SD) (n = 3) and assessed by one-way ANOVA (**, P < 0.01). (C) Block of RIG-I mitochondrial translocation by HSV-1 requires γ134.5. MEF cells were mock-infected or infected with wild type HSV-1 or Δγ134.5 (10 pfu/cell). At 8 h post infection, whole lysates, cytoplasmic and mitochondrial fractions were collected. Samples were processed for Western blot analysis with antibody against RIG-I, LDHA, COX IV, γ134.5 and β-actin. The experimental data are representative of results from three independent experiments.
Fig 7.
The γ134.5 protein precludes formation of the RIG-I-14-3-3ε complex required for mitochondrial translocation and IRF3 activation.
(A) The γ134.5 protein prevents the interaction of RIG-I and 14-3-3ε. HEK-293T cells were transfected with Myc-RIG-I together with vector plasmid or Flag-γ134.5 or lag-Ns3-pro (DENV). At 24 h posttransfection, cells were treated with SeV (100HA/ml) for 24 h. Whole-cell lysates (WCLs) were subjected to immunoprecipitation (IP) with anti-Myc antibody. Precipitated proteins and WCLs were probed with antibodies against Flag, Myc, 14-3-3ε and β-actin. (B) The γ134.5 protein dose not interact with 14-3-3ε. HEK-293T cells were co-transfected with Myc-14-3-3ε along with vector plasmids, Flag-mCherry, Flag-γ134.5 and Flag-Ns3-pro (DENV) for 36 h. Cells were then harvested and subjected to immunoprecipitation (IP) with anti-Myc antibody. Precipitated proteins and WCLs were probed with antibodies against Flag, Myc, and β-actin. (C) Inhibition of the RIG-I-14-3-3ε complex formation by HSV-1 requires γ134.5. MEFs were infected with wild type HSV-1 or Δγ134.5 (10 pfu/cell). At 8 h postinfection, cells were processed for immunoprecipitation (IP) with anti-RIG-I antibody. Whole-cell lysates and precipitated proteins were probed with antibodies against RIG-I, 14-3-3ε, γ134.5, ICP27 and β-actin. (D) Block of RIG-I mitochondrial translocation by γ134.5 inhibits IRF3 phosphorylation. MEFs were infected with wild type HSV-1 or Δγ134.5 (10 pfu/cell). At 8h postinfection, cells were harvested for cytoplasmic and mitochondrial fractionation. Samples were processed for Western analysis with antibodies against 14-3-3ε, RIG-I, LDHA, COX IV MAVS, phosphorylated IRF3, IRF3, ICP27, γ134.5 and β-actin. The data are representative of results from three independent experiments.
Fig 8.
The γ134.5-RIG-I interaction influences HSV-1 replication.
(A) Viral replication in Rig-I+/+ or Rig-I-/- MEFs. Cells were infected with wild-type HSV-1 or the γ134.5 deletion virus (Δγ134.5) at a MOI 0.01. At 48 h postinfection, virus yields were determined on Vero cells by plaque assay. (B) Kinetics of viral growth in Rig-I+/+ or Rig-I-/- MEFs. Viral infection was performed as described for panel (A) and viral yields were measured at the indicated time points. (C) Viral replication in control and RIG-I knockdown human lung fibroblasts cells. shCtrl (control) or shRIG-I (RIG-I knockdown) HEL cells were infected with wild type HSV-1 or Δγ134.5 (0.01 pfu/cell). At 48 h postinfection, virus yields were determined by plaque assay. (D) Kinetics of viral growth in control and RIG-I knockdown cells. Viral infection was performed as described in panel (C) and viral yields were measured at the indicated time points. The data are representative of results from three experiments with triplicate samples. Differences between the selected groups were statistically assessed by one-way ANOVA (A and C) or a two-tailed Student’s t test (B and D) (**, P < 0.01).