Identification of a herpes simplex virus 1 gene encoding neurovirulence factor by chemical proteomics

Figures

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  Fig. 1. Identification of previously unidentified HSV-1 CDSs by B chemical proteomics. a Diagram illustrating the procedure of AHA labeling experiments for BONCAT-based MS-analyses (n = 2). In each of the two experiments, MS-analyses were performed twice. b Ion-intensity of viral polypeptides relative to that of viral and host polypeptides. Each value is the mean ± SEM of four MS-analyses. c Total numbers of peptides detected in HSV-1-infected cells at 4 h and 12 h post-infection and mapped to known or previously unidentified HSV-1 CDSs in the experiments shown in Fig. 1a and Supplementary Fig. 1a. The number is shown above the bars. d Venn diagrams showing the results of two independent AHA-labeling experiments by 4 h post-infection using wild-type HSV-1(F)-infected cells as described in Fig. 1a. The numbers indicate the number of peptides derived from HSV-1 CDSs. e Venn diagrams showing the results of five independent AHA-labeling experiments by 12 h post-infection using wild-type HSV-1(F) infected cells as described in Fig. 1a (n = 2) and Supplementary Fig. 1a (n = 3). The numbers indicate the number of peptides derived from HSV-1 CDSs. f Schematic diagram of the genome of wild-type…

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  Fig. 2. Expression of piUL49 in HSV-1-infected cells. a Schematic diagram of the genome of wild-type HSV-1 (line 1) and the locations of the UL49 (VP22) and iUL49 (piUL49) CDSs (line 1). The domain(s) of iUL49 detected by MS-analyses (line 3) or used to generate antibodies to piUL49 (line 4). The nucleotide and amino-acid sequences of VP22 (line 5) and piUL49 (line 6) are indicated. b, c Vero cells mock-infected or infected with Venus-iUL49 (b), Venus-iUL49ΔM (b), MEF-iUL49 (c), or MEF-iUL49ΔM (c) for 12 h at an MOI of 10 were analyzed by immunoblotting with antibodies to GFP (b), VP23 (b), Flag (c), gB (c), or β actin (b, c). d Vero cells mock-infected or infected with wild-type HSV-1(F), iUL49ΔM, iUL49ΔM-rep, VP22ΔM, or VP22ΔM-rep for 12 h at an MOI of 10 were analyzed by immunoblotting with antibodies to piUL49, VP22, ICP8, gB, or β actin. e, f Vero cells mock-infected or infected with wild-type HSV-1(F) (e, f), HSV-1(17) (e), HSV-1(KOS) (e) or HSV-1(VR3) (e), ΔVP22/ΔiUL49 (f), or ΔVP22/ΔiUL49-rep (f) for 12 h at an MOI of 10 were analyzed by immunoblotting with antibodies to piUL49, VP22, or β actin. Digital images are representative of three independent experiments (b–f).

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  Fig. 3. piUL49 is an HSV-1 virulence factor. a–f Vero cells were infected with wild-type HSV-1(F) (a–f), iUL49ΔM (a, d), iUL49ΔM-rep (a, d), VP22ΔM (b, c, e, f), VP22ΔM-rep (b, c, e, f), ΔVP22/ΔiUL49 (c, f), or ΔVP22/ΔiUL49-rep (c, f) at an MOI of 10 (a–c) or 0.01 (d–f). Total virus titers in cell culture supernatants and infected cells at 21 (a–c) or 48 h (d–f) post-infection were assayed. Each value is the mean ± SEM of 4 (a–c) or 5 (b–f) independent experiments. The indicated P-values were obtained using one-way ANOVA followed by Tukey’s test. n.s. not significant. g, h Thirty plaque diameters (g) and representative images (h) of Vero cells infected with each of the indicated viruses were measured at 48 h post-infection. Each value is the mean ± SEM for each group. The indicated P-values were obtained using one-way ANOVA followed by Tukey’s test. n.s. not significant. Scale bar, 200 μm. i–k Three-week-old female ICR mice were infected intracranially with serial 10-fold dilutions of iUL49ΔM-rep (i), iUL49ΔM (i), VP22ΔM-rep (j), VP22ΔM (j), ΔVP22/ΔiUL49-rep (k), or ΔVP22/ΔiUL49 (k) in groups of six mice per dilution and monitored for 14 days. LD50 values were determined by the…

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  Fig. 4. piUL49 interacts with vdUTPase and functions as an activator of the viral enzyme. a Confocal microscope images of Vero cells infected with wild-type HSV-1(F) or MEF-iUL49 for 12 h at an MOI of 10 and stained with Hoechst33342 or antibodies to Flag and vdUTPase. Scale bar, 10 μm. b Vero cells were infected with HSV-1(F) or SE-vdUTPase for 12 h at an MOI of 10, lysed, precipitated with StrepTactin-sepharose and analyzed by immunoblotting with antibodies to iUL49, vdUTPase and UL12. c vdUTPase activity of 0.3 μM purified 6xHis-SUMO-vdUTPase or 6xHis-SUMO-vdUTPaseD97A with 74.07 μM dUTP in the absence or presence of 0.3 μΜ of purified SE-piUL49 or SE-piUL49R83L. The release of PPi upon dUTP hydrolysis by 6xHis-SUMO-vdUTPase was followed by measuring luciferase signaling and relative luminescence units (RLUs) were normalized by subtracting the value of vdUTPaseD97A from that of vdUTPase. Each value is the mean ± SEM of five independent experiments. The indicated P-values were obtained using one-way ANOVA followed by Tukey’s test. n.s. not significant. d–g Kinetic analyses of vdUTPase activity using purified 6xHis-SUMO-vdUTPase and 6xHis-SUMO-vdUTPaseD97A in the absence or presence of…

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  Fig. 5. piUL49 is required to compensates for low cellular dUTPase activity. a, b Viral titers of sh-Luc-HEp-2 or sh-hDUT-HEp-2 were infected with wild-type HSV-1(F), iUL49ΔM, iUL49ΔM-rep or iUL49ΔM/F-hDUTn for 36 h at an MOI of 10 (a) or for 60 h at an MOI of 0.01 (b), respectively, and assayed. Each value is the mean ± SEM of four independent experiments. The indicated P-values were obtained using one-way ANOVA followed by Tukey’s test. n.s. not significant. c, d Viral titers in the brains (c) (n = 16) or LD50 values (d) of 3-week-old female ICR mice intracranially infected with iUL49ΔM-rep, iUL49ΔM, or iUL49ΔM/F-hDUTn were determined as described in Fig. 3i–l. Each value is the mean ± SEM for each group. The indicated P-values were obtained using one-way ANOVA followed by Tukey’s test. n.s. not significant. Dashed line indicates the limit of detection. e dUTPase activities (dpm/μg of total protein) in eyes, TGs and brains of nine 4-week-old female ICR mice. Each value is the mean ± SEM of four independent experiments. The indicated P-values were obtained using one-way ANOVA followed by Tukey’s test. n.s. not significant. f–h Four-week-old female ICR mice were infected ocularly with 3…