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Viperin catalyzes methionine oxidation to promote protein expression and function of helicases

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Science Advances  28 Aug 2019:
Vol. 5, no. 8, eaax1031
DOI: 10.1126/sciadv.aax1031
  • Fig. 1 KSHV helicase and cellular DNA helicase MCM7 are evolutionarily conserved and interact with viperin.

    (A) Cladogram demonstrating the conservation between KSHV helicase and other helicases of the taxa in the evolutionary tree. Data represent a small portion of phylogenetic analysis of helicases and helicase-associated proteins covering human, mice, yeast, zebrafish, and herpesviruses. KSHV helicase is boxed in red. (B) Cladogram illustrating the evolutionary relationships between 13 human helicases and KSHV helicase amino acid sequences. The tree is drawn to scale, and its branch lengths represent evolutionary distances. KSHV helicase is boxed in red. (C) Venn diagram describing the overlap of all proteins identified via mass spectrometry in three different immunoprecipitation groups. (D) Reciprocal coimmunoprecipitation (co-IP) assays to examine physical interactions between viperin and KSHV helicase in 293T cells. HA, hemagglutinin. (E) In vivo co-IP assays to detect the interaction between KSHV helicase and viperin. RGB-FLAG cells were induced with doxycycline (2 μg/ml) for 72 hours. Whole-cell lysates (WCLs) were precipitated with anti-FLAG (KSHV helicase). Precipitated proteins and WCLs were analyzed by immunoblotting. IgG, immunoglobulin G. (F) Glutathione S-transferase (GST) pulldown, with GST or GST-viperin purified from Escherichia coli and the KSHV helicase–FLAG translated in vitro, was analyzed by immunoblotting.

  • Fig. 2 Viperin promotes methionine oxidation of KSHV helicase.

    (A) Mass spectrometry analysis of methionine oxidation in 293T cells transfected with KSHV helicase with or without viperin expression. The M401 was oxidized (in red) under viperin overexpression. m/z, mass/charge ratio. (B) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were harvested and analyzed by immunoblotting. eGFP, enhanced green fluorescent protein. (C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, RNA was extracted, and complementary DNA (cDNA) was prepared to determine KSHV helicase mRNA by qPCR analysis. The data are expressed as means ± SEM; n = 3; ns, not significant; CON, control. (D) RGB-FLAG cells were transfected with siRNA as indicated. At 6 hours after transfection, cells were induced with doxycycline (2 μg/ml) for 72 hours. WCLs were then analyzed by immunoblotting. (E) 293T cells were transfected with plasmids containing indicated genes. At 24 hours after transfection, cells were treated with cycloheximide (CHX) (10 μg/ml). WCLs were then analyzed by immunoblotting. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 3 Viperin-induced methionine-401 oxidation enhances the stability and function of KSHV helicase.

    (A) Diagram illustrating five peptides of KSHV helicase examined to evaluate methionine oxidation (top) and depicting the 11 KSHV helicase mutants (bottom). WT, wild type. (B and C) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (D and E) RGB-FLAG and RGB-mutated FLAG cells were induced with doxycycline for 72 hours. WCLs were then analyzed by immunoblotting. (F and G) RGB-FLAG and RGB-mutated FLAG cells were transfected with siRNA as indicated. At 6 hours after transfection, cells were induced with doxycycline for 72 hours. WCLs were then analyzed by immunoblotting. (H) RGB-FLAG and RGB-mutated FLAG cells were induced with doxycycline for 72 hours. Extracellular viral DNA was extracted and analyzed by qPCR. (I) RGB-FLAG and RGB-mutated FLAG cells were transfected with siRNA as indicated. At 6 hours after transfection, cells were induced with doxycycline for 72 hours. Extracellular viral DNA was extracted and analyzed by qPCR. The concentration of doxycycline is 2 μg/ml. For (H) and (I), the data are expressed as means ± SEM; n = 3.

  • Fig. 4 Viperin associates with lipid droplets that are required to induce the methionine oxidation of KSHV helicase.

    (A) In vivo immunofluorescence assay to detect KSHV helicase and viperin colocalization with lipid droplets in iSLK-BAC16 KSHV helicase-FLAG (GFP deletion) cells. Cells were induced with doxycycline for 72 hours and immunostained with a mouse anti-FLAG antibody against KSHV helicase (pink) and a rabbit anti-viperin antibody (red). Lipid droplets (green) and nuclei (blue) were labeled with BODIPY and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Cells were analyzed by Leica confocal microscopy and representative images were shown. The magnified panel was derived from the red box in the merged panel. Scale bars, 10 μm (merged panel) and 5 μm (magnified panel). (B and C) RGB-FLAG cells were treated with 1 μΜ T.C or dimethyl sulfoxide (DMSO) as a negative control for 3 hours, and then cells were induced with doxycycline for 72 hours. (B) WCLs were analyzed by immunoblotting; (C) viral DNA was extracted and analyzed by qPCR. (D to F) RGB-FLAG and RGB-mutated FLAG cells were treated with 1 μΜ T.C or DMSO as a negative control for 3 hours, and then cells were induced with doxycycline for 72 hours. (D and E) WCLs were analyzed by immunoblotting; (F) extracellular viral DNA was extracted and analyzed by qPCR. (G) 293T cells were treated with 1 μΜ T.C or DMSO as a negative control for 3 hours, and then cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. The concentration of doxycycline is 2 μg/ml. For (C) and (F), the data are expressed as means ± SEM; n = 3; ****P < 0.0001.

  • Fig. 5 Viperin-induced methionine oxidation stabilizes MCM7 and promotes DNA replication.

    (A) Reciprocal co-IP assays to examine physical interactions between viperin and MCM7. (B) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (C) 293T cells were transfected with plasmids containing indicated genes. At 24 hours after transfection, cells were treated with CHX (10 μg/ml). WCLs were then analyzed by immunoblotting. (D) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (E) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, cells were harvested, cross-linked, and used for ChIP assay to determine relative binding of MCM7 wild type and MCM7-TA at cellular c-Myc replication origin. (F) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, cells were labeled with EdU (5-ethynyl-2′-deoxyuridine), total DNA was extracted, and Click-iT procedure was used to isolate DNA from the active sites of replication. DNA was then analyzed by qPCR at cellular c-Myc replication origin. (G) 293T cells were transfected with plasmids containing indicated genes after transfection for 6 hours with siRNA, as indicated. At 48 hours after transfection, cells were harvested, cross-linked, and used for ChIP assay to determine relative binding of MCM7 wild type and MCM7-TA at cellular c-Myc replication origin. (H) 293T cells were transfected with plasmids containing indicated genes after transfection for 6 hours with siRNA, as indicated. At 48 hours after transfection, cells were labeled with EdU, total DNA was extracted, and Click-iT procedure was used to isolate DNA from the active sites of replication. DNA was then analyzed by qPCR at cellular c-Myc replication origin. (I) In vivo co-IP assays to detect the interaction between MCM7 and viperin. The WCLs of 293T were precipitated with anti-viperin. Precipitated proteins and WCLs were analyzed by immunoblotting. (J) 293T cells were transfected with siRNA, as indicated. At 48 hours after transfection, WCLs were then analyzed by immunoblotting. (K) 293T cells were transfected with siRNA, as indicated. At 48 hours after transfection, cells were harvested, cross-linked, and used for ChIP assay to determine relative binding of endogenous MCM7 at cellular c-Myc replication origin. (L) 293T cells were transfected with siRNA as indicated. At 48 hours after transfection, cells were labeled with EdU, total DNA was extracted, and Click-iT procedure was used to isolate DNA from the active sites of replication. DNA was then analyzed by qPCR at cellular c-Myc replication origin. For (E) to (H), (K), and (L), the data are expressed as means ± SEM; n = 3; **P < 0.01, ***P < 0.001, and ****P < 0.0001.

  • Fig. 6 Methionine oxidation catalyzed by viperin increases the stability and function of RNA helicase RIG-I.

    (A) Reciprocal co-IP assays to examine physical interactions between viperin and RIG-I. (B) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (C) 293T cells were transfected with plasmids containing indicated genes. At 24 hours after transfection, cells were treated with CHX (10 μg/ml). WCLs were then analyzed by immunoblotting. (D) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. (E) 293T cells were transfected with plasmids containing indicated genes. At 24 hours after transfection, cells were transfected with poly(I:C) for 16 hours. RNA was extracted, and cDNA was prepared to determine CXCL10 and IFN-β mRNA expression by qPCR analysis. (F) Reconstituted MEF cells were infected with VSV [multiplicity of infection (MOI), 0.1], and viral titer in the supernatant was determined by plaque assay. pfu, plaque-forming units. (G) 293T cells were transfected with plasmids containing indicated genes after transfection for 6 hours with siRNA, as indicated. At 24 hours after transfection, cells were transfected with poly(I:C) for 16 hours. RNA was extracted, and cDNA was prepared to determine CXCL10 and IFN-β mRNA expression by qPCR analysis. (H) Reconstituted MEF cells were infected with VSV (MOI, 0.1) after transfection for 24 hours with siRNA, as indicated, and viral titer in the supernatant was determined by plaque assay. (I) 293T cells were transfected with siRNA, as indicated. At 24 hours after transfection, cells were transfected with poly(I:C) for 16 hours. RNA was extracted, and cDNA was prepared to determine CXCL10 and IFN-β mRNA expression by qPCR analysis. (J) MEF cells were infected with VSV (MOI, 0.1) after transfection for 24 hours with siRNA, as indicated, and viral titer in the supernatant was determined by plaque assay. (K) Viperin wild type (Viperin-WT), viperin-Del, and RIG-I were purified separately from 293T cells. Oxidative reaction was performed in vitro, and methionine oxidized peptides were quantitatively determined by mass spectrometry analysis. (L) 293T cells were transfected with plasmids containing indicated genes. At 48 hours after transfection, WCLs were analyzed by immunoblotting. For (E to K), the data are expressed as means ± SEM; n = 3; ***P < 0.001 and ****P < 0.0001.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/8/eaax1031/DC1

    Fig. S1. KSHV helicase and cellular DNA helicase MCM7 are evolutionarily conserved.

    Fig. S2. KSHV helicase is critical for viral DNA replication.

    Fig. S3. Viperin is critical for viral DNA replication and KSHV-encoded RTA binds to viperin promoter to up-regulate viperin expression.

    Fig. S4. Viperin promotes methionine oxidation of KSHV helicase.

    Fig. S5. Viperin-induced methionine-401 oxidation enhances the stability and function of KSHV helicase.

    Fig. S6. Viperin associates with lipid droplets that are required to induce the methionine oxidation of KSHV helicase.

    Fig. S7. Viperin-induced methionine oxidation stabilizes MCM7 and promotes DNA replication.

    Fig. S8. Viperin interacts with several DNA helicases.

    Fig. S9. Methionine oxidation catalyzed by viperin increases the stability and function of RNA helicase RIG-I.

    Table S1. Amino acid sequence alignment for each human DNA helicase with KSHV helicase.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. KSHV helicase and cellular DNA helicase MCM7 are evolutionarily conserved.
    • Fig. S2. KSHV helicase is critical for viral DNA replication.
    • Fig. S3. Viperin is critical for viral DNA replication and KSHV-encoded RTA binds to viperin promoter to up-regulate viperin expression.
    • Fig. S4. Viperin promotes methionine oxidation of KSHV helicase.
    • Fig. S5. Viperin-induced methionine-401 oxidation enhances the stability and function of KSHV helicase.
    • Fig. S6. Viperin associates with lipid droplets that are required to induce the methionine oxidation of KSHV helicase.
    • Fig. S7. Viperin-induced methionine oxidation stabilizes MCM7 and promotes DNA replication.
    • Fig. S8. Viperin interacts with several DNA helicases.
    • Fig. S9. Methionine oxidation catalyzed by viperin increases the stability and function of RNA helicase RIG-I.
    • Table S1. Amino acid sequence alignment for each human DNA helicase with KSHV helicase.

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