Journal: The Journal of Biological Chemistry

Article Title: Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

doi: 10.1016/j.jbc.2022.101597

Figure Lengend Snippet: NPL4-VCP complex interacts with Japanese encephalitis virus (JEV)-NS4B. A , pull-down assay of JEV-NS4B with VCP and NPL4. The 293T cells were cotransfected with OSF-VCP or OSF-NPL4 with Myc-JEV-NS4B. Bait and prey proteins in the bound fraction were detected using the anti-Myc ( top panel ) or anti-FLAG antibodies ( middle panel ). Prey proteins in the lysate (Input) were detected ( bottom panel ). B , immunoprecipitation of JEV-NS4B with endogenous NPL4 using 293T cells infected with JEV (MOI = 1.0) or mock for 48 h. Bait and prey proteins bound to anti-JEV-NS4B or control antibody–conjugated beads detected using the anti-NPL4 ( left top panel ) or anti-JEV-NS4B ( left bottom panel ) antibodies. Black and gray arrows indicate immunoglobulin bands and NS4B, respectively. Prey proteins in the input lysate (Input) fraction were detected using the anti-NPL4 ( right top panel ) or anti-JEV-NS4B ( right bottom panel ) antibodies. C , NPL4 expression promotes the interaction between VCP and NS4B. The 293T cells were cotransfected with OSF-tagged VCPs and Myc-tagged JEV-NS4B with or without Myc-NPL4. Bait and prey proteins in the bound fraction were detected using the anti-Myc ( top panel ) or anti-FLAG ( middle panel ) antibodies. Prey proteins in the input lysate (Input) fraction were detected using the anti-Myc antibodies ( bottom panel ). D , effect of NPL4 knockdown on the interaction between VCP and NS4B. Schematic presentation of pull-down HiBiT assay is shown in the left panel . 293T cells were transfected twice with siRNA, followed by the OSF-VCP and NS4B-HiBiT expression plasmids. After cell lysis and pull-down with Strep-Tactin Sepharose beads, HiBiT-dependent NanoLuc luciferase activity (HiBiT activity) in the bead-bound fraction was measured (graph). Prey proteins in the input lysate were detected using the anti-NPL4 ( top panel ) antibody, anti-FLAG ( middle panel ) antibody, or HiBiT blotting ( bottom panel ). E , two-successive pull-down experiments for NS4B–NPL4–VCP ternary complex. Schematic presentation of two-successive pull-down HiBiT assays is shown in the left panel . 293T cells were transfected with NS4B-HiBiT, with or without Myc-NPL4 and OSF-VCP. After cell lysis and pull-down with the Strep-Tactin Sepharose beads, HiBiT activity was measured in the bead-bound fractions ( second panel ). After eluting bead-bound proteins by adding destiobiotin, the eluted fractions were mixed with anti-Myc antibody–conjugated Sepharose beads. HiBiT activities were then measured in the bead-bound fractions ( third panel ). HiBiT activities in the input fractions were measured ( right panel ). F and G , colocalization of NPL4 and JEV-NS4B in JEV-infected cells or cotransfected cells. Post infection (24 h), JEV (MOI = 1.0)-infected HeLa cells were transfected with OSF-NPL4 and fixed. The levels of OSF-NPL4 and NS4B were detected with the anti-FLAG ( green ) or anti-JEV-NS4B ( red ) antibodies ( F ). HeLa cells were cotransfected with OSF-NPL4 ( green ) and JEV-NS4B-Myc ( red ) ( G ). The numbers under each image indicate Mander’s colocalization coefficient R. The scale bar represents 20 μm ( F and G ). H , schematic structure of NPL4 and its fragments used in ( I ). UBD, “ubiquitin-like” domain; NZF, Npl4 zinc finger or RanBP2/Nup358 zinc finger. I , yeast two-hybrid assay for examining the interaction between NPL4 and JEV-NS4B fragments (2393–2447 aa). Each pair of plasmids includes one plasmid that expresses AD fused to the fragments of NPL4 and one plasmid that expresses DBD fused to fragments derived from JEV polypeptide containing the cytoplasmic region. Plasmids were transformed into the yeast strain AH109. Cells were plated on the control medium ( right panel , Trp − and Leu − ) or the selective medium ( left panel , Trp − , Leu − , His − , and Ade − ). The numbers in each column indicate the amino acid regions expressed in the yeast cells. VCP, valosin-containing protein.

Article Snippet: For HiBiT blotting, the protein-bound Immobilon-P polyvinylidene difluoride membrane was treated with the bacterially expressed recombinant LgBiT and NanoLuc substrate of the Nano-Glo Luciferase Assay System (Promega).

Techniques: Virus, Pull Down Assay, Immunoprecipitation, Infection, Control, Expressing, Knockdown, Transfection, Lysis, Luciferase, Activity Assay, Ubiquitin Proteomics, Y2H Assay, Plasmid Preparation, Derivative Assay, Transformation Assay

Journal: The Journal of Biological Chemistry

Article Title: Flavivirus recruits the valosin-containing protein–NPL4 complex to induce stress granule disassembly for efficient viral genome replication

doi: 10.1016/j.jbc.2022.101597

Figure Lengend Snippet: VCP ATPase is important for early stage of viral propagation . A , effect of NPL4 and/or VCP knockdown on JEV propagation. 293T cells were transfected with siRNA against NPL4 and/or VCP twice for 24 h. The cells were then infected with JEV (MOI = 0.3). Virus titers in the supernatant 48 h post infection were measured ( top graph ). ∗∗ p < 0.0001; ns, not significant (Tukey's multiple comparison test). The levels of VCP ( second panel ), NPL4 ( third panel ), and α-tubulin ( bottom panel ) were determined using Western blotting. B , effect of NPL4 and/or VCP knockdown on Dengue virus (DENV) genome replication. 293T cells were transfected with siRNA against NPL4 and/or VCP twice for 24 h, followed by transfection with NanoLuc expressing the DENV subgenomic replicon RNA-expressing plasmid. NanoLuc-dependent luciferase activities were measured in the cell lysate at 72 h post transfection ( top graph ). ∗∗ p < 0.001; ns, not significant (Tukey's multiple comparison test). The levels of VCP ( second panel ), NPL4 ( third panel ), and α-tubulin ( bottom panel ) were determined using Western blotting. C , effect of DBeQ on JEV propagation. The 293A cells were infected with JEV (MOI = 0.3) for 2 h and treated with 10 μM of DBeQ for 4 h. Post infection (48 h), the infectious virus titer in the culture supernatant was measured using a focus forming assay. ∗∗ p < 0.01 (Student’s t test). D and E , time of addition assay. D , schematic diagram of DBeQ treatment. The 293A cells were infected with or without JEV (MOI = 0.3) and treated with 10 μM of DBeQ or 10 μM of Eer1 for 4 h at the indicated time points. Virus inoculation ( white box ) and drug treatment ( gray box ) time frames are indicated. E , results of time of the addition assay. All supernatants were collected 48 h post infection to determine the JEV infectious titer using a focus forming assay. JEV, Japanese encephalitis virus; MOI, multiplicity of infection; VCP, valosin-containing protein.

Article Snippet: For HiBiT blotting, the protein-bound Immobilon-P polyvinylidene difluoride membrane was treated with the bacterially expressed recombinant LgBiT and NanoLuc substrate of the Nano-Glo Luciferase Assay System (Promega).

Techniques: Knockdown, Transfection, Infection, Virus, Comparison, Western Blot, Expressing, Plasmid Preparation, Luciferase, Focus Forming Assay