tbev  (ATCC)

Journal: PLoS Pathogens

Article Title: Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

doi: 10.1371/journal.ppat.1001255

Figure Lengend Snippet: SW13 (A, B) or Huh7.5 (C–E) cells were seeded in equal numbers into plates and transduced with V1 vector expressing GFP (GFP, filled circles), V1 vector expressing full-length human DNAJC14 (FL, open circles) or V1 vector expressing truncated (aa 305–702) human DNAJC14 (NT1, triangles). After 2 days, the cells were challenged (moi = 5) with YFV 17D (A), YFV Asibi (B), Kunjin virus (C), Langat virus (D) or were challenged (moi = 0.1) with HCV Jc1FLAG2/p7-nsGluc2A. The medium (A–D) or cells (E) were harvested at the indicated times for quantification of virus replication. For A and B, a single separate well was utilized for each time point, and virion production was enumerated by plaque assay. In both cases, there were fewer than 100 plaque-forming units at 12 h post infection. The dashed line indicates the sensitivity of the plaque assay. Pfu, plaque forming units. For C and D, duplicate wells were infected and the medium was harvested and replaced at each timepoint. Virion production since the prior time point was enumerated by focus forming assay as described in . Data points represent the mean titer; error bars indicate the range. Similar results were obtained in an independent experiment for both Kunjin and Langat viruses. FFU, focus forming units. For E, cells were harvested at the indicated times after infection for measurement of luciferase activity as described in . Data points represent mean values obtained from triplicate wells; error bars indicate the standard deviation. RLU, relative light units.

Article Snippet: After 4 d the monolayers were fixed with 100% methanol and plaques were visualized by incubation with polyclonal mouse antibody cross-reactive to Langat (hyperimmune mouse ascites fluid, clone Russian Spring Summer Encephalitis VR79; ATCC) or polyclonal mouse anti-West Nile virus E protein (obtained from Dr. Robert Tesh, World Reference Center for Emerging Viruses and Arboviruses) followed by secondary goat anti-mouse peroxidase-labelled polymer (DAKO Envision Systems) and application of peroxidase substrate containing 0.4 mg/ml 3,3′ diaminobenzidine and 0.0135% hydrogen peroxide in PBS.

Techniques: Transduction, Plasmid Preparation, Expressing, Virus, Plaque Assay, Infection, Focus Forming Assay, Luciferase, Activity Assay, Standard Deviation

Journal: PLoS Pathogens

Article Title: Identification and Characterization of the Host Protein DNAJC14 as a Broadly Active Flavivirus Replication Modulator

doi: 10.1371/journal.ppat.1001255

Figure Lengend Snippet: (A) SW13 cells were left untransduced (top row) or were transduced (lower 2 rows) with the noninhibitory V1-hDNAJC14 mutants FL-H471Q or CT1 as indicated. Two d later the cells were mock treated (left panels) or were challenged with YFV (moi = 5, right 3 panels). After an additional 2 d, the cells were fixed and immunostained with rabbit anti-YFV NS3 polyclonal antibodies (NS3), and mouse anti-calnexin antibody (calnexin) or mouse anti-myc monoclonal antibody (myc) as indicated. AF488-conjugated anti-mouse IgG and AF594-conjugated anti-rabbit IgG antibodies were used as secondary antibodies. The cells were analyzed by confocal microscopy and representative images are shown. Calnexin or DNAJC14 mutants are shown in green, YFV NS3 is shown in red, and the merged images are shown on the right. (B) SW13 cells were left untransduced or were transduced with the V1-hDNAJC14-CT1 mutant (CT1-myc) as indicated and were infected 2 d later with YFV (moi = 1). After 2 d of infection, myc-tagged DNAJC14-CT1 was immunoprecipitated using anti-myc antibody. Western blots were performed using antibodies against NS3, calnexin and the myc epitope tag as indicated. (C) SW13 cells were left uninfected or were infected with YFV (moi = 1) as indicated. The cells were fixed 1 d later and analyzed by confocal microscopy for endogenous DNAJC14 (red) and double stranded RNA (dsRNA, green). The merged image is shown on the right. Arrows indicate several areas of colocalized DNAJC14 and dsRNA.

Article Snippet: After 4 d the monolayers were fixed with 100% methanol and plaques were visualized by incubation with polyclonal mouse antibody cross-reactive to Langat (hyperimmune mouse ascites fluid, clone Russian Spring Summer Encephalitis VR79; ATCC) or polyclonal mouse anti-West Nile virus E protein (obtained from Dr. Robert Tesh, World Reference Center for Emerging Viruses and Arboviruses) followed by secondary goat anti-mouse peroxidase-labelled polymer (DAKO Envision Systems) and application of peroxidase substrate containing 0.4 mg/ml 3,3′ diaminobenzidine and 0.0135% hydrogen peroxide in PBS.

Techniques: Confocal Microscopy, Transduction, Mutagenesis, Infection, Immunoprecipitation, Western Blot