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1.
Fig. 1.

Fig. 1. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

NSs downregulates p62 in whole-cell lysates. (A) 293 cells were mock infected or infected with MP-12 at an MOI of 3 and harvested at the indicated time points postinfection. Whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, and anti-β-actin antibodies (from top to bottom). The asterisk denotes a nonspecific band. (B) 293 cells were transfected with in vitro-synthesized RNA for GFP or NSs and harvested 8 h posttransfection. Whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, anti-GFP, and anti-β-actin antibodies (from top to bottom). Representative examples of at least 3 independent experiments are shown.

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.
2.
Fig. 5.

Fig. 5. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

The proteasome is involved in p62 downregulation. (A) 293 cells were pretreated with 50 μM lactacystin (lac) or 5 μM MG132 for 30 min to inhibit proteasomal degradation before being mock transfected or transfected with in vitro-synthesized RNA for NSs. Cells were harvested at 8 h posttransfection, and whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, anti-ubiquitin, and anti-β-actin antibodies (from top to bottom). [Ub]n denotes high-molecular-weight polyubiquitin conjugates. (B) 293 cells were mock transfected or transfected with in vitro-synthesized RNA for NSs. Immediately following transfection, cells were treated with 100 μM chloroquine to inhibit lysosomal degradation. Cells were harvested at 8 h posttransfection, and whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, and anti-β-actin antibodies (from top to bottom).

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.
3.
Fig. 6.

Fig. 6. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

NSs interacts with p62. (A) Schematic representation of the S segment of the recombinant virus (rMP-12-NSs-SF) used in the experiments whose results are shown in panel B. SF, Strep-Tag II–FLAG tag. (B) 293 cells were mock infected or infected with rMP12-NSs-SF at an MOI of 3. At 5 h.p.i., NSs-SF was affinity precipitated with Strep-Tactin magnetic beads. Whole-cell lysates and the precipitated proteins (STREP) were analyzed by Western blotting with anti-p62, anti-FLAG, and anti-β-actin antibodies (from top to bottom). Representative examples of at least 3 independent experiments are shown.

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.
4.
Fig. 7.

Fig. 7. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

p62 is degraded in the nucleus of infected cells. (A) 293 cells were mock infected or infected with MP-12 at an MOI of 3. Immediately after infection, cells were treated with 20 nM leptomycin B (LMB) where indicated. Cells were harvested at 8 h.p.i., and whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, and anti-β-actin antibodies (from top to bottom). (B) 293 cells were mock infected or infected with MP-12 at an MOI of 3 and treated with 5 μM MG132 at 3 h.p.i. or 20 nM LMB at 0 h.p.i. where indicated. Cells were harvested at 8 h.p.i. and fractioned into cytoplasmic and nuclear compartments, followed by analysis by Western blotting with anti-p62, anti-RVFV, anti-GAPDH, and anti-lamin B antibodies (from top to bottom). (C) 293 cells were mock treated or treated with 20 nM LMB for 7 h before fixation and permeabilization. RanBP1 localization was visualized by immunostaining. Representative examples of at least 3 independent experiments are shown.

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.
5.
Fig. 4.

Fig. 4. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

Shutoff of host transcription or translation does not induce p62 downregulation. (A) 293 cells were mock infected or infected with MP-12 at an MOI of 3 or treated with 5 μg/ml of actinomycin D (ActD) for 9 h and harvested at 8 h.p.i. p62 mRNA expression levels were analyzed by RT-PCR using relative quantification normalized to the level of GAPDH. Error bars represent the standard deviations (SD) of the results of three independent experiments. Statistical analysis was carried out using Student's t test (n.s., not significant). (B) 293 cells were mock treated, treated with 5 μg/ml ActD for 9 h, or infected with MP-12 at an MOI of 3 for 8 h. Whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, and anti-β-actin antibodies (from top to bottom). (C) 293 cells were mock transfected or transfected with in vitro-synthesized RNA for GFP or NSs. Immediately after transfection, cells were treated with 100 μg/ml cycloheximide (CHX) where indicated. Cells were harvested at 8 h posttransfection, and whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, anti-GFP, and anti-β-actin antibodies (from top to bottom). Representative examples of at least 3 independent experiments are shown.

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.
6.
Fig. 2.

Fig. 2. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

NSs induces host transcriptional suppression. (A) 293 cells were mock infected (a to c) or infected with MP-12 (d to f) at an MOI of 3. To label newly synthesized RNA, cells were treated with 1 mM ethynyluridine (EU) from 15 to 16 h.p.i., followed by fixation and permeabilization. Labeled RNA was subsequently detected by click chemistry with an Alexa Fluor 594-coupled azide. The expression of viral proteins was visualized by immunostaining with a polyclonal anti-RVFV antiserum followed by an Alexa Fluor 488-labeled secondary antibody. (B) 293 cells were mock transfected (a to f) or transfected with in vitro-synthesized RNA for GFP (g to i) or NSs (k to m) and treated with 1 mM EU for 1 h before fixation at 16 h posttransfection. Cells shown in panels d to f were treated with 5 μg/ml actinomycin D (ActD) for 1 h concurrently with the EU treatment. Labeled RNA was subsequently detected by click chemistry with an Alexa Fluor 594-coupled azide. The expression of GFP and NSs was visualized by immunostaining with anti-GFP (g and i) or anti-RVFV (k and m) antibodies followed by an Alexa Fluor 488-labeled secondary antibody. (C) Magnification of a mock-transfected cell (a to c) and an NSs-transfected cell (k to m) from the experiment whose results are shown in panel B to visualize RNA synthesis in the nucleoli. Representative examples of at least 3 independent experiments are shown.

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.
7.
Fig. 3.

Fig. 3. From: NSs Protein of Rift Valley Fever Virus Promotes Posttranslational Downregulation of the TFIIH Subunit p62.

NSs induces host transcriptional suppression. (A) 293 cells were mock infected (mock) or infected with MP-12 at an MOI of 3 and treated with 0.5 mM EU for 1 h at the indicated time points of infection. As a control for transcriptional suppression, cells were treated with 5 μg/ml ActD concurrently with the EU labeling (ActD). Cells were harvested, fixed, and permeabilized immediately after the labeling step. EU-labeled RNA was then detected by click chemistry with an Alexa Fluor 647-coupled azide. The expression of viral proteins was visualized by immunostaining with a polyclonal anti-RVFV antiserum followed by an Alexa Fluor 488-labeled secondary antibody. Cells were then analyzed by flow cytometry. (B) To better demonstrate the progressive loss of transcriptional activity in MP-12-infected cells, data from the experiment whose results are shown in panel A were gated on anti-RVFV-positive cells, and the fluorescence of labeled RNA is displayed as a histogram. (C) Data from the experiment whose results are shown in panel A were gated on anti-RVFV-negative cells, and the fluorescence of labeled RNA in mock-infected and ActD-treated cells is displayed as a histogram. (D) 293 cells were mock infected or infected with either MP-12 or an MP-12 mutant carrying a large deletion in its NSs gene that renders the NSs protein nonfunctional (C13type). Cells were harvested at 12 h.p.i., and whole-cell lysates were analyzed by Western blotting with anti-p62, anti-RVFV, and anti-β-actin antibodies (from top to bottom). (E) 293 cells were mock infected (mock) or infected with either MP-12 or C13type at an MOI of 3 and were treated with 0.5 mM EU from 12 to 13 h.p.i. Cells were harvested, fixed, and permeabilized, and EU-labeled RNA was detected by click chemistry with an Alexa Fluor 647-coupled azide. The expression of viral proteins was visualized by immunostaining with a polyclonal anti-RVFV antiserum followed by an Alexa Fluor 488-labeled secondary antibody. Cells were then analyzed by flow cytometry.

Birte Kalveram, et al. J Virol. 2011 July;85(13):6234-6243.

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