Enhanced Replication of Mouse Adenovirus Type 1 following Virus-Induced Degradation of Protein Kinase R (PKR)

Figures

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  FIG 1. Viral DNA yield is increased in PKR−/− mouse embryonic fibroblasts. PKR WT MEFs (WT), N-PKR−/− MEFs (N-), and C-PKR−/− MEFs (C-) were infected with MAV-1 at an MOI of 1 and collected at 48 and 72 hpi. DNA was purified from cell pellets and analyzed for MAV-1 genome copies by qPCR. The graph is representative of results from three experiments (14 biological replicates per cell line per time point). Error bars represent standard errors of the means (SEM). *, P ≤ 0.05; ***, P ≤ 0.0002; ****, P ≤ 0.0001.

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  FIG 2. Mouse PKR is depleted during MAV-1 infection. (A) Cells (indicated at left) were infected with MAV-1 (MAV) at an MOI of 10 or were mock infected (mock). Cell lysates were collected at the indicated times and analyzed by immunoblotting with antibodies for PKR (B-10 for C57BL/6 MEFs and D-20 for C57BL/6 primary peritoneal macrophages and CMT93 cells) and actin. Blots are representative of results from a minimum of three independent experiments per cell line. (B) Densitometry quantitation of five of the C57BL/6 MEF immunoblots represented in panel A. Error bars represent SEM. n = 5 for 24 hpi, n = 4 for 48 hpi, and n = 2 for 72 hpi. **, P ≤ 0.01. (C) Untreated SV40 MEFs or kinase-dead (K271R) SV40 MEFs were infected with MAV-1 at an MOI of 10. Cell lysates were immunoblotted as described for panel A with PKR D-20. The numbers below the blots represent the proportion of PKR protein for each time point, normalized to actin, and the mock PKR protein levels from the corresponding time point.

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  FIG 3. MAV-1 does not cause PKR depletion by reducing steady-state levels of PKR mRNA at times when the protein levels are already reduced. MEFs (A) or isolated primary peritoneal macrophages (B) were harvested and infected with MAV-1 at an MOI of 10 or mock infected. The cell pellets were collected, and RNA was isolated. cDNA was generated from the RNA, and qPCR was used to quantitate PKR mRNA levels. Each graph represents 5 to 7 replicates for each time point from three pooled experiments. Error bars show the SEM. *, P ≤ 0.05; **, P ≤ 0.01.

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  FIG 4. MAV-1 infection does not affect PKR translation. (A) C57BL/6 MEFs were infected with MAV-1 (MAV) at an MOI of 5 or were mock infected (mock) and collected at 48 hpi. Cells were lysed, and cleared lysates from three 10-cm-diameter plates were layered onto 25% sucrose and centrifuged to pellet ribosomes. RNA was purified from the pellets, cDNA was generated from the RNA, and qPCR was used to quantitate the PKR mRNA levels. The graph represents 9 replicates for each time point, pooled from 3 independent experiments. Error bars show the SEM. (B) C57BL/6 MEFs were infected with MAV-1 (MAV) at an MOI of 2 or were mock infected (mock). Cells were collected at 25 hpi and lysed; cleared lysates were layered onto 10%-to-50% sucrose gradients and centrifuged. Gradients were collected from the top and pumped through a UV spectrophotometer, and 34 fractions were collected. The gradients are displayed with the bottom fractions indicated to the right. The UV trace of the first 10 fractions (including 40S and 60S ribosomal subunits) is not shown. (C) RNA was purified from each fraction of the gradients represented in panel B. cDNA was generated from the RNA, qPCR was used to quantitate PKR mRNA…

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  FIG 5. PKR is not depleted by lysosomal degradation during MAV-1 infection. (A) CMT93 cells were infected with MAV-1 (MAV) at an MOI of 10 or were mock infected (mock) and treated with 10 mM ammonium chloride or 60 µM chloroquine to inhibit lysosomal degradation (or with water as a control). Cell lysates were analyzed by immunoblotting with antibodies for PKR (D-20) and actin. Blots are representative of results from three independent experiments. (B) Inhibitors were tested for activity using a DQ BSA assay; the DQ BSA molecule fluoresces only if lysosomal degradation is functional. Uninfected cells were treated as indicated and imaged by fluorescence microscopy.

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  FIG 6. PKR is depleted by proteasomal degradation during MAV-1 infection. (A) C57BL/6 MEFs were infected with MAV-1 (MAV) at an MOI of 10 or were mock infected (mock) and treated with DMSO (vehicle for inhibitors), 1 µM MG132, or 1 µM bortezomib. Cell lysates were then analyzed by immunoblotting with antibodies for PKR (D-20) and actin. Blots are representative of results from four independent experiments. (B) Densitometry quantitation of four independent experiments. Treatment with bortezomib significantly inhibited PKR depletion in MAV-1-infected cells. *, P ≤ 0.05.

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  FIG 7. PKR is actively depleted early in infection. (A) CMT93 cells were infected with MAV-1 (MAV) at an MOI of 10 or were mock infected (mock) (top), or uninfected cells were treated with 50 µg/ml cycloheximide (CHX, bottom) to inhibit elongation of protein synthesis. Cell lysates were analyzed by immunoblotting with antibodies for PKR (D-20) and actin. Blots are representative of results from five independent experiments. (B) Densitometry quantitation of five independent experiments; **, P ≤ 0.01. (C) CMT93 cell lysates from the experiment performed as described in the panel A legend were analyzed with a second immunoblotting step with antibodies for E1A and actin. Blots are representative of results from four replicates from two independent experiments. The E1A blot image was uniformly adjusted to a brightness value of 30 and a contrast value of 5 in Adobe Photoshop.

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  FIG 8. Early gene expression is required for PKR depletion by MAV-1. (A) Cells (as indicated at left) were infected with WT MAV-1 (WT MAV) or UV-inactivated MAV-1 (UV MAV) at an MOI of 10 or were mock infected (mock). Cell lysates were analyzed by immunoblotting with antibodies for PKR (D-20) and actin. Two independent wells were infected for each condition at both time points. (B) CMT93 cells were infected with WT MAV-1 (MAV) at an MOI of 10 or were mock infected (mock). Infected cells were also treated (+) or not (-) with 20 µg/ml cytosine arabinoside (AraC), an inhibitor of DNA synthesis. Cell lysates were analyzed with antibodies for PKR and actin.