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Results: 6

1.
FIG. 1.

FIG. 1. From: Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly .

Antipoxviral agents identified by high-throughput screening. Thirteen compounds emerging from the screen that passed secondary screens for potency and cytotoxicity are listed in the far-left column. The secondary- and tertiary-assay methods are discussed in the text. Exemplary assays and results are shown for mitoxantrone in Fig. 2 and 4.

Liang Deng, et al. J Virol. 2007 December;81(24):13392-13402.
2.
FIG. 3.

FIG. 3. From: Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly .

Mitoxantrone does not affect viral gene expression. BSC40 cells were infected with the vaccinia-EGFP virus at an MOI of 10. The cells were pretreated for 12 h with 1 μM mitoxantrone and maintained in mitoxantrone thereafter (mitoxantrone). Control cells were infected in the absence of drug. The cells were examined by bright-field microscopy (top) and fluorescence microscopy (bottom) of the same fields at 12 h postinfection. Mock-infected cells were analyzed in parallel.

Liang Deng, et al. J Virol. 2007 December;81(24):13392-13402.
3.
FIG. 6.

FIG. 6. From: Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly .

Mitoxantrone-resistant vaccinia viruses. Wild-type vaccinia WR (WT) and two mitoxantrone-resistant variants (MX-R1 and MX-R2) obtained after forced passage in the presence of drug and two rounds of plaque purification were tested for plaque formation on BSC40 cell monolayers. The infected cells were overlaid with medium containing either 0.5 μM mitoxantrone (+ drug) or no additive (no drug). Photographs of the crystal violet-stained cell monolayers are shown. The chemical structure of mitoxantrone is depicted at the bottom.

Liang Deng, et al. J Virol. 2007 December;81(24):13392-13402.
4.
FIG. 5.

FIG. 5. From: Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly .

Mitoxantrone blocks formation of IMV. BSC40 cells were infected with the vaccinia WR at an MOI of 10. The cells were pretreated for 12 h with 1 μM mitoxantrone prior to infection and maintained in mitoxantrone thereafter (+ mitoxantrone). Control cells were infected in the absence of drug. At 24 h postinfection, cells were washed with PBS and then overlaid with fixative solution containing glutaraldehyde and paraformaldehyde. The fixed cells were dislodged by scraping and then collected by centrifugation. The specimens were dehydrated and then embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate for visualization by transmission electron microscopy. Middle, arrows indicate sporadic immature virions containing a smaller enveloped membrane; right, arrows indicate half-moon particles.

Liang Deng, et al. J Virol. 2007 December;81(24):13392-13402.
5.
FIG. 2.

FIG. 2. From: Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly .

Mitoxantrone inhibits vaccinia virus replication. (A) Virus yield from a low MOI. BSC40 cells were infected with vaccinia WR at an MOI of 0.01 and then overlaid with medium containing mitoxantrone. Virus yield at 48 h (log PFU) is plotted as a function of mitoxantrone concentration. (B) Synchronous infection in the presence of drug. Cells were pretreated for 12 h with medium containing 1 μM mitoxantrone or no drug and then infected at an MOI of 3 in the presence or absence of drug. Virus yield (log PFU) is plotted as a function of time postinfection. (C) Antiviral effect does not require preincubation with mitoxantrone. Cells were pretreated with medium containing 1 μM mitoxantrone for 12, 8, 4, or 0 h prior to infection at an MOI of 3. The infected cells were then overlaid with medium containing 1 μM mitoxantrone. Virus yield at 30 h postinfection (log PFU) is plotted as a function of the time of preincubation and compared to the yield from a control infection in the absence of drug. (D) Cells infected at an MOI of 3 were overlaid with medium containing 1 μM mitoxantrone at 0, 2, 4, 6, or 8 h after removal of the inoculum. Virus yield at 30 h postinfection (log PFU) is plotted as a function of the time of drug addition and compared to the yield from a control infection in the absence of drug. (E) Cells infected at an MOI of 3 were overlaid with drug-free medium or medium containing 1 μM mitoxantrone immediately after removal of the inoculum. Cells were harvested at 24 h or 48 h postinfection as specified. One of the infected monolayers was incubated with mitoxantrone-containing medium for 24 h and then washed three times with drug-free medium and incubated for another 24 h in the absence of drug prior to harvest.

Liang Deng, et al. J Virol. 2007 December;81(24):13392-13402.
6.
FIG. 4.

FIG. 4. From: Identification of Novel Antipoxviral Agents: Mitoxantrone Inhibits Vaccinia Virus Replication by Blocking Virion Assembly .

Mitoxantrone inhibits proteolytic processing of virion structural proteins. (A) BSC40 cells were infected with vaccinia WR at an MOI of 10. The cells were pretreated for 12 h with 1 μM mitoxantrone prior to infection and maintained in mitoxantrone thereafter (mitoxantrone). Control cells were infected in the absence of drug. At the specified times postinfection, medium was removed, and cells were washed with methionine-free MEM and then overlaid with methionine-free MEM containing 30 μCi/ml of [35S]methionine (1,135 Ci/mmol) with or without mitoxantrone. The medium was removed after 30 min and the cells lysed in situ in SDS sample buffer. Aliquots of the lysates were analyzed by SDS-PAGE. An autoradiograph of the gel is shown. The positions and sizes (in kilodaltons) of prestained marker polypeptides are indicated on the left. Synthesis of late viral proteins (indicated by ◂) is evident at 6 to 12 h postinfection in the presence or absence of mitoxantrone. (B) Cells infected in the presence of 1 μM mitoxantrone and control infected cells were pulse-labeled for 30 min with [35S]methionine at 12 h postinfection as described above. The cells were washed twice with MEM after removing the labeling medium and then either lysed immediately (lanes marked −) or overlaid with fresh medium containing unlabeled methionine with or without mitoxantrone. The monolayers were returned to incubate at 37°C and then lysed in situ after 4 or 8 h. Aliquots of the lysates were analyzed by SDS-PAGE. An autoradiograph of the gel is shown. Mitoxantrone blocked the conversion of major structural protein precursors into mature forms migrating at the positions denoted by asterisks.

Liang Deng, et al. J Virol. 2007 December;81(24):13392-13402.

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