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. 2007 Aug;6(8):2360-70.
doi: 10.1158/1535-7163.MCT-07-0108.

Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma associated herpesvirus

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Development of a fluorescence-based assay to screen antiviral drugs against Kaposi's sarcoma associated herpesvirus

Tamara K Nun et al. Mol Cancer Ther. 2007 Aug.

Abstract

Tumors associated with Kaposi's sarcoma-associated herpesvirus infection include Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Virtually all of the tumor cells in these cancers are latently infected and dependent on the virus for survival. Latent viral proteins maintain the viral genome and are required for tumorigenesis. Current prevention and treatment strategies are limited because they fail to specifically target the latent form of the virus, which can persist for the lifetime of the host. Thus, targeting latent viral proteins may prove to be an important therapeutic modality for existing tumors as well as in tumor prevention by reducing latent virus load. Here, we describe a novel fluorescence-based screening assay to monitor the maintenance of the Kaposi's sarcoma-associated herpesvirus genome in B lymphocyte cell lines and to identify compounds that induce its loss, resulting in tumor cell death.

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Figures

Figure 1
Figure 1
Validation of the assay. A, cells from three B lymphocyte cell lines (BJAB, KSHV-BJAB, and BCBL-1) were incubated in RPMI containing test or control samples. Samples were initially screened for loss of fluorescence in the KSHV-BJAB cells, suggestive of loss of virus. To verify loss of virus from the cells, additional assays were done including real-time PCR viral load assays and immunofluorescence assays for LANA. B, incubation of KSHV-BJAB cells with a known inhibitor of KSHV latency, glycyrrhizic acid, leads to a dose-dependent decrease in mean fluorescence as compared with an ethanol control. C, digital images of the KSHV-BJAB cells incubated with glycyrrhizic acid (2 and 6 mmol/L) or an ethanol control. Top, fluorescence images; bottom, merged fluorescent and bright-field images. D, real-time QPCR using primer sets to amplify viral (vGPCR) and cellular (U6) regions of DNA isolated from BCBL-1 cells incubated with glycyrrhizic acid (GA) or an ethanol (EtOH) control. Dose-dependent increases in the cycle threshold for viral (○) and cellular (◆) template as compared with the ethanol control were evident.
Figure 2
Figure 2
Initial screening for antiviral activity is based on loss of fluorescence. Mean fluorescence of live B lymphocyte cultures incubated with potential antiviral compounds is measured every 2 to 3d for 5 wks. A, extracts A05810 ( formula image), A05830 ( formula image), and A05898 ( formula image) caused accelerated loss of mean fluorescence in KSHV-BJAB cultures as compared with the DMSO control ( formula image), eventually establishing a new plateau level of fluorescence ≥50% less than the DMSO control. B, after incubation with extracts A05831 ( formula image), A05853( formula image), and A05901 ( formula image) fluorescence declined steadily and without leveling off, suggesting continuous reduction of the viral load.
Figure 3
Figure 3
Microscopic evaluation of KSHV-BJAB cells incubated with potentially antiviral plant extracts. Digital images of KSHV-BJAB cells incubated with representative plant extracts that exhibited an accelerated loss of fluorescence as compared with a DMSO control, suggesting loss of the viral genome. Left, fluorescence images; right, merged fluorescent and bright-field images. A, extracts A05810, A05830, and A05898, which achieved a new plateau level of fluorescence, as compared with DMSO. B, digital images of KSHV-BJAB cells incubated with extracts A05831, A05853, and A05901, which steadily reduced fluorescence as compared with the DMSO control.
Figure 4
Figure 4
Five extracts exhibited cytotoxicity to all three B lymphocyte cell lines. A, mean fluorescence graph representative of acute and delayed cytotoxic samples, A05814 (○) and A05854 (□), respectively. B, digital images of KSHV-BJAB cells incubated with acutely cytotoxic sample A05854 or delayed cytotoxic sample A05814. Left, fluorescence images; right, merged fluorescent and bright-field images.
Figure 5
Figure 5
Real-time QPCR allows potential hits to be stratified based on selectivity and specificity. Real-time QPCR was done using viral (vGPCR) and cellular (U6) primer sets. For uninfected BJAB (A) and naturally infected BCBL-1 cells (B), cell viability of extract-treated cells was normalized to the DMSO control using the equation: 1.9−ΔCT(U6). C, cellular and viral DNA in extract-treated KSHV-BJAB cells were first normalized to DMSO-treated KSHV-BJAB cells. Then, the selectivity index was calculated as the ratio of changes in viral DNA to changes in cellular DNA.
Figure 6
Figure 6
Indirect immunofluorescence assays for LANA investigate possible mechanisms of interference with viral genome maintenance. BCBL-1 cells incubated with plant extract or a DMSO control were spotted on slides and fixed with precooled acetone. Incubations with anti–KSHV ORF-73(LANA) monoclonal antibody followed by TRITC-conjugated antirat IgG were used to detect the presence of LANA. Left, bright-field images; right, fluorescent staining of the KSHV LANA. A, DMSO control; B, extract A05807; C, extract A05831; and D, extract A05853.

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References

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