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Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions - PubMed

  • ️Mon Jan 01 2007

Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions

Denise Whitby et al. Int J Cancer. 2007.

Abstract

Kaposi's sarcoma (KS) and its causative agent, Kaposi's sarcoma associated herpesvirus (KSHV/HHV-8), a gamma2 herpesvirus, have distinctive geographical distributions that are largely unexplained. We propose the "oncoweed" hypothesis to explain these differences, namely that environmental cofactors present in KS endemic regions cause frequent reactivation of KSHV in infected subjects, leading to increased viral shedding and transmission leading to increased prevalence of KSHV infection as well as high viral load levels and antibody titers. Reactivation also plays a role in the pathogenesis of KSHV-associated malignancies. To test this hypothesis, we employed an in vitro KSHV reactivation assay that measured increases in KSHV viral load in KSHV infected primary effusion lymphoma (PEL) cells and screened aqueous natural product extracts from KS endemic regions. Of 4,842 extracts from 38 countries, 184 (5%) caused KSHV reactivation. Extracts that caused reactivation came from a wide variety of plant families, and extracts from Africa, where KSHV is highly prevalent, caused the greatest level of reactivation. Time course experiments were performed using 28 extracts that caused the highest levels of reactivation. The specificity of the effects on viral replication was examined using transcriptional profiling of all viral mRNAs. The array data indicated that the natural extracts caused an ordered cascade of lytic replication similar to that seen after induction with synthetic activators. These in vitro data provide support for the "oncoweed" hypothesis by demonstrating basic biological plausibility.

(c) 2006 Wiley-Liss, Inc.

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Figures

FIGURE 1
FIGURE 1

Time course experiment with 28 extracts that caused the highest increases in KSHV viral load in the initial screen. The names and country of origin of the extracts are listed in Table III. The results are shown relative to the viral load observed in sodium butyrate induced cells. The standard deviation was calculated from the results of day 1 for all samples. Black dashed lines indicate one and a half standard deviations above and below the normalized average unstimulated control values for each day.

FIGURE 2
FIGURE 2

(a) Raw CT values for three cellular mRNAs, namely actin (blue squares), gapdh (red triangles) and c-myc (black circles). CT values are depicted on the vertical axis and correspond to the level of mRNA on a logarithmic scale. Higher CT values indicate lower mRNA levels. Hence, a CT = 40, for the negative nontemplate control (NTC) lanes indicated no amplification. We used BCP-1 cells as a positive control. As expected, gapdh mRNA is more abundant than actin, which in turn is more abundant than c-myc mRNA, as indicated by their relative CT values. Moreover, a 1:2 dilution of the input cDNA pool resulted in the same, reproducible and significant increase in CT for each mRNA, affirming the quantitative nature of our analysis. (b) dCT values for 2 KSHV mRNAs, K14 (red circles) and ORF57 (blue squares) and 2 cellular mRNAs, actin (black open circles) and c-myc (black triangles) at days 0, 1, 2, 3 and 4 after exposure to natural product extracts. Lower CT values correspond to higher mRNA levels on a log 2 scale, and 0 indicates mRNA levels equivalent to gapdh mRNA.

FIGURE 3
FIGURE 3

Heatmap representation of hierarchical clustering of dCT values for 73 KSHV mRNAs. Gapdh mRNA is present at an intermediate level, indicated by the black color. High abundance mRNAs are in red. The topmost group of viral mRNAs was present at high levels (indicated by red color in each sample) and changed very rapidly and significantly. A second group changed from low abundance (blue) to medium abundance (black). The third group of KSHV mRNAs increased slowly over time and only at the last time point exhibited levels above the median (red). In this depiction, black indicates equivalency to the level of gapdh, because individual genes are not normalized, which preserves information on the relative levels of each mRNA to one another. Primer pairs that amplified splice site specific mRNAs were excluded because the relative PCR efficiency was not comparable.

FIGURE 4
FIGURE 4

KSHV virion production in supernatant from BCP-1 cells stimulated with extracts from C. dependens, E. coccinea, C. polystachyus and D. fragrans. Concentrated cell supernatants from untreated BCP-1 cells and extract exposed cells were run on a denaturing polyacrylamide gel and the proteins were transferred to a PVDF membrane. Immunoblotting for reactivity of the late lytic glycoprotein K8.1A/B demonstrated KSHV virion production in the cells treated with all 4 extracts but not in the untreated BCP-1 cells.

FIGURE 5
FIGURE 5

Electron microscopy of BCP-1 cells stimulated with extracts from C. dependens, E. coccinea, C. polystachyus and D. fragrans demonstrated the production of typical herpesvirus virions. Immature and mature particles were observed in cells treated with all 4 extracts as well as the positive control (sodium butyrate). Panels A and B show immature particles assembled in the nucleus. Panel C shows mature particles in a cytoplasmic vacuole. Panel D shows an extracellular mature particle. The scale bar represents 200 nm.

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