A total of 4,842 extracts from 38 countries were tested for their ability to induce KSHV reactivation in BCP-1 cells. Most were from land plants but extracts from 86 marine plants, 97 marine animals and 87 species of fungi were included. Usable results were obtained from 3,703 extracts. A total of 2,958 extracts were inactive, 561 reduced levels of spontaneous KSHV replication and 184 (5.0%) induced viral reactivation above the level of sodium butyrate, which is the most potent chemical inducer of KSHV reactivation. The degree of viral reactivation by natural products was significantly higher than that obtained by host biological response modifiers such as interferon-gamma.25,26
More than half (2,477) of the extracts with usable results were from African land plants and 126 (5.1%) of these were activators, compared with 58 (4.7%) from other or unknown geographical regions (). Activators included 3 marine plants, 1 marine animal and 1 fungal extract.
Natural Products Causing KSHV Reactivation by Geographical Region
Using likelihood ratio χ2 tests, African plants were more likely to cause reactivation than non-African plants (p = 0.024: likelihood ratio χ2 = 7.442, 2 df). Of the activating extracts, the magnitude of activation was greater for those from Africa (log10 mean = 8.41) compared with those from elsewhere (log10 mean = 8.21, p = 0.044, t-test). These associations were only weakly statistically significant. There were no activators from Europe or Asia, although only a small number of extracts from these regions were tested. Somewhat surprisingly, the proportion of extracts from Central America and the Caribbean and from The Philippines and Indonesia causing reactivation was higher than for African plants (5.6% and 7.2%, respectively vs. 5.1%), although the numbers of extracts were relatively small.
Viral activators belonged to a wide variety of plant families (), although consistent with sampling based only on geography; for many families only a few extracts were tested. For example, 2 out of 3 samples from the Hypericaceae and 3 out of 7 samples from the Pittosporaceae were classified as activators. Between 7 and 15% of the extracts from the Acanthaceae, Boraginaceae, Celastraceae, Leguminosae, Rutaceae and Solanaceae were classified as activators compared with 5% or less for other families. The 28 natural products that had the highest reactivation levels in the screening experiment are listed in , along with the family to which they belong, country of origin and the induced KSHV level expressed as log10 KSHV copies per 105 cells.
Plant Families from which 5% or More Extracts Produced KSHV Reactivation
Name, Family and Country of Origin of Natural Products from which Aqueous Extracts Produced the Highest Levels of KSHV Reactivation in Initial Screen
Apparent inhibition of KSHV replication was observed with 555 extracts (15%).These extracts included 525 land plants, 11 marine plants, 9 marine animals and 10 fungi. We considered it unlikely that these extracts were specifically inhibiting KSHV replication. More likely, the reduction in KSHV load that was observed with these extracts was probably caused by toxic effects of the extracts specific to BCP-1 cells, even though we had excluded extracts known to cause cytotoxicity in other cell lines. Some of these extracts may cause specific inhibition of KSHV replication but, since this was not the focus of this study, we did not attempt to characterize these further, with the exception of 3 extracts selected for the viral transcription array experiment.
We further characterized the dynamics of reactivation by retesting the 28 most potent extracts from the initial screen in a 4-day time course experiment over 4 days. Using the K6 real-time quantitative PCR assay, we confirmed the initial screening results for 22 of the extracts by showing KSHV viral load increases over time (). To further elucidate the mechanism of reactivation, RNA was extracted from duplicate cell pellets for viral gene expression analysis using a previously reported viral array18
that queries the entire KSHV transcriptome by real time quantitative PCR. Four extracts causing high levels of reactivation, Dracaena fragrans, Chasmanthera dependens, Cleistanthus polystachyus
and Emilia coccinea
, were chosen for RNA analysis as well as 3 causing apparent inhibition: Uvaria angalensia, Keetia carmichailli
and Garcinia species
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 . The results are shown relative to the viral load observed in sodium (more ...)
No mRNAs could be isolated from pellets in experiments using extracts, U. angolensia and Garcinia species, which caused apparent inhibition, consistent with our assumption that the natural product extract killed the cells early on. The other results fell into 2 categories; E. coccinea, C. dependens and D. fragrans caused only minimal changes in levels of 3 different cellular mRNAs over time, suggesting that the extract did not affect cell viability or basic cellular transcription (). In contrast, extracts from K. carmichailli and C. polystachyus caused a rapid decrease in cellular mRNA levels between 24 and 48 hr post exposure, suggesting a deleterious effect on viability or down regulation of basic cellular transcription. On the basis if this result, we normalized all data to the level of gapdh for each sample at each time point.
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 (more ...)
To determine whether the extracts were specifically causing KSHV reactivation, we first quantified the levels of 2 early viral mRNAs: K14 and ORF57 (). All 3 of the extracts causing apparent decrease of KSHV replication exhibited significant signs of cellular toxicity. We were therefore unable to demonstrate any specific effects on KSHV replication by these extracts. In contrast, for extracts causing activation, E. coccinea, C. polystachyus, C. dependens and D. fragrans, the 2 viral mRNAs showed a different pattern than the cellular mRNAs. Levels for K14 and ORF57 increased over time up to and beyond the level of gapdh, which is indicative of KSHV lytic reactivation. By comparison, the 2 cellular mRNAs, c-myc and actin showed little or no change, indicating that the effect was specific for viral transcripts. shows fold induction relative to mock treated cells for selected mRNAs, representing the different kinetic classes of genes. All classes were induced relative to mock treatment.
Maximum Fold Induction by Natural Product Extracts for Selected mRNAS
To evaluate the effects of E. coccinea, C. polystachyus, C. dependens and D. fragrans extracts on all KSHV mRNAs, we performed cluster analysis of the viral array data as shown in . Most KSHV mRNAs were induced upon exposure to the natural products and with similar kinetics, as shown by the gradual shift from blue (low levels) to black (median level) to red (high levels) over time. This suggested that the transcriptional changes were the result of an ordered cascade of lytic reactivation rather than activation of random viral promoters by the extract. The RNA transcription data indicate that the increases in viral load observed with our real time PCR assay are a consequence of specific viral reactivation as a result of exposure to the natural product extracts.
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 (more ...)
To determine whether viral reactivation by natural products resulted in virion production, we filtered and concentrated supernatants from untreated BCP-1 cells and cells treated with C. dependens, E. coccinea, C. polystachyus
and D. fragrans
for 96 hr. Immunoblotting of the KSHV late-lytic viral protein K8.1 A/B detected the expected 68–72 kDa broad band of virion-associated glycoprotein27
in samples from cells exposed to the 4 extracts, suggesting natural product reactivation induced a full lytic cycle of the virus ().K8.1 was not detected in the supernatant from the untreated BCP-1 cells. Cells from this experiment were processed for electron microscopy. Immature and mature virions were present in cells treated with all 4 extracts as well as the positive control. Representative virions are shown in .
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 (more ...)
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 (more ...)