Eradicating latently infected cells represents the ultimate goal in the therapy of KSHV-associated malignancies, in which loss of the viral episome expectedly leads to tumor cell death. Currently, treatment of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease typically includes chemotherapeutic agents that target all replicating cells, failing to distinguish between virally infected and uninfected cells. Such regimens are associated with severe side effects, including myelotoxicity and pancytopenia, which can become life-threatening in an already immunocompromised population. Because KSHV infection remains in a latent state in the majority of infected tumor cells, drugs that target latent viral proteins may be more effective than current regimens at both preventing and treating disease and may have an added benefit of fewer side effects.
This report describes an assay designed to identify samples that induce viral episome loss, irrespective of the specific mechanism, and without generalized cytotoxicity. The design hinges on a two-step screen. The first step identifies samples that cause loss of the latent virus in a cell line (KSHV-BJAB) that does not depend on the virus for viability. The second step validates those hits in a cell line (BCBL-1) that does depend on the virus for survival.
The initial screening step employs a B lymphocyte cell line (KSHV-BJAB) carrying the KSHV-BAC and expressing GFP. KSHV-BJAB cells are incubated in medium containing test samples and are monitored for loss of fluorescence (i.e., loss of the viral episome). Seven percent of 81 screened plant extracts consistently reduced fluorescence by ≥50% in KSHV-BJAB cells as compared with a DMSO control. An additional 6% of the extracts were cytotoxic to all B lymphocyte cell lines tested, regardless of their KSHV infection status. Eighty-seven percent had no significant effect (data not shown).
Because nonspecific promoter silencing could diminish fluorescence in KSHV-BJAB cells, virus-specific effects were verified by real-time QPCR for the viral genome. Using primer sets for both a viral (νGPCR) and a cellular (U6) gene and DNA templates isolated from BJAB, KSHV-BJAB, or a primary effusion lymphoma cell line, BCBL-1, after incubation with the test samples, the cytotoxicity and selectivity of each extract was assessed. Six extracts, identified as potential hits (≥50% reduction of fluorescence) in the first screening step, were tested. Extracts A05831 and A05853 showed selective activity against latent virus, as the viral episome was lost from a model infection (KSHV-BJAB) at least 10 times more efficiently than host chromosomal DNA (corresponding to cell death). Furthermore, both extracts resulted in at least a 50% reduction in cell viability from naturally infected tumor cells (BCBL-1), as compared with a DMSO control. Extract A05831 receives higher priority for further study because it is relatively nontoxic in uninfected BJAB cells, whereas extract A05853 may prove too nonselectively toxic in its unrefined state.
In principle, compounds that interfere with viral genome maintenance may target cellular or viral proteins required to maintain latency. Samples exerting their effects by targeting viral proteins are preferred because a specific antiviral effect may be less toxic to other highly replicating cells and, presumably, would have fewer side effects than currently available chemotherapeutics. KSHV LANA is a likely viral target for antiviral samples because it is essential for KSHV genome maintenance.In a complex with multiple cellular proteins (37
), LANA tethers the viral genome to the cellular chromosome, ensuring that the two are replicated coincidentally and are segregated equally to each daughter cell. Samples that target LANA may accomplish their antiviral effect by one or more means, including (a
) transcriptional down-regulation, (b
) degradation or posttranslational modification, (c
) sequestration outside the nucleus, (d
) interference with binding to the host chromosome or the viral genome, or (e
) similarly targeting cellular proteins that complex with LANA. Additionally, other viral latent proteins may also be involved in viral genome maintenance and may also be targeted.
To explore the mechanism(s) by which extracts A05831 and A05853 propel episomal loss, a LANA immunofluorescence assay was done on BCBL-1 cells incubated with the two lead extracts, identified by the initial fluorescence-based screening step and verified by QPCR. Both extracts resulted in near-background levels of LANA immunofluorescent staining. Thus, these plant extracts may contain one or more compounds that down-regulate the transcription of LANA, accelerate its degradation, or cause posttranslational modifications that render it undetectable by this antibody. Therefore, extracts A05831 and A05853 are prime candidates for further experimentation. Because each extract likely contains hundreds of compounds, future studies will employ a bioactivity-directed fractionation strategy to purify and identify the antiviral constituent(s).
Loss of fluorescence mediated by culturing KSHV-BJAB cells with antiviral samples is dependent on two factors: loss of the KSHV-BAC, which contains the GFP gene and degradation of GFP that is made prior to loss of the gene. GFP has a reportedly long half life, ranging from 26 to 80 h in eukaryotic cells (44
), which contributed to the length of the initial screen, which was further extended to 5 weeks in order to assess cumulative cytotoxicity. Analysis of the data, however, showed that the most potent inhibitors already displayed a significant effect by day 20, and the second screening step allowed for the earlier detection of cumulative cytotoxic effects.
In modern medicine, many highly effective therapeutic agents such as camptothecin and Taxol/paclitaxel, were first isolated from plant extracts and have revealed novel targets and mechanisms for antitumor drug action. Although certain herbal extracts were recently found to reactivate KSHV (47
), the novel assay described in this report identifies plants as a rich source for antiviral compounds that may cure KSHV infection by interfering with latent viral episome maintenance. Although a relatively small sample set was tested, the results are representative of the discovery potential for samples with therapeutic promise, as an overall hit rate of 2% is consistent with other natural product screens, in which hit rates typically range from 0.5% to 5%, regardless of the biological target. Indeed, small molecule libraries derived from plant or other natural product sources may prove to be repositories for antiviral agents with varied targets and activities against a breadth of currently incurable viral infections.