A team of US researchers has developed a fluorescence-based screening assay to monitor the maintenance of Kaposi’s sarcoma-associated herpes virus in B-cell lines, and to identify compounds that induce its eradication in tumor cells and prompt cell death.
The team, led by researchers at the University of North Carolina-Chapel Hill’s Lineberger Cancer Center, said the assay can identify compounds that target latent KSHV viral proteins, which may be an important mechanism of action for treating existing tumors or preventing new tumors from developing.
To arrive at their results, the investigators introduced a KSHV-green fluorescent protein into a KSHV-negative B-cell line (BJAB) that does not require KSHV infection for its survival. They then incubated these transfected cells with a screening set of 81 plant extracts, and identified two compounds, dubbed A05831 and A05853, that caused a loss of the latent virus.
The researchers validated their findings using a primary effusion lymphoma cell line (BCBL-1) that is dependent on KSHV for survival.
Virus-specific effects were verified by real-time PCR for the KSHV genome. To elucidate the antiviral mechanism of A05831 and A05853, the researchers used an indirect immunofluorescence assay against the KSHV latency-associated nuclear antigen, or LANA.
The researchers found that the two hits resulted in near-background levels of LANA immunofluoresent staining, and concluded that A05831 and A05853 had somehow reduced the amount of LANA present.
The findings appear in the August issue of Molecular Cancer Therapeutics.
Blossom Damania, an associate professor of microbiology and immunology at the Lineberger Cancer Center and the corresponding author on the paper, spoke with Cell-Based Assay News about the assay and its potential use for screening additional libraries of natural and synthetic compounds.
Can you give me a little background on your work?
We work on a virus called Kaposi’s sarcoma-associated herpes virus, or KSHV. This virus is a member of the gammaherpes virus family. KSHV is associated with several different types of cancer in humans, including all forms of Kaposi’s sarcoma, primary effusion lymphoma, and the plasmablastic variant of multicentric Castleman’s disease.
Primary infection with KSHV is usually asymptomatic. It is mostly in immunodeficient populations, such as HIV-infected persons or transplant patients, that this virus rears its ugly head, so to speak, because it causes cancer in these populations. Those who are not immunosuppressed can still develop these cancers, but they are seen much more frequently in the immunodeficient groups.
Can you explain how the technology on which this assay is based works?
We wanted to develop an assay that we could use to identify chemicals, drugs, or extracts that actually eradicate the virus from cells. We took the KSHV virus and we made a recombinant virus tagged with a green fluorescent protein tag. We introduced the recombinant virus into a KSHV-negative B-cell line — BJAB — to create a KSHV-BJAB cell line.
We then incubated this cell line with different extracts over a period of four to six weeks. We were looking for an extract that would result in diminishing green fluorescence over time, meaning that the drug or extract is actually inducing loss of the virus. However, the most likely cause of loss of fluorescence is cell death, because many extracts are generally cytotoxic.
We wanted an assay that would distinguish between an extract that is generally cytotoxic and an extract that only kills the virus without killing the cell. We measured not only loss of green fluorescence, but also cell viability over time. We selected only those extracts that resulted in loss of fluorescence while maintaining good cell viability.
We found that out of 81 extracts, 79 of them either had no effect or were generally cytotoxic. Only two extracts, A05831 and A05853, showed a specific antiviral effect.
Although many KSHV-associated cancers depend on the virus being present for their survival, we chose to infect a B-cell line that could survive independently of KSHV. The use of such a cell line allowed us to distinguish between samples that caused loss of the latent viral episome and those that were generally toxic to the host cell.
We used real-time quantitative PCR and found that viral load in the BJAB cells cultured with either of the two extracts gradually diminished over time. We then wanted to identify the mechanism by which this was occurring.
A viral protein called the latency-associated nuclear antigen, or LANA, allows the virus to remain in the cell. We found that A05831 and A05853 somehow reduced the expression of LANA. We do not know exactly how, but the net result was that the expression of LANA was reduced in the presence of these extracts.
After the initial screen to identify the extracts that cause loss of the latent virus in the KSHV-BJAB cell line, we validated the two hits in a cell line — BCBL-1 — that does not depend on KSHV infection for survival. The BCBL-1 cells died, which is exactly what we expected to occur and the reason why we did the initial screen using cells that were independent of the virus.
Are plant extracts a resource for potential lead compounds?
Yes. Several anticancer therapies, such as paclitaxel, are derived from plants. We thought that a good starting point for us would be to figure out what other natural products we can use as anticancer chemotherapies.
However, the nice thing about this assay is that it can also be used to screen libraries of synthetic compounds, which we plan to do. It can be used to screen other types of extracts such as fungal extracts, and other libraries out there.
What would be the next step in your research?
As I mentioned, we are planning to screen other extracts and synthetic compounds. The thing is, you can find an extract that works in vitro, but once you take it in vivo, it might not be practical for several reasons. For example, the compound may not be very soluble, or may be hepatotoxic or cardiotoxic.
Our goal with this assay is to get as many potential candidates as possible and put them through a pipeline to determine if they really work, both in vitro and in vivo. It is likely that identifying two compounds will not be enough, because they may not necessarily work in vivo for the reasons that I just mentioned.
Each of the two extracts that we have identified in our current study probably comprises at least 10 components. The next step would be to fractionate each extract to identify the active component that results in loss of the virus. In parallel, we will be screening additional libraries to determine if there are other targets out there.
Is this something that you plan to commercialize?
Yes, if we get a good hit!