Researchers discover new mechanism of drug that alters genetic makeup of viruses

December 04, 2000

Researchers at Penn State have discovered a new mechanism for an existing antiviral drug that could permit the design and production of a new class of antiviral agents to treat RNA viruses. Such viruses, a family that includes poliovirus and hepatitis C, use RNA as both their core genetic material and also to direct protein synthesis.

A paper published in the December 2000 issue of Nature Medicine, by a team led by Craig Cameron, assistant professor of biochemistry and molecular biology at Penn State, reveals that ribavirin, a synthetic compound that inhibits RNA viruses by working at the cellular level, also possesses an ability to alter the structure of the viruses at the genetic level. Researchers used poliovirus as the experimental model for their work.

"Our results indicate the antiviral effects of ribavirin come from its direct incorporation into the viral RNA," Cameron said. "When that happens, it changes the behavior of the base pairs of the RNA and the virus no longer produces faithful copies of itself. In that manner, ribavirin effectively shifts the internal balance of the virus and the virus suffers from a genetic meltdown."

While most organisms use DNA as their genetic material and RNA to direct protein synthesis, RNA viruses use RNA for both functions. When an RNA virus infects a cell, it directs the synthesis of proteins used to make copies of the original RNA and then uses those copies to build the chromosomes of the virus. Many RNA viruses can be stopped by intervention from the immune system or with the help of vaccinations. Others adapt, developing their own "quasispecies" so rapidly that neither the immune system nor vaccinations provide relief.

In general, RNA's instability--when compared to DNA--means it works well as a virus because it changes form, or mutates, often enough to prevent the immune system from providing effective antiviral activity. With ribavirin acting at the genetic level, researchers have discovered a way to use the mutations against the virus. Ribavirin capitalizes on the mutations and stops the virus by altering its genome, upsetting its delicate balance, and forcing it to collapse upon itself.

Typically, an RNA molecule consists of a long chain of phosphates in which the sugar is ribose and the bases are adenine (A), cytosine (C), guanine (G), and uracil (U). Those bases rest on the sugar backbone, resembling one side of a zipper. When RNA pairs, as it does when it is copied, two separate zipper halves come together as the bases connect. Using an assay developed in his laboratory, a symmetrical primer/template substrate referred to as "sym/sub," Cameron and his colleagues utilized ribavirin as a substrate for the poliovirus polymerase.

"When an RNA virus copies its RNA, it does so with an RNA-dependent RNA polymerase--and that represents our target," Cameron said. "It is that enzyme that allows the antiviral effect of ribavirin by incorporating it into the RNA genome."

When the poliovirus polymerase incorporated ribavirin into the RNA, inappropriate genetic base-pairings occurred. Instead of the viral RNA pairing together in typical C-G or G-C and A-U or U-A combinations, it produced altered, or "mutant," combinations as it copied its RNA genome. Because ribavirin actually changed the structure of the poliovirus' RNA, the virus could not adapt or build an immunity to the treatment.

Researchers believe the findings represent an important addition to existing knowledge about ribavirin's antiviral potency. According to its accepted mechanism, ribavirin--in its monophosphate state, known as RMP--inhibits a specific cellular enzyme, inosine monophosphate dehydrogenase (IMPDH), thereby reducing guanosine triphosphate (GTP) pools in cells, which in turn diminishes the synthesis of viral proteins and limits the replication of viral genomes. For Cameron and his colleagues, the RMP part of the equation was only the beginning. Because ribavirin in its triphosphate state, RTP, accumulates in cells after treatment with ribavirin, the researchers theorized ribavirin's antiviral effect required direct incorporation into viral RNA. So, using what could be considered the leftovers from the typical ribavirin mechanism, researchers worked to incorporate RTP.

"It's not just inhibiting the IMPDH that's important, and we've proven that with good science," Cameron said. "But, we're not excluding the existing model. While what we've found might be different, it should also be considered unifying. It makes sense. You could see how inhibiting the cellular enzymes could even increase the ability of RTP to be utilized because you're going to have a greater concentration of it and it's going to be in a less competitive environment."

Although effective treatments for poliovirus do exist, Cameron and his colleagues used that virus for their research because of the availability of their "sym/sub" assay as well as other biochemical and genetic approaches. Their work included initial tests with purified polymerase, purified RNA, and purified ribavirin in order to prove ribavirin would be incorporated into the viral RNA. After that, tests included use of the poliovirus itself in tissue cultures as the researchers attempted to determine whether the virus that came out of the cells was different from what was introduced to the cells.

In every instance, the relationship between the presence of ribavirin and the alterations made to the viral RNA was obvious--and the antiviral effects increased in proportion to the amount of ribavirin used. With that strong ribavirin-to-antiviral correlation in poliovirus, Cameron hopes to have crafted a template that can be utilized to address other RNA viruses. Along with poliovirus and hepatitis C, members of the RNA virus family include: rhinovirus, the common cold; coxsackie virus, summer flu; and foot-and-mouth disease.

"If we use this template as a unifying factor, we might have found the Achilles' heel of these types of viruses," Cameron said. "Together, a unified model really seems to explain what's happening because every time we saw mutations in the viral RNA we saw antiviral activity--and the more mutations there were, the higher the antiviral activity."
-end-
This research was supported by the National Institutes of Health. Collaborators with Cameron were: Jamie Arnold and David Maag from Penn State; Raul Andino and Shane Crotty from the University of California at San Franciso; and Zhi Hong, Johnson Lau, and Weidong Zhong from Schering-Plough Research Institute in Kenilworth, New Jersey.

CONTACTS:
Craig Cameron, 814-863-8705, cec9@psu.edu
Steve Sampsell (PIO), 814-865-1390, sws102@psu.edu

Penn State

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