University at Buffalo - The State University of New York

Published: June 19, 2003

By LOIS BAKER
Contributing Editor

Molecular biologists at UB have discovered a novel way to inhibit the replication of poxviruses, the group that includes smallpox virus, by interfering with messenger RNA synthesis necessary for the viruses to reproduce in a host organism.

The discovery, which has a patent pending, could lead to drugs that could be available to treat the potentially deadly disease if there were a bioterrorism-related outbreak.

Such drugs also would be effective against related poxviruses such as monkeypox, which recently has infected dozens in the U.S who came in contact with animals imported from Africa, where monkeypox is indigenous.

"Any success that results in a treatment is a success for everyone," said Edward Niles, professor of microbiology and biochemistry, and primary discoverer of the new antireplication mechanism. "We need something."

Niles noted that work that would lead to new drugs is in the early stages.

There is no effective treatment for smallpox or other poxviruses. Smallpox was declared eradicated in 1977 after a worldwide vaccination campaign. The U.S. and Russia maintain the only authorized repositories of the virus, but virologists acknowledge that the virus may exist outside these sites.

Existing vaccines, which could be used to protect against smallpox bioterrorism, have a high incidence of side effects and may not be administered to certain segments of the population, notably pregnant women, persons with compromised immune systems due to disease or medications, persons with a history of eczema and children under one year of age.

Drugs developed using this novel approach could be stockpiled for use if an outbreak occurs, said Niles. If a new smallpox vaccination campaign were undertaken, such drugs also could be available to treat persons who have serious reactions to the vaccine.

Niles' discovery, achieved working with vaccinia virus, which causes cowpox, exploits a peculiar aspect of poxvirus biochemistry: Instead of creating copies of itself in the nucleus of the infected cell, as with other DNA viruses such as the herpes virus, poxviruses replicate in the cell's cytoplasm, the gel-like material surrounding the nucleus.

"Since poxviruses replicate in the cytoplasm, they can't use hosts enzymes present in the nucleus to make their proteins," said Niles. "These viruses have evolved in a manner that allows them to produce their own enzymes to express their genes and permit their replication."

"This quirk in the poxvirus replication process should make it possible for scientists to design drugs targeted to those unique viral enzymes without interrupting normal cellular functions," he said.

Vaccinia virus is the virus strain used for immunization against smallpox. The initial interest of Niles and colleagues was to understand the basic process in the early stage of poxvirus gene expression (virus gene expression takes place in three stages: early, intermediate and late).

"The early phase is unique in that for transcription (mRNA synthesis) to proceed, it requires an initiating event at a site on the DNA called a promoter," he said. "Another signal, called a terminator, is required to stop the early gene transcription. We wanted to know what that terminator signal does."

To study this mechanism, the UB researchers synthesized a short RNA fragment, or oligonucleotide, that contained the known termination signal. They then added the fragment to a test tube transcription reaction, and measured RNA synthesis.

"We expected the oligonucleotide to inhibit the termination reaction," said Niles, "but instead of stopping it, the presence of the oligonucleotide stimulated premature termination. This resulted in the synthesis of truncated RNA molecules, which would be unable to direct the synthesis of normal proteins.

"This termination mechanism is unique to poxviruses, and this method of inhibition of gene expression should work on all poxviruses," he said. "If this oligonucleotide could be delivered as a drug, it would inhibit synthesis of all poxvirus proteins early in infection and stop the virus from replicating in the host."

The work is in its very early stages, Niles noted, with many steps that must be accomplished before a viable drug can be developed.

"We have to identify the most active compounds in vitro, test their activities on virus replication in tissue culture, and then figure out how best to deliver it in an animal model before we can even begin to test it in humans."

Mohamed Ragaa Mohamed, postdoctoral fellow working in Niles' laboratory, collaborated on the research.

The work was funded by the National Institute of Allergies and Infectious Diseases of the National Institutes of Health.