Antimicrobial agents in the physician's armamentarium are usually better at stopping bacterial than viral infections. But scientists have had some success finding antiviral medications based on the body's natural defenses against viruses.
Most antiviral proteins in the cell that scientists know about are set into action by interferon. Interferon activates certain proteins, which mostly fight infection by inhibiting the production of the virus's proteins inside the host cell. Physicians take advantage of the cell's innate defenses when they try to slow the spread of hepatitis C with doses of interferon.
But Dr. Stephen Goff believed other defense proteins against viruses exist inside our cells, besides those activated by interferon, and developed a method to find them. Once identified, these proteins could be used as the foundation for new types of antiviral therapies. Using his technique, Dr. Goff, Higgins Professor in Biochemistry and Molecular Bio-physics and Microbiology, recently reported success in finding rZAP, a unique retroviral defense protein in rodents, in the Sept 6 issue of Science.
Previously, the search for additional antiviral proteins was hampered by inefficient methods. Genetic techniques, for example, for finding antiviral proteins depend on identifying rare mutant cells that resist viral infection. Once a resistant cell is found, researchers then tried to find the mutated protein involved in the resistance. But a virus can often fail to infect a cell, even if the cell is not particularly resistant. Techniques that then try to isolate the virus-resistant cells by killing sensitive cells leave a lot of normal survivors in the background. Consequently, few antiviral proteins beyond those connected with interferon have been found.
Dr. Goff improved the selection process so fewer normal cells survive. For every one true resistant cell, only about 100 normal cells survive. He also took advantage of a good cDNA library that covers a significant part of the expressed genome. Researchers have successfully used similar methods to search for cancer-causing genes and signal transduction genes, but Dr. Goff is the first to apply the new method to the search for viral defense proteins.
To start, the lab made 500,000 lines of rat cells. Each line produced a different rat protein, or protein fragment, in excessive amounts from a cDNA insert. The researchers then infected all the lines of cells with a mouse retrovirus to see which of the overexpressed proteins fought off the infection.
The retrovirus used to infect the cells does not kill the cells so Dr. Goff used genetic engineering to be able to detect cells resistant to the virus. He created a retrovirus that had an extra gene that coded for thymidine kinase, which when expressed would kill the cell in the presence of the drug trifluorothymidine. He infected all the cells with the recombinant retrovirus and added the drug. When a retrovirus successfully infected a cell, the thymidine kinase gene was ex-pressed and the cell became sensitive to the drug and died. When a cell resisted infection, the drug did nothing, and the cell survived.
Two hundred cells survived the drug, but only one cell turned out to be truly resistant to the retrovirus. Dr. Goff's team located the gene responsible for resistance on the rat's chromosome 6. They dubbed it rZAP, for rat Zinc-finger Antiviral Protein.
Further experiments by Dr. Goff showed the protein seems to be protecting the cell from the retrovirus in a unique way. "The viral mRNA is made in the nucleus of the resistant cell, but rZAP seems to either keep the mRNA in the nucleus or destroys it if it reaches the cell's cytoplasm," Dr. Goff says. "If the virus can't get its mRNA into the nucleus, it can't produce new virus." Most known viral defense proteins, Dr. Goff says, do not interfere with viral mRNA, but instead interfere with the translation of mRNA into viral proteins.
"We think our method to find those proteins in cells resistant to a virus is a good scheme," Dr. Goff says "and we're going to use it again to search for proteins that can fight infection by human retroviruses, including HIV."
The research was supported by a Public Health Service grant from the National Cancer Institute. Dr. Stephen Goff is a Howard Hughes Medical Institute investigator.