The research appears may point towards important answers to the unsolved puzzle of how this alternate form of DNA functions and may lead to the development of anti-viral compounds that are effective against smallpox, one of the deadliest of human diseases. A report on the research by two teams of scientists from the Massachusetts Institute of Technology and Arizona State University is forthcoming in the May 19 Online Early Edition of the Publication of the National Academy of Sciences. Entitled "A role for Z-DNA binding in vaccinia virus athogenesis," the paper is authored by Yang-Gyun Kim, Ky Lowenhaupt, and Alexander Rich from MIT and Maneesha Muralinath, Teresa Brandt, Matthew Pearcy, Kevin Hauns and Bertram L. Jacobs from ASU. Z-DNA, discovered by MIT's Rich in 1979, is an alternate zig-zig-shaped form (or "conformation") that DNA sometimes assumes instead of the familiar double helix conformation (known as B-DNA). Certain sequences of DNA in the normal B conformation will "flip" into the Z form (which is less stable), apparently in response to genes being transcribed immediately "downstream" on the molecule's sequence. Coiled to the left instead of to the right, the Z-DNA conformation is a significantly different arrangement of the molecule, but the functional role of this major difference has remained largely unclear.
In the report, the researchers find clear evidence that a critical pox virus protein (one known to be necessary for the virus to disable animal cell defenses) works by binding to Z-DNA and apparently interfering in its operation.
The researchers aimed a variety of experiments towards understanding the functionality of vaccinia virus protein E3L, a protein previous experiments have shown be produced by vaccinia in order to cause disease, and that can also be disabled by modifying the protein's active site.
Noting the similarity of E3L's active site to a site on a protein known as ADAR1 that has been proven to bind to Z-DNA, the researchers first replaced the active site in E3L with the ADAR1 site and found that viruses containing this modified protein were still lethal.
Next, the team mutated the modified protein and found that mutations that affected the active site and the protein's ability to bind to Z-DNA in the test tube diminished the virus's lethality, much as mutations to E3L's active site had earlier been proved to disable the virus. They also found that non-lethal forms of the virus containing a similar protein that does not bind with Z-DNA could be made lethal by mutating the protein to make its active site capable of Z-DNA binding.
"We have very good evidence now that in order for vaccinia virus to kill a mouse, it has to have a Z-DNA binding protein," said Jacobs. "This is how E3L works."
Specifically how Z-DNA binding affects mouse cells defenses against viruses still remains to be seen, said Jacobs, but the current experiments' results point to a way to find out.
"The fact that we've got a Z-DNA binding protein that's critical for whether a virus kills an animal or not gives us tools to start asking what is Z-DNA really doing. One possibility is that Z-DNA plays a role in regulating the transcription of specific anti-viral genes," said Jacobs. "Maybe you have some cellular proteins binding to the Z-DNA and that increases transcription of the anti-viral genes. Maybe what the virus has done is make another protein that binds to the Z-DNA and stops the process...
"The beauty of our system is that we can now ask those questions. What genes are induced if you infect with a wild-type virus, as opposed to what genes are induced if you infect with a virus that doesn't contain the Z-DNA binding protein? We're starting to examine these issues," he said.
A potential by-product of the research is the development of new anti-viral drugs that could be effective against smallpox. Smallpox is very similar to the vaccinia virus (live vaccinia virus is the key component in smallpox vaccine) and smallpox contains a gene that is essentially the same as the gene that produces the Z-DNA binding E3L protein. From the group's understanding of the similarities between E3L and ADAR1, Jacobs believes that a molecule could be designed that would block the Z-DNA binding site of E3L and thus disable the disease-causing capabilities of both vaccinia and smallpox.
"Dr. Rich has solved the crystal structure for this Z-DNA binding domain from ADAR1, so we know what it looks like," Jacobs said. "We think that the Z-DNA binding domains on the viral proteins are similar to this, so we can potentially make a small molecule that will fit into this binding site on the viral protein. This would keep the viral protein from binding to Z-DNA, and this thus prevent smallpox from causing disease."
Source: Bert Jacobs, 480-965-4684
Journal
Proceedings of the National Academy of Sciences