For years researchers have been trying to understand how a few HIV-infected patients naturally defeat a virus that otherwise overwhelms the immune system. Last year, a research team at the University of Rochester Medical Center confirmed that such patients, called long-term non-progressors, maintain higher than normal levels of the enzyme called APOBEC-3G (A3G) in their white blood cells, which function to stave off infections. Now, the same group has teamed up with a structural biologist to provide the first look at the A3G structure. Such information represents an early step toward the design of a new class of drugs that could afford to all the same natural protection enjoyed by few, according to a study published today in The Journal of Biological Chemistry.
Researchers believe that A3G works by mutating or "editing" the HIV genetic code every time the virus copies itself. Editing introduces errors until the virus can no longer reproduce. At the same time, HIV has also evolved to counter A3G with its own defense protein, the viral infectivity factor (Vif), which holds firmly to A3G and tricks the white blood cell into destroying it. The results of the current study suggest how the physical form of A3G leads to its role in the immune system, and what parts of it may need to be protected so that it can continue to protect the body.
"Keeping A3G in action represents a new way to attack HIV," said Joseph E. Wedekind, Ph.D., associate professor in the Department of Biochemistry and Biophysics at the University of Rochester Medical Center. Wedekind, along with Harold C. Smith, Ph.D., professor in the same department, led the study. "This first, rough glimpse of A3G's physical structure gives us a map to follow in the search for a new class of AIDS treatments," Wedekind said.
For two decades, Smith and his team have worked to determine how "editing enzymes" like A3G make necessary changes to human genetic material. As the human immune system evolved, it recognized the ability of these enzymes to cause rapid genetic change and unleashed them on viral DNA. Last September, Smith's laboratory published work in the Journal of Virology that found higher levels of A3G closely correspond to lower HIV viral levels. After confirming that the A3G plays a key role in the body's fight with AIDS, Smith sought out Wedekind for a collaboration to determine its structure.
Wedekind is an expert in structural biology, the branch of molecular biology concerned with the study of the molecular shape and properties of proteins and nucleic acids, the molecules that make up the body's structures and carry out its life functions. Improved understanding of both protein and nucleic acid architecture has revolutionized medicine in recent years and has contributed to the design of current leading AIDS drugs. In seeking to determine the structure of A3G, however, the team was unable to use standard methods to start.
For instance, X-ray crystallography, Wedekind's area of expertise since 1989, involves aiming a high-energy X-ray beam at a sample of protein or nucleic acid that has been crystallized to form a repeating lattice of the molecule. The beams reflect off the atoms within a crystal, a camera records the reflected pattern and the data are reconstructed into a 3-D electron map by computers. The technique gives high-resolution images of the positions of atoms within a molecule, but only if researchers can first crystallize the molecule of interest. The team is making progress on crystallizing A3G, but wanted complementary, structural information in the meantime.
To achieve immediate results, the researchers elicited the help of Richard Gillilan, Ph.D., staff scientist at the Cornell High-Energy Synchrotron Source (CHESS) in Ithaca, N.Y., and second author on the JBC manuscript. Gillian has expertise in an imaging method called small-angle X-ray scattering (SAXS), which does not require the sample analyzed to be crystalline. While less detailed than crystallography, SAXS provides the general shape of a molecule, the spatial relationship of its parts to one another and hints about the function of each part.
Implications of Shape
Within infected human cells, viral DNA chains must temporarily unzip their two attached chains into single strands to be read and copied, and they must be copied if the virus is to infect more cells. Past work had established that A3G edits single strands of HIV DNA exposed while the virus copies itself. The newly determined structure of A3G suggests how it is able to crawl down HIV DNA chains, introducing mistakes wherever the chains are unzipped and skipping over zipped-up, double stranded regions. Researchers now believe A3G is capable of this because its structure is surprisingly different from other enzymes in its class.
The study also confirmed that A3G has two forms -- one that actively disrupts viral reproduction by editing as a free protein, and another in which the enzyme is inactive due to the presence of mRNA. The new SAXS results show what both forms look like, and suggest new ways in which HIV or the cell itself may turn off A3G.
HIV, along with deploying Vif, may also create a surplus of molecules that force A3G into its inactive form, researchers said. The theory is that HIV infection disrupts the normal process of making proteins, creating a surplus of free messenger RNAs that force A3G to become inactive. Messenger RNAs, copies of DNA that serve as a templates for the building of proteins, thus, may be natural regulators of whether A3G remains active or not. Preventing this mRNA interaction with A3G may represent yet another new avenue of attack on HIV.
"We are the first group to be able to say 'here are the parts of the molecule that need to be protected to keep A3G active in its age-old, ongoing war against viruses" Smith said. "We believe this work will lead to the development of a new treatments that enable patients to better harness their own natural defense mechanisms."