Hybrid Method May Block HIV Progression to AIDS

Researchers seek to develop drugs that block the Nef protein that is associated with HIV progressing to AIDS.

A hybrid method has been developed to study the HIV protein Nef, which is involved in the progression of HIV to AIDS, with the primary goal of blocking the protein.

Nef is a shape-changing protein that travels to the membrane of the infected cell, and tricks it into destroying the cell’s own immune system signaling receptors, allowing the infected cell to evade the immune system. Furthermore, Nef is able to hijack cellular communication to make it easier for the virus to reproduce.

“Nef is a protein essential for AIDS,” said Mike Kent, a researcher at Sandia National Laboratories' Biological and Engineering Sciences Center. “It accomplishes its missions by altering signaling and receptor trafficking. It binds to critical immune system receptors and then signals your cells to destroy them. If you know how this protein works, you have a better shot at developing drugs to stop it.”

To find out how Nef is able to change structures to perform its functions, researchers at Northeastern University combined 2 known biophysical techniques called neutron reflectometry and hydrogen-deuterium exchange mass spectrometry.

Neutron reflectometry is a technique that obtains nanometer-scale structural information about films and biological membranes. This technique was used in the study to compare the global structure of Nef in its membrane-bound form to its inactive, membrane-free form.

The technique hydrogen-deuterium exchange mass spectrometry measures the local structure and flexibility of proteins. This was used in the study to obtain information on the local structure and dynamics of Nef when it’s bound to the membrane.

“People have been studying Nef for a long time and there was a model of what people thought the protein might look like and might do,” Kent said. “Nef is a difficult protein to study because you can only crystalize the folded part of the protein, and about half of the protein is unstructured. In addition, you can’t study the membrane-bound form of crystallography.”

However, when researchers used these techniques together, it provided a more complete picture of Nef and its structural changes.

“It’s the first time anybody has measured these kinds of structural changes and the results were consistent with the hypothetical model,” Kent said. “Details of these shape changes provide important new molecular insights into how Nef functions.”

In order for researchers to combine these 2 different techniques they needed to make a special apparatus that contained a flat lipid monolayer made of saturated fats, which mimicked the biological membrane. Furthermore, it needed to be integrated with equipment at neutron sources for neutron reflection measurements, and have rapid exchange of the watery support layer for they hydrogen-deuterium exchange experiments.

In infected cells, Nef is tagged with a special lipid called myristate group, which anchors Nef to the cell membrane. In order for researchers to correctly produce the Nef protein, it needed to contain this essential lipid.

Now that researchers have this unique apparatus and hybrid method, they are looking for fund to help answer further questions about Nef.

“We studied it alone; now we want to study it with its binding partners, with the host proteins and the complexes that it forms, and in the presence of drug molecules or inhibitors,” Kent said. “Stopping it from binding with its partners or inhibiting it from adopting the conformation that leads to receptor degradation would have important medical implications.”

Kent noted that he hopes to apply the hybrid method to other structural problems of membrane-associated proteins, including virus maturation; the workings of bacterial toxins such as botulinum, tetanus, and diphtheria; and cell-signaling dysfunctions ranging from cholesterol levels to cancer.

“There is a lot of potential for combining these 2 techniques in a more general sense,” Kent said. “There are no other ways to get this kind of specific, direct information about essential membrane proteins. This is a significant niche of biological problems that could not be addressed before our work, and we’ve made some big steps forward. The future benefit depends on how broadly we can apply the method beyond just this one HIV protein.”

Currently, researchers are screening for potential drugs that may block the actions of Nef.