Study reveals previously unknown role for viral protein integrase in HIV's life cycle

The tiny shell protecting the HIV virus resembles a slightly rounded ice cream cone, but there is nothing sweet about it. 

More than 40 million people worldwide live with AIDS because of this virus, and treatments must continually evolve as HIV mutates. During the acute stage of infection, a single human cell can produce as many as 10,000 new HIV particles. 

At the University of Delaware, Professor Juan R. Perilla and his research team in the Department of Chemistry and Biochemistry have spent over a decade probing the structure and function of HIV's protective shell, or capsid, and the proteins packed inside. Their goal is to identify new targets for drugs that could stop HIV in its tracks.

Now, in a surprising discovery, published Feb. 18 in Nature, Perilla and collaborators in the U.S. and the United Kingdom have revealed a previously unknown role for the viral protein integrase. Scientists already knew that integrase helps HIV insert itself into human DNA. But this new study provides the first direct evidence that integrase plays a critical structural role earlier on in HIV's life cycle - when the virus matures into an infectious force. 

Using high-resolution cryo-electron microscopy (cryo-EM), the research team found that integrase proteins form gluey filaments that line the inside of the capsid. Each segment of the filament slots neatly into the capsid's hexagon-shaped tiles, while gripping tightly to HIV's RNA genome. This zipper-like arrangement organizes and packs the virus, preparing it to hijack a cell and start making copies of itself.

Integrase plays a structural role inside the HIV capsid - nobody expected that. This protein forms filaments that anchor the RNA to the capsid. Without these filaments, the virus is non-infective." 

Professor Juan R. Perilla, University of Delaware

Peeking into an HIV particle

Seeing inside HIV is no small feat. The capsid is only about 120 nanometers wide -roughly 1/800th the thickness of a human hair. It is incredibly small, fragile, densely packed and constantly changing, Perilla said. 

To reveal its hidden architecture, the researchers relied on deep collaboration and a combination of sophisticated microscopy, molecular modeling and experimentation. 

Cryo-EM imaging - performed at the Francis Crick Institute in a facility some 20 meters below ground to safeguard against vibrations and magnetic fields - requires samples to be frozen in milliseconds, kept colder than outer space, and imaged with beams of electrons instead of light to capture fine structural details at the atomic level. This method produces millions of 2D images of frozen particles, which must be sorted, averaged and aligned into a 3D model to visualize individual proteins - work that just wouldn't be possible without high-performance computing, Perilla said.

Once the overall shapes were identified, molecular modeling provided the chemical details. Researchers built atom-by-atom models that fit the cryo-EM data, revealing how the virus's components lock together. 

Previous experiments designed to inhibit the virus added even more insight. The team used specialized inhibitors called ALLINIs to disrupt the formation (oligomerization) of larger assemblies of the integrase, which are now understood to also break the integrase-capsid interactions. While some pre-clinical inhibitors affect these interactions, Perilla said, no existing FDA-approved drugs target this newly discovered structural role of integrase - providing a promising new frontier for drug development.

A collaborative effort with global impact

"The thing with HIV is that people are chronically living with it," Perilla said. "Treatments are effective, but patients always need new therapeutics. We want to help develop the next generation of inhibitors and hope to have a significant contribution." 

Perilla emphasized that this research was made possible by long-time collaborations and public funding. He has worked with co-authors Peter Cherepanov at the Francis Crick Institute and Alan Engelman at Harvard for nearly a decade. Additional partners from the Francis Crick Institute, Dana-Farber Cancer Institute, University of Oxford, Birkbeck College, Harwell Science and Innovation Campus and Imperial College London also contributed to the study. 

University of Delaware students, including Ph.D. candidate Juan S. Rey in Perilla's lab, also have played a key role over the years, with many going into careers in the pharmaceutical industry to continue advancing biomedical research.

"If it was not for public funding - from the U.S National Science Foundation, National Institutes of Health and U.S. Department of Energy - we would not have this work," Perilla said. "Without public funding, our research wouldn't exist."