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Over the years, HIV has proved a tricky target. No one could definitively show where in the cell it assembled, or when it was released. Certainly no one knew how long it took a single virus to be born. And so much of what’s known about HIV and other viruses has been pieced together through experiments that rely on inference: microscopic and chemical probing of cells frozen in different states of viral infection provide only information about what was happening in that cell at a particular moment in time. Now researchers have been able to watch as hundreds of thousands of molecules assemble inside a cell to create a single particle of HIV.

“No one’s ever actually observed virus particles assembling before,” says Paul Bieniasz, a virology researcher at Rockefeller University and the Aaron Diamond AIDS Research Center, and one of the scientists involved in the project. Their study marks the first time that scientists have been able to observe a virus–any virus–being built, and it holds the potential to revolutionize the relationship that scientists have with the viruses they study.

The research is a collaboration between Bieniasz, an HIV specialist, and Sanford Simon, a biophysicist at Rockefeller who studies how large molecules enter and leave the cell. The scientists used a suite of inventive imaging techniques to record each step of the process, allowing them to watch as the virus assembled and then gradually budded off of its host cell. The entire process can occur in as few as six minutes.

At the heart of the research is an often overlooked microscopy trick called total internal reflection. This technique takes advantage of light’s ability to bend. When light is shined through glass onto a cell’s surface at a very steep angle, it begins to bend. The steeper the angle, the greater the bend, until the angle is so sharp that light reflects back into the glass and illuminates only the very thin area along the surface of the cell–an area otherwise impossible to visualize.

By homing in on this outer membrane, and tagging one of the virus’s major structural proteins, called Gag, with a fluorescent protein, the researchers were able to watch as the molecules aggregated to form a single virion. Visually, it showed up as little bright spots appearing and disappearing, “like little stars appearing in the sky,” Simon says. “It was really beautiful.”

In order to make sure that what they were seeing really was the virus assembling, Simon and Bieniasz then tagged the Gag proteins with fluorescent molecules that change color when in close proximity to one another–something that would indicate that the proteins were assembling into a tightly packed structure. Sure enough, the fluorescent tags reacted, and their color change confirmed that Gag proteins were coming together to form a virus.

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Credit: Nolwenn Jouvenet, Paul Bieniasz, and Sanford Simon.

Tagged: Biomedicine, imaging, virus, HIV, cells

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