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.
Once the researchers had determined that the Gag molecules were gathering in tight groups, the next thing they had to do was show that these assemblages were budding off from the cell surface to form completely independent virions. If that were happening, they reasoned, nothing should be visibly transferring back and forth between the newly formed virus particles and the inside of the cell. To test this, Simon and Bieniasz attached a different fluorescent protein to Gag, one that reacted to acid in its environment. When they acidified the cell’s interior with a brief pulse of carbon dioxide, Gag molecules still connected to and exchanging protons with their parent cell should react quite quickly. Virions that had already pinched off should have a much slower response, proving themselves now to be independent entities no longer dependent on the host cell.
“It really is an important step forward,” says Wesley Sundquist, a biochemist at the University of Utah who studies the life cycle of HIV. (He was not involved in the research.) “This is the first time we’ve been able to look at the behavior of real virion particles, and to do so in real time.”
Unlike most techniques in biology, in which a scientist has to infer what’s happening based on his or her observations, “here we could directly observe the process of assembly, and unequivocally show where it occurs and how long it takes,” Bieniasz says.
In the future, HIV researchers may also be able to use these techniques to create far more precise therapeutics. Now that they have the means to better investigate individual steps of viral assembly, they could potentially figure out ways to interfere with the process. Bieniasz says that researchers could also use the same approach to investigate other viruses.