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.