Catching HIV budding from cells: it all comes down to ALIX

The secrets of the AIDS virus may all come down to a protein named ALIX.

Researchers have devised a way to watch newly forming AIDS particles emerging or "budding" from infected human cells without interfering with the process.

The method shows that a protein named ALIX (which stands for "alg-2 interacting protein x") gets involved during the final stages of virus replication, not early on, as was believed. ALIX assists in separating new virus buds from a cell. These buds repeat the replication process and further infect their host.

"We watch one cell at a time" and use a digital camera and special microscope to make movies and photos of the budding process, says virologist Saveez Saffarian, a scientist at the University of Utah, and co-author of a paper on HIV budding published this week in the journal PLOS ONE.

"We saw ALIX recruited into HIV budding for the first time," he says. "Everybody knew that ALIX was involved in HIV budding, but nobody could visualize the recruitment of ALIX into the process."

The finding has no immediate clinical significance for AIDS patients because ALIX is involved in too many critical functions like cell division to be a likely target for new medications, Saffarian says.

"We know a lot about the proteins that help HIV get out of the cell, but we don't know how they come together to help the virus emerge," he says. "In the next 10 to 20 years, we will know a lot more about this mechanism."

Saffarian conducted the research with the paper's first author Pei-I Ku, as well as researchers Mourad Bendjennat, Jeff Ballew and Michael Landesman. All are with the University of Utah.

The research was funded by the National Science Foundation (NSF).

"This project has led to the development of an important technique in basic research in cell biology and virology," says Parag Chitnis, director of NSF's Division of Molecular and Cellular Biosciences.

"It's uncovering a new understanding of the viruses involved in human diseases," says Chitnis. "This is an excellent example of how purely basic research can lead to the fundamental understanding of topics of societal need."

Watch, don't touch, as HIV buds

Biochemical methods used for years involve collecting millions of viruses in lab glassware and conducting analyses to reveal the proteins that make up the virus--for example, by using antibodies that bind to certain proteins and using other proteins to make the first proteins fluoresce so they can be seen.

"You're not doing it one virus at a time," Saffarian says. "The problem is that you don't see the differences among similar viruses. And you don't see the timing of how various proteins come and go to help the virus get out of the cell."

Other methods freeze or otherwise fix cells as new HIV particles emerge, and use an electron microscope to photograph freeze-frame views of viral replication.

Saffarian employs technology known as "total internal reflection fluorescence microscopy" that looks at the dynamic processes in cells.

The method has been used to make images of the budding of HIV and a similar horse virus, EIAV.

But Saffarian says that the EIAV study didn't show ALIX becoming involved in HIV budding, and that it wrongly indicated that ALIX got involved early in the EIAV budding process, suggesting it did the same in HIV budding.

Ku, Saffarian and colleagues combined their microscopy method with an improved way of genetically linking a green fluorescent "label" to ALIX proteins in cloned cells so they could see the proteins without harming their normal function.

The researchers tried numerous so-called "linkers" and found the one that let them see the ALIX proteins as they became involved in HIV budding.

Neither the microscope technology nor labeling proteins with green fluorescence are new, but "what we did that is new is to connect these fluorescence proteins to ALIX using many different kinds of linkers," says Saffarian, to find one that let the ALIX protein function properly.

The problem with research that indicated ALIX was involved early in the budding process was that only one linker was used, and it impaired ALIX's normal function, the scientists say.

Looking at proteins forming HIV

When HIV replicates inside a human cell, a protein named Gag makes up most of the new particles--there are 4,000 copies of the Gag protein in one HIV particle--although other proteins get involved in the process, including ALIX.

Experiments like those by Saffarian use "virus-like particles," which are HIV particles stripped of their genetic blueprint or genome so they don't pose an infection risk in the lab.

"Virus-like particles maintain the same geometry and same budding process as infectious HIV," Saffarian says.

During budding, Gag proteins assemble on the inside of a cell membrane--along with ALIX in the late stages--and form a new HIV particle that pushes its way out of the cell--the process by which AIDS in an infected person spreads from cell to cell.

To look at the budding process, Ku and Saffarian placed human cells containing the particles in a small amount of liquid growth medium in a petri dish and placed it under the microscope, which is in a glass chamber kept at body temperature so the cells can remain alive for more than 48 hours.

A solid-state blue laser was aimed at the sample to make the green-labeled ALIX and red-labeled Gag proteins glow or fluoresce so they could be seen as they assembled into a virus particle.

With red-labeled Gag proteins and green-labeled ALIX proteins, "we could see ALIX come in at the end of the assembly of the virus particle," says Saffarian. Some 100 ALIX proteins converged with the roughly 4,000 Gag molecules and assembled into a new HIV particle.

Enter ALIX

ALIX then brought in two other proteins, which cut off the budding virus particle from the cell when it emerged. ALIX's position during the pinching off of new particles hadn't been recognized before.

The researchers watched the virus particles bud one cell at a time: about 100 particles emerged during a two-hour period. Most of the ALIX proteins left when HIV assembly was complete and returned to the liquid inside a cell.

Saffarian says the discovery that ALIX doesn't get involved until the late stages of HIV budding suggests the existence of a previously unrecognized mechanism that regulates the timing of ALIX and other proteins in assembling new HIV particles.

"We discovered that the cellular components that help with the release of the virus arrive in a much more complex timing scheme than predicted based on the biochemical data," he says.

"The outcome of this study is promising because it uncovers a new regulatory mechanism for recruitment of cellular components to HIV budding sites, and opens the door to exciting future studies on the mechanism of HIV budding."