After infecting a susceptible cell, the human immunodeficiency virus hijacks that cell's normal machinery to produce carbon copies of itself. New HIV particles roll off the cellular assembly lines, burst like bubbles out of the cell, and float off to invade other cellular factories. Vanderbilt University Medical Center investigators have now identified an early step in HIV particle assembly.
The findings, published March 11 in Cell, could lead to new drugs that combat HIV infection by shutting down the virus's assembly lines.
For several years, Paul W. Spearman, M.D., associate professor of Pediatrics and Microbiology & Immunology, and colleagues have been studying the assembly of HIV particles, specifically the distinct steps HIV structural proteins take in order to come together and create a viral particle.
"The assembly process is just one part of the whole HIV life cycle," Spearman noted, "but it's an important part in that each step along the way is required to make an infectious viral particle."
Spearman's team has focused on a protein called "Gag," the major HIV structural protein. In recent years, Spearman said, it has become apparent that Gag moves to a compartment in the cell called the multivesicular body, or late endosome. In some cell types, Gag and the HIV viral envelope protein form particles in the multivesicular body; in other cell types, Gag makes its way from this site to the cell membrane before assembling into particles.
Although many studies have demonstrated that Gag is present in the late endosome and have focused on particle assembly at that point, none have tackled how the Gag protein gets there in the first place. The current work fills this gap.
Spearman and colleagues used the HIV-1 Gag protein as bait to "fish" for Gag binding partners. They identified several known and novel interacting proteins and selected one, the delta subunit of AP-3, for further evaluation. AP-3 is an "adaptor protein complex," a group of four proteins known to sort cargo proteins to specific compartments in the cell.
Through a series of experiments, the group demonstrated that the AP-3 delta subunit interacts with Gag to direct it to the multivesicular body. Disruption of the interaction, using a specific piece of the AP-3 subunit, eliminated Gag trafficking to the multivesicular body and diminished HIV particle formation in cells.
"The significance of this paper comes from really identifying how Gag gets to the multivesicular body and in demonstrating that if you block that trafficking step specifically, you block production of particles," Spearman said. "That says this is not a dead-end pathway, but that it is part of the normal, productive assembly pathway."
The newly identified early step in the HIV assembly process could be a target for a new generation of drugs to combat the virus. No existing anti-HIV drugs disrupt particle assembly or the movement of Gag in the cell.
"We have hopes of identifying compounds that inhibit this Gag-AP-3 interaction and that may lead to new efforts to treat HIV infection," Spearman said.
Such drugs should be very specific, he said, since it should be possible to block Gag's interaction with AP-3 without disrupting AP-3 function in the cell. And by targeting an early step in HIV particle assembly, an inhibitor of this sort would be expected to block viral replication in cells that complete assembly at the multivesicular body as well as those that assemble particles at the cell membrane.