Structure of dueling proteins points to HIV origins, treatment avenues

When HIV infects human immune cells, a protein known as A3G fights back by thwarting the virus’s attempts to replicate itself. But HIV retaliates with a protein called Vif, which marks these A3G proteins for elimination by the cell’s own machinery. The genes for these two proteins coevolved in a molecular arms race, explains biochemist John Gross of the University of California, San Francisco. “Each side accumulates mutations to gain the upper hand,” says Gross. “One side is always winning, and it goes back and forth throughout millions of years of primate evolution.”

Now, Gross and colleagues have uncovered the physical structure of a human A3G protein bound together with an attacking Vif protein. The findings, recently published in Nature, point to a possible drug target and reveal in greater detail how HIV evolved to infect humans in the first place.  

HIV, like all retroviruses, replicates by reverse-transcribing its own viral RNA into viral DNA, which is then inserted into the host cell’s genome. There, it’s transcribed and translated to produce more virus. A3G blocks this process by mutating the viral genome so that it can’t replicate.

The virus circumvents this defense by using Vif to attach a ubiquitin protein to A3G. Ubiquitin acts as a signal, telling the cell to degrade the protein. The infected cell proceeds to destroy its own defense.  

Previous genetic work by study coauthor Michael Emerman of the Fred Hutchinson Cancer Research Center in Seattle, WA, showed that Vif had an important role in helping the precursor to HIV jump from monkeys to chimpanzees—and ultimately humans. Essentially, the Vif protein evolved to be able to attack the chimpanzee version of A3G.

But although this evolutionary history had been well characterized, the actual physical form that these dueling proteins take when locked together remained elusive. “We really wanted to understand the structural basis of how viruses like HIV-1 are born,” says Gross.

To find out, UCSF structural biologist Yifan Cheng and UCSF postdoc in pharmaceutical chemistry Yen-Li Li sought to reveal the Vif-A3G interaction via single particle cryogenic electron microscopy (cryo-EM). They uncovered the 3D structure of the interaction by capturing numerous images of individual sets of these proteins locked together while frozen in a thin layer of ice. Computational processing combined the 2D images into a high-resolution 3D structure.

As expected, the resulting structure shows Vif bound to A3G along with other cellular proteins that the virus co-opts to help with attaching the ubiquitin. Unexpectedly, however, it shows a single strand of RNA locked in place between Vif and A3G. The authors suspect that this RNA belongs to the cells of the fall armyworm insect, which the team used as a cost-effective vessel for generating the human and HIV proteins for the study. During an actual HIV infection, they hypothesize, this RNA would belong to the virus itself, since A3G is known to bind to viral RNA as it’s attacking the virus. This RNA strand then works as a “molecular glue” that helps Vif attach to A3G, explains Gross.

“Based on previous studies, the A3G–Vif interaction was expected to be purely protein based,” says computational imaging scientist Alberto Bartesaghi at Duke University in Durham, NC. “Instead, this work shows that they form a ribonucleoprotein complex,” notes Bartesaghi, who was not involved in the work.  

Previous genetic work by Emerman’s lab identified two key locations on Vif and A3G that were involved in the evolutionary arms race. The present structural analysis shows that the proteins at these two sites are indeed in direct contact with one another, rather than being separated by RNA.

The researchers then used a computer model based on this structure to explore how Vif and A3G would have interacted in the red-capped mangabey monkey before jumping to chimpanzees and, ultimately, to humans. Their model suggests that the evolution of Vif at one direct point of interaction with A3G may have allowed Vif to overcome the chimpanzee’s A3G defense. Meanwhile, the points on each of these proteins that bind to RNA have remained relatively unchanged over long evolutionary timescales. “These findings may help suggest routes for A3G escape from Vif targeting,” says Bartesaghi, “and potentially open new avenues for therapeutic development against HIV-1 and other lentiviruses.”

Right now there are no HIV drugs on the market that target Vif, notes Sebla Kutluay, a molecular virologist at Washington University School of Medicine in St. Louis, MO, who was also not involved in the study. “If we target this interaction, VIF will no longer be able to block A3G from doing its antiviral function,” she says.

Still, Kutluay cautions that HIV could eventually find ways to mutate its Vif gene to evade any drugs that block its function. The arms race, she says, could very well continue.