He Who Hesitates …

Anton 2 Simulations Identify Critical Pause in Flu-Virus Protein Motion; May Be a Target for Future Therapy

Viruses such as influenza and HIV take a heavy toll, both in human life and in dollars. But these viruses are shape-shifters, changing their outer proteins via mutation so human immunity and antiviral drugs have a hard time keeping up. A team from Rice University and Baylor College of Medicine has used the Anton 2 supercomputer at PSC to simulate the role that a portion of a critical protein called “hemagglutinin (HA)” plays in the flu virus merging with host cells and injecting its genes. They found that the protein’s “stem domain,” HA2, pauses in the process—a critical step that may offer a target for future therapy that can outsmart the viral transitions. The work offers a completely new approach to therapy since it focuses on the critical injection mechanism rather than the virus’s outward structures.

Why It’s Important:

In the 2018-19 flu season, the influenza virus killed more than 57,000 people in the U.S. The flu costs the country between $2 billion and $6 billion a year in health care costs and economic loss. HIV, the virus that causes AIDS, killed over three quarters of a million people world-wide in 2018. Across the globe, AIDS treatment and losses cost half a trillion dollars between 2005 and 2015.

Despite many years of hard work by scientists, the flu vaccine remains imperfect, with its effectiveness varying from year to year. And there still is no HIV vaccine. This is because the viruses’ outer coat proteins, which they use to attach to human cells, are in essence shape-shifters. These proteins mutate between virus generations so rapidly that they are an ever-changing target for both vaccines and drugs.

<Two_fold_states.jpg>HA2, initially covered by HA1, sits in the outer membrane of the virus (left). When it encounters the host cell membrane, HA1 falls off, revealing the folded version of HA2 (center). The HA2 protein then unfolds, enabling its fusion peptides (top of right-hand molecule) to fuse the viral and cell membranes and release the virus’s genes into the cell’s interior.

But these viruses need to do more than just attach to host cells to invade them. They must also merge their outer membranes with the host cell membrane so they can inject their genes into the target cell. José Onuchic and colleagues at Rice University and Baylor College of Medicine wondered whether that second step might be a better target for attacking the viruses. To figure this out, they have been studying the HA2 “stem domain” of the outer coat protein of the influenza virus, which causes the viral and host membranes to fuse.

“The idea here is if you are the flu virus, you have to go inside the cell. The question is how this happens. There is this HA2 protein that has to undergo a complete conformational change. During this transition there are some peptides in HA2 that we call the fusion peptides that have to get out of the protein and connect to the cell membrane … It’s a protein-folding problem; HA has to go from one particular shape to another.”—José Onuchic, Rice University

The Rice-Baylor team focused on the process by which HA2 components called fusion peptides reach out between the viral and host membranes, like arms pulling the membranes together. Other scientists had proposed this happens in a “spring-loaded” way, with the protein refolding to connect the two membranes in one quick snap. But Onuchic’s grad student Xingcheng Lin and postdoctoral fellow Jeffrey Noel had carried out theoretical work indicating that the HA2 protein wouldn’t refold in one go like that. There is no way to test the motion of the protein in the lab directly. So the scientists turned to simulations on the D.E. Shaw Research Anton 2 supercomputer hosted at PSC and operationally funded by the National Institutes of Health.

How PSC Helped

Simulating the motions of HA2 was a challenge. It’s a huge protein, far bigger than current computers can simulate in a reasonable amount of time. “Modeling” HA2 successfully would mean simulations of the molecule in parts and more or less as a whole. The team would have to use Anton 2 to simulate systems with as many as 698,495 atoms, including the protein, the membrane lipids, water molecules and other components—at the limit even for the abilities of Anton 2 at PSC. To make matters worse, scientists estimate that in real life the critical step of the HA2 membrane fusion process takes milliseconds (thousandths of a second) to happen. In computer simulation time, that is also huge.

Anton 2, which was specifically designed to simulate the motions of large molecules, was the only computer that could handle both the size of the system and the length of the simulation. Working with PSC’s Marcela Madrid over a period of two years, Lin and Noel were able to carry out some 40 microseconds (millionths of a second) of simulations on Anton 2 that helped nail down what HA2 was doing. Jianpeng Ma and Qinghua Wang at Baylor College of Medicine also played vital roles in the work.

“This was really a perfect Anton simulation. You needed several microseconds of this gigantic system; it wouldn’t have been possible to do it any other way, both based on size and on time.”—José Onuchic, Rice University

The simulation agreed with the earlier theoretical work. The virtual HA2 in the computer only refolded halfway; then it paused before completing its motion. It turned out that an amino-acid component of HA2, called “threonine 59,”was hesitating before entering a coil called an “alpha helix” in the final protein fold. This was because threonine, a “water-loving” amino acid, is more strongly attracted to the water in the protein’s surroundings than the other “water-hating” amino acids in the alpha helix.


Just before it fuses the viral and host cell membranes, the HA2 protein transitions from a folded-up shape (left) into a disordered, “random loop” shape before snapping into its unfolded, “coiled coil” shape (right). The Rice University team’s Anton 2 simulations suggest that the process pauses at the random loop, giving time for the fusion peptides (light blue? Purple?) to pull the two membranes together and saving some of the unfolding energy to drive the membrane fusion. From Lin et al. (2018) Atomistic simulations indicate the functional loop-to-coiled-coil transition in influenza hemagglutinin is not downhill. PNAS 115(34):E7905-13. © 2018 National Academy of Sciences.

The pause in the refolding makes a lot of sense biologically. For one thing, it halts the protein’s movement to give the fusion peptides time to reach out to the host membrane. For another, it means the energy released by the refolding is held in check long enough for the protein to use it to drive the fusion of the viral and host membranes.

“There are two reasons why spring loading couldn’t work. If HA2 switches its form too fast, you don’t give enough time for the fusion peptides to capture the target membrane. You also don’t have enough free energy to pull the two membranes together.”—José Onuchic, Rice University

Best of all the pause is a promising target for therapy. If scientists could learn how to lock the HA2 protein at this half-way point, the virus can’t merge with the host membrane and can’t inject its genes into the cell. Better yet, threonine 59 is present in all known strains of the group 2 influenza viruses, which represent half of flu viruses. This may mean its function in the pause is so critical the virus won’t be able to mutate away from therapies designed to target it. An important objective going forward for the Rice-Baylor team will be to compare different strains of the flu virus to reconstruct the evolution of HA2—particularly threonine 59. They want to see what parts of the protein are critical for the pause to happen in order to identify drugs and other therapeutic means to lock it into place.