Researchers uncover clues to flu's mechanisms

The influenza hemagglutinin protein reconfigures itself as it targets host cells to infect them. Until new analysis by Rice University and Baylor College of Medicine researchers, nobody had been able to study the intermediate states of the protein-refolding process that may be vulnerable to treatment with drugs. Credit: Jeffrey Noel/Rice University

A flu virus acts like a Trojan horse as it attacks and infects host cells. Scientists at Rice University and Baylor College of Medicine have acquired a clearer view of the well-hidden mechanism involved.

Their computer simulations may lead to new strategies to stop influenza, perhaps even a one-size-fits-all vaccine.

The discovery detailed this week in the Proceedings of the National Academy of Science shows the path taken by hemagglutinin, a glycoprotein that rides the surface of the influenza virus, as it releases fusion peptides to invade a .

The release mechanism has been the subject of many theories, but none have explained experimental observations as well as the new work led by biophysicist José Onuchic at Rice and biochemists Qinghua Wang at Baylor and Jianpeng Ma, who has a joint appointment at the two institutions.

The Rice-Baylor team applied protein-folding algorithms developed by Onuchic and his colleagues to analyze how hemagglutinin reconfigures itself as it infects a cell.

Hemagglutinin is completely folded at the start of the process of interest to researchers who study viral infection, Ma said. "It may be the only case known to human beings where a protein starts at a fixed point and literally completely refolds," he said.

Proteins are the molecular motors that spring from DNA and perform tasks essential to life, and they are the prime focus of study for Onuchic and his colleagues at Rice's Center for Theoretical Biological Physics (CTBP). The researchers use their energy landscape theory to determine the path an unfolded strand of amino acids takes as it collapses into a final, functional protein. That involves calculating the energetic preferences of every acid in the chain as well as the influence of the surrounding environment as folding progresses.

The influenza hemagglutinin protein may take two paths as it reconfigures while attaching a virus to a host cell: It can send all its fusion peptides to the host or split them between the host and the virus. Researchers at Rice University and Baylor College of Medicine hope to identify a point during reconfiguration at which drugs could inactivate the peptides. Credit: Nathanial Eddy/Rice University

When Ma met Onuchic a few years ago, he recognized an opportunity. "I told him there's a very important feature of the viral system that would be ideal for his energy landscape approach." Ma said.

Researchers have long observed hemagglutinin's initial and final structures through X-ray crystallography. But because the change happens so quickly, it has been impossible to capture an image of the glycoprotein in transit. Ma said the key to stopping the flu could be to attack these intermediate structures.

Energy landscape theory predicts how a protein will fold no matter how fast it happens. In the case of hemagglutinin, the unfolding and refolding happens in seconds. During the process, part of the protein "cracks" and releases fusion peptides.

"The fusion peptides are the most important part of the molecule," said Rice postdoctoral researcher and co-author Jeffrey Noel. "The hemagglutinin is attached to the viral membrane, and when these peptides are released, they embed themselves in the target cell's membrane, creating a connection between the two."

"The purpose of hemagglutinin is to poke a hole between the two membranes," Ma said. "They have to fuse so the genetic material will be injected into the human cell."

Hemagglutinin is recognized by polysaccharide receptors on host cells and is absorbed when the cells engulf it. Initially, part of the protein forms a cap that protects the segments inside.

Acidic conditions cause the cap to fall off, and the protein begins to reconfigure itself. "The release of the fusion peptide, which is initially hidden inside hemagglutinin, is triggered by that giant conformational change," Ma said.

"When the cap is on, the whole protein is stable," Noel said. "What we see in the simulation is that the hydrophobic pocket where the fusion peptides are buried is very unstable and wants to crack as soon as the cap comes off."

By using the experimental structural information from X-ray crystallography to approximate the full energy landscape of hemagglutinin, the researchers can now capture a rough picture of the steps involved in its reconfiguration, including the point at which the peptides are released. "We now, for the first time, have mapped out the entire process, going from state A to state B, and the energetics along the way," Ma said.

Ma said frequent mutations to the cap help the virus avoid antibodies; this is the reason people need flu shots every year. But he suspects the inner part of the is more highly conserved. "We're targeting the part that the virus cannot afford to change. Therefore, it provides more hope for developing therapeutic agents," he said. Such agents could lead to a that would last a lifetime.

He said the membrane fusion mechanism is widely shared among many biological systems, which makes influenza a good model for studying other diseases. "HIV has one. Ebola has one. And it's also shared by intercell transport in the nervous system," Ma said.

He noted the work could not have been done without CTBP, which moved to Rice from the University of California, San Diego, three years ago to take advantage of collaborations with Texas Medical Center researchers – one of Rice's Priorities for the New Century. "This demonstrates a very interesting collaboration between TMC and Rice," Ma said. "We're very happy with that."

More information: Xingcheng Lin, Nathanial R. Eddy, Jeffrey K. Noel, Paul C. Whitford, Qinghua Wang, Jianpeng Ma, and José N. Onuchic. "Order and disorder control the functional rearrangement of influenza hemagglutinin." PNAS 2014 ; published ahead of print July 31, 2014, doi:10.1073/pnas.1412849111. www.pnas.org/content/early/201… /1412849111.abstract

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thingumbobesquire
5 / 5 (3) Aug 01, 2014
Very important work here leading to pathway for universal flu vaccination.
JVK
1 / 5 (3) Aug 01, 2014
Re: "Ma said frequent mutations to the cap help the virus avoid antibodies; this is the reason people need flu shots every year."

I thought nutrient-dependent amino acid substitutions that stabilize the virus in an ever-changing ecological environment were the reason people need flu shots every year.

http://www.scienc...abstract
"The molecular basis of antigenic drift was determined for the hemagglutinin (HA) of human influenza A/H3N2 virus. From 1968 to 2003, antigenic change was caused mainly by single amino acid substitutions..."

I also thought that mutations perturb protein-folding, and would like to learn the difference between what some researchers call a mutation and what I think is a nutrient-dependent ecological adaptation.
JVK
not rated yet Aug 06, 2014
The only mention of mutations in the journal article was in this context:

"Although experiments have probed the fusion mechanism through mutation (8) and provided measures of fusion kinetics (9), there is a lack of structural information about how HA2 transitions from the prefusion to postfusion conformations."

However, the mutation appears to be loosely linked to mutation-initiated natural selection and evolution: "The dual-funneled landscape of HA2 has evolved to avoid this fate."

If the antigenic change from 1968 to 2003 was attributed to single amino acid substitutions, why are these researchers still reporting results in terms used by evolutionary theorists?.

http://www.scienc...abstract
anonymous_9001
5 / 5 (1) Aug 06, 2014
Substitutions are the result of mutations.

http://scholar.go...t=1%2C14
JVK
not rated yet Aug 07, 2014
Mutations perturb the protein folding that amino acid substitutions stabilize, which is why theorists are now trying to make it look like they really meant that epimutations initiate natural selection that leads to the evolution of biodiversity, despite the clarity of the fact that amino acid substitutions link ecological variation to ecological adaptations in species from microbes to man via conserved molecular mechanisms.

Thanks for the link to so many irrelevant published articles. They exemplify how pervasive pseudoscientific nonsense can be -- enough to influence at least 3 generations of theorists who never asked is there a model for that, but accepted what they were taught to believe in and became the idiot minions and anonymous fools their teachers wanted them to become.