Site-Specific Labeling Offers Novel Flu Analysis

Michael Vahey, PhD

As the influenza (flu) virus varies in composition in humans, it has long been challenging to study the number and location of proteins on individual virus strains.

But a new study utilizing site-specific labeling allowed investigators to take a more detailed look at flu viruses, to determine whether the variation seen in individual flu viruses might be adaptive and help the virus to spread infection.

Study author Michael Vahey, PhD, assistant professor in the School of Engineering & Applied Science at Washington University in St. Louis, suggested the study intended to assess under what circumstances is the flu virus adaptive, and how so.

"This is a first step toward understanding that. But it's not a complete picture,” Vahey said in a statement.

Through his method of site-specific labeling, Vahey inserted sequences of 5-10 amino acids into the proteins that make up Influenza A virus—the most common strain, and also the most dangerous to humans.

After inserting these short sequences, he and his team introduced enzymes and small amounts of fluorescent dyes. These enzymes take different dye molecules and connect them to the engineered viral proteins, giving investigators the ability to see individual proteins without disrupting their function.

Specifically, investigators examined the proteins hemagglutinin (HA) and neuraminidase (NA). HA is responsible for allowing a flu virus to attach to a cell, and NA is responsible for decoupling the virus from the cell so that it can go on to infect others.

"Once we have the ability to label individual viruses, we can image them and quantify how much of each protein they have per particle, and what the size of that particle is," Vahey explained.

Investigators found viruses that are smaller or have more NA are more resistant to the NA inhibitor, thus being more likely to have the ability to detach from a cell that has been challenged with oseltamivir (Tamiflu) and go on to infect more cells.

Additionally, viruses with more HA, or that are larger, were found to have the ability to bind more strongly to cells. Under any circumstance, Vahey said, this range may be most beneficial to patients.

"In the case of Tamiflu treatment, you're inhibiting NA such that the viruses that happen to have more NA and also happen to be smaller now have a little bit of a leg up,” Vahey said. "If you have an environment that is changing rapidly over time, if you were reliant on genetic adaptations, you might be in some trouble, because it takes a certain amount of time for mutations to accumulate."

That said, Vahey noted that phenotypic diversity quickly generates change. When a virus replicates, the next generation varies, with elements potentially being more suited to its current environment.

In terms of future research, Vahey acknowledged that the importance of phenotype may have an impact on the development of vaccines.

"Typically in the development of a flu vaccine, you're concerned about how genetic changes in the virus may reduce the effectiveness of the vaccine," Vahey said. "This could be an additional consideration, how variation in viral phenotype may contribute."

The study, “Low-Fidelity Assembly of Influenza A Virus Promotes Escape from Host Cells,” was published online in the journal Cell.