Two different genes play opposite roles in influenza virus evolution

The influenza virus that causes influenza is a major public health problem, infecting millions of people and causing an estimated $10 billion in direct medical costs
in the United States each year.
Like most viruses, the flu virus mutates rapidly as it spreads, making it difficult
to vaccinate against every possible strain.
Every year there is a great deal of effort to determine which strain is likely to be most prevalent in order to produce a vaccine
that provides the best protection against that season.

"It's a high-stakes guessing game
.
We're basically sitting here trying to guess what variants
will come up next.
I think it underscores how important it is to better understand the basic rules that govern the evolution of viruses and how they escape our immune system," said
Chris Brooke (IGOH), associate professor of microbiology at the University of Illinois at Urbana-Champaign.

Brooke explained that most of the research on flu vaccines has focused on the evolutionary potential of the surface protein hemagglutinin, because this is the main target protein
of our immune system.
Hemagglutinin binds to receptors on the surface of our cells, allowing the influenza virus to enter and begin to replicate
.
However, another surface protein called neuraminidase has largely been
overlooked.
NA is important later in the viral life cycle because it destroys receptors originally used to enter, divides cells and releases virions
inside.
Although HA and NA act in opposite directions, they are functionally highly correlated with each other because they are both necessary for
viral infection and transmission of cells.

This paper, published in Cell Host & Microbe, Brooke's team explores how changes in NA activity affect the evolutionary potential
of HA.
To do this, they used a wild-type flu strain, as well as two identical strains to the wild-type, except for a mutation
that reduced NA activity.
Next, in collaboration with Nicholas Wu (IGOH/MMG), assistant professor of biochemistry at the University of Illinois, the team used a process called deep mutation scanning to essentially create a library
of HA mutations of their three flu strains.
Researchers can then measure HA activity and the fitness
of mutant strains.

"This is a high-throughput method that introduces each possible amino acid substitution into a specific region of interest and then measures the effect
of these substitutions on relative fitness," Brooke said.
We can then quantify the fitness impact
of a particular replacement based on the paired NA genes.
”

Through this process, Brooke's team determined that viral strains with reduced NA activity had higher mutation tolerance in changes in HA
.
In other words, when influenza strains with lower NA receptor binding are paired with mutations that also bind less to HA receptors, their fitness is largely undiminished compared to wild-type strains, which tend to have a decrease
in fitness.
Brooke explains that this may be due to the opposite function of HA and NA, with the former invading cells and the latter escaping cells
.

"If the replacement of NA reduces its activity, we see that the compensatory replacement of HA also reduces its relative activity, bringing them back into equilibrium
.
" If only one of them rises or falls, the cells lose their balance, reducing overall adaptability
.
So, you'll see a compensatory substitution that brings it back to the optimal range
.
”

"I think we were all surprised to see that," said Tongyu Liu, a graduate student in Brooke's lab and the paper's first author
.
"The textbook view of mutations is that they are mostly harmful
.
But in our experiments, we demonstrated that the interaction between HA and NA can reshape the fitness effects of mutations, from primarily harmful to primarily neutral
.
”

The researchers also cultured the same strain with reduced NA activity in the presence of neutralizing antibodies targeting HA to see what mutations
would occur.
This experiment basically mimics what happens in the human body, when immune cells target and attack the HA protein of flu cells to clear the infection
.
By looking at which HA variants are selected to evolve in this environment, the researchers found more evidence that the pathways by which HA evolved to escape immune stress were highly dependent on the function of
NA.

In flu vaccine research, the evolution of HA has mostly been done in isolation from other genes, but Brooke said this data points to the need to study the interactions between HA and other protein genes, such as NA, in order to better predict how HA will evolve
based on mutations in these other genes in different strains.
It is hoped that this research will be used to improve the accuracy of predicting major genotypes in the future and to develop vaccines
against these strains.

"Every year we try to pick targets for next year's vaccine from many different co-circulating flu strains, and if we choose the wrong one, it can lead to more serious infections and deaths
," Brooke said.
Therefore, it is important to understand the rules that govern the evolution of the virus so that we can better predict its specific path
.
”

The evolutionary potential of influenza A virus hemagglutinin is highly constrained by epistatic interactions with neuraminidase