Hiding in plain sight

EDINBURGH, Scotland—A group of researchers in the UnitedKingdom and the United States, notable among them University of Edinburgh Prof.Paul Digard, have located and described a previously unknown influenza genethat, while it may not be a total game changer, sheds an important light on thedynamics of flu infections.

The results, published in the July 13 issue of Sciencemagazine, prompted Discover magazine to gush that the finding is "likesomeone took the text of Macbeth, put the spaces in different places, and got Hamlet."Digard tells ddn that's a bit of hyperbole, and it's probably moreaccurate to say that it's like finding an excellent sonnet hidden in the textof a short play.

As Digard summarizes the process, he and other researchersdiscovered a new gene in the influenza virus that helps the virus control thebody's response to infection and, while this control is exerted by the virus itself,the surprising part is that it reducesthe impact of the infection—a finding that can help other researchers betterunderstand how flu can cause severe infections and guide them in research fornew treatments.

Speaking more technically, the abstract for the Sciencearticle says, "We report that segment 3 of the virus contains a second openreading frame ("X-ORF"), accessed via ribosomal frameshifting. The frameshiftproduct, termed PA-X, comprises the endonuclease domain of the viral PA proteinwith a C-terminal domain encoded by the X-ORF and functions to repress cellulargene expression. PA-X also modulates IAV virulence in a mouse infection model,acting to decrease pathogenicity. Loss of PA-X expression leads to changes inthe kinetics of the global host response, which notably includes increases ininflammatory, apoptotic and T lymphocyte-signaling pathways."

The ultimate importance of this finding is yet to bedetermined, though the feat of finding a new gene after so many decades ofinfluenza research is nothing to sniff at, notes Digard, a professor oflivestock immunology at the Roslin Institute at the University of Edinburgh.

"Knowing the gene is there and showing it matters in a mousemodel of infection with one strain of virus is the first step," says Digard,who says his current job title doesn't do a good job of explaining what hedoes, being a molecular virologist with some 25 years of experience withinfluenza and formerly a senior lecturer on influenza at the University ofCambridge. "It gives us more clues about virus disease mechanisms, but it'sgoing to be a lot more work to understand whether all flu strains behave thesame way and to work out whether we can exploit this understanding for bettercontrol of the virus in humans or animals."

So, while he says he doesn't want to oversell the finding,and there are no immediate implications for therapeutic or diagnosticbreakthroughs, Digard emphasizes that it "reiterates the fact that when youhave severe disease, a lot of the damage comes from inappropriate immuneresponse rather than directly from the virus, because when you knock out thatgene, we found that you don't reduce viral load. It's just that at the pointmice should be beginning to clear out the virus, things go wrong. So it pointsto the fact that in treating severe flu or any other disease, it's not justabout clearing the virus but about making sure the immune system is doing theright thing."

Digard notes this might be a cautionary note in particularfor those who want to treat the flu with immune modulators, noting, "you needto get it right if you start altering immune response, as it may not necessarilymake things better."

Digard explains that finding this gene has been in part ahappy accident and in part diligent teamwork between his lab and researchers atthe Universities of Cambridge, Cork, Edinburgh and Utah, the Institute ofSystems Biology in Seattle and the U.S. National Institutes of Health.

"Students in my lab had worked out that there was ahost-cell shut off activity in segment 3 some time ago, and we could havewritten something up as long as six years ago," Digard says. "We didn't becausethere were some inexplicable bits of data that made it clear we didn't properlyunderstand what was going on. Around the same time we got our first sight ofthe ORF. Of course, I tried to link the two things, but couldn't put the piecestogether in a way that fit properly—this took a collaborator with betterknowledge of unusual events during protein translation, Andrew Firth, topropose the ribosomal frame shifting mechanism that allows PA-X expression. Thethird piece of the jigsaw came from collaboration with Jeff Taubenberger,through the happy link of a joint grad student, Brett Jagger, that facilitatedthe animal and microarray experiments to show the hidden gene matters duringinfection."

Reportedly, Jagger first noticed something odd about thegene when he realized that one part of it was incredibly similar acrossdifferent flu strains. Because flu viruses evolve so quickly, anything thatremained constant, he thought, must have some significant importance.

Researchers found when the virus gene PA-X was active, miceinfected with flu subsequently recovered. When the PA-X gene did not workproperly, the immune system was found to overreact, thus making the infectionworse, and did not help destroy the virus any quicker.

"It really was a collaborative effort to pull this storytogether, and none of us could have done it without the others," says Digard,who will soon be publishing an article on yet another hidden flu gene that wasdiscovered and characterized almost solely in his lab. Of that, virus, he says,"we figured it out ourselves," but adds that based on what he's seen so far,this newly discovered gene offers "no evidence that it affects pathogenicity."

Commenting on the PA-X gene in the Discover articleabout the recently published findings, Wendy Barclay, a flu researcher fromImperial College London who has worked with Digard before, asked rhetorically,"How can we have missed it?"—a question that could probably be asked of thesecond hidden flu gene Digard will soon write about—then added, "It justemphasizes how compact these genomes are."