New flu gene found hiding in plain sight, and affects severity of infections

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I could write the entire genome of a flu virus in around 100 tweets. It is just 14,000 letters long; for comparison, our genome has over 3 billion letters. This tiny collection of genetic material is enough to kill millions of people. Even though it has been sequenced time and time again, there is still a lot we don’t know about it.

A new study beautifully illustrates the depths of our ignorance. Brett Jagger and Paul Digard from the University of Edinburgh have discovered an entirely new flu gene, hiding in plain sight among the 12 we knew about. It’s like someone took the text of Macbeth, put the spaces in different places, and got Hamlet.

This new gene, known as PA-X, affects how the virus’s host responds to the virus. Oddly, it seems to reduce the severity of infections. “This is indeed an exciting finding in the flu field,” says virologist Ron Fouchier. “How can we have missed it?” asks Wendy Barclay, a flu researcher from Imperial College London who has worked with Digard before. “It just emphasizes how compact these genomes are.”

Most influenza viruses belong to the influenza A group – these are the ones behind all the big pandemics, the seasonal strains that sweep the world every year, and the mutant strains that have caused such a stir recently. Each influenza A virus is a shell containing eight strands of RNA, a genetic molecule related to DNA. But some of these strands encode multiple genes, each of which produces a different protein. Until recently, we thought that the eight strands contain 12 different genes, and the new study raises that number to 13. The influenza genome, it turns out, is absolutely packed with overlapping instructions.

Here’s how it works. RNA is made from building blocks called nucleotides, denoted by the letters A, C, G and U. Every set of three letters corresponds to an amino acid, which string together to form proteins. For example, GCA equals alanine, while AGU equals serine. Using this code, you can build a protein from a sequence of RNA, by translating its letters into a chain of amino acids. But of course, it all depends on where you start.

Consider this short sequence: AGUCCAAGGUAUG. If I start translating it from the first A, I get Serine-Proline-Lysine-Tyrosine and one letter left at the end. But if I start translating from the second letter (G), I get a completely different chain: Alanine-Glutamine-Glycine-Methionine. The same sequence can be parsed in up to three different ways, known as ‘reading frames’. This is how the flu virus can double-dip its genetic material to get two genes out of the same sequence.

The new gene that Jagger discovered is another double-dip. It’s found in the virus’ third RNA strand, which was traditionally thought to only contain the PA gene. PA helps the virus copy its genome. Jagger first noticed something weird about the gene when he found that one part of it was incredibly similar across different flu strains. Flu evolves at a breakneck pace, so any island of constancy amid this sea of change must mean something. Jagger discovered that this conserved region contains a second gene called PA-X.

RNA is translated into amino acids by molecular factories called ribosomes. When ribosomes reach the conserved region of PA, they encounter the letters CGU – one of the rarest of all triplets. This stalls the ribosomes long enough that some of them slip forward slightly. They start reading the gene from one letter along, producing an entirely different chain of amino acids. This is PA-X.

Fouchier notes that “the conservation of PA-X in flu virus genomes certainly suggests that [it] is important under normal circumstances.” But while its sister gene PA allows the virus to copy itself, PA-X has a different role.

It cuts up bits of RNA from the virus’s host, stopping it from activating its own genes. This process, known as host-cell shut-off, is a win-win strategy for the virus. It stops the host from mounting an effective defence against the virus, and it means that the host is more likely to manufacture proteins using the virus’s genetic instructions, rather than its own destroyed RNA.

To understand how this helps the virus, Jagger took the strain of flu behind the 1918 pandemic and mutated it so that the PA-X gene no longer worked properly. Without the ability to shut down the host cell’s response, you’d expect that these mutant viruses would be cleared away more easily. But not so – the mutant virus actually proved to be more deadly than the normal 1918 strain, causing greater weight loss in infected mice, and killing more of them.

“At first sight, it is paradoxical,” says Digard. It seems that without PA-X, the infected cells activate immune genes more intensely and much earlier in the course of infection. This triggers a similar response from nearby uninfected cells, leading to an overly vigorous counter-attack and, ironically, more severe illness. These experiments suggest that PA-X is something of a viral ambassador. It manipulates the host’s genes to control how it responds to the virus.

Beyond that, the new study leaves us with many questions. “Does genetic variation in PA-X in nature account for some different outcome of disease?” asks Barclay. Can we produce better treatments for flu by targeting the gene? PA apparently influences how well bird flu viruses can reproduce in mammal cells, so does PA-X help the virus cross the species barrier? And why do pig flu viruses have a shorter version of PA-X than those from other animals? It staggers the mind that after decades of research, scientists can still open up such a miniscule genome and find a treasure trove of unanswered questions.

And given that we’ve just found a new flu gene, Barclay asks, “Are there any more? I bet Digard is looking.” He most certainly is. “I’m working on a manuscript today that describes another one,” he told me.

Reference: Jagger, Wise, Kash, Walters, Wills, Xiao, Dunfee,Schwartzman, Ozinsky, Bell, Dalton, Lo, Efstathiou, Atkins, Firth, Taubenberger & Digard. 2012. An Overlapping Protein-Coding Region in Influenza A Virus Segment 3 Modulates the Host Response. Science

Image by Doug Jordan, CDC

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