Comprehensive approaches to understand the interaction of H1N1 strains with human cells

Dec 18 2009

Scientists have generated hundreds of new leads in the fight against the H1N1 flu pandemic, according to two new studies published online December 17th in the journal Cell, a Cell Press publication. Both research teams took comprehensive approaches to understanding the interaction of H1N1 strains with human cells, yielding results that point toward new targets for therapy and perhaps also new tools to speed vaccine production, the researchers say.

One study took a "multilayered" approach to understand the physical interactions between the virus and human host cell as well as changes in the host as it is manipulated by and responds to viral infection. Aviv Regev and Nir Hacohen of The Broad Institute of MIT and Harvard said their goal wasn't to drill down into any specific interaction; rather their findings provide the research community a global "roadmap" -a bird's eye view of the human-virus interaction - that can now help guide future studies.

"Navigating with a map is very different than without it," Regev said. "Without a map, you might explore a small patch and stay very close to that. There might be a protein you know something about and you'll look around there. With a global map, you may find uncharted territory. It's like discovering America. You still have a lot to learn, but at least you know it's there."

"This approach generates questions we didn't have before," Hacohen added. "It allows us to stumble upon things we didn't know we needed to figure out."

Their findings reveal how the virus alters the expression of host proteins involved in the detection of viral RNA and in the inflammatory response. Their data also suggest roles for some unanticipated host and viral proteins in viral infection and the host response, including a network of proteins that bind RNA, components of a signaling pathway involved in cell proliferation, and subunits of the viral polymerase that were previously only thought to be responsible for replication of the viral genome.

The approach they took included assays to examine the direct physical interaction of viral and host proteins along with genome-wide expression profiling of infected human cells. Those studies led them to more than 1,700 candidate genes with potentially important roles to play in the infection with influenza or the host response to that infection. They then depleted each of those genes one by one in human lung cells to find those that affect viral replication.

In providing a big picture of the interaction, the researchers also show that the virus's 10 proteins "pack a lot of functions into each one," Hacohen said. "We were impressed by how many host proteins interact with each viral protein," Regev said. "The viral proteins contact the same [human] pathways again and again." That gives the virus many ways to manipulate important pathways, although the researchers don't understand the consequences of those interactions just yet.

In the second study led by Stephen Elledge of Harvard Medical School, researchers went in search of host cell modifiers of influenza A H1N1 viral infection. They used RNA interference to turn human genes off one by one to find out which of them change the way that the flu propagates in cells. That analysis landed them 120 key genes that they refer to as influenza A virus-dependency factors.

One family of related proteins in particular, known as IFITM, caught the team's eye as a particularly important early player that works to stop flu and they decided to explore it in more detail.

"Our protein is induced by interferon and it blocks virus at entry," Elledge said, explaining that interferon is produced as an alarm signal to other cells when a cell senses that it has been invaded by a virus. "If you get rid of IFITM, the virus is replicated five to 10 times more efficiently. It blocks 80 to 90 percent of the virus just by itself."

He said IFITM is a particularly small protein that sits on the surface of vesicles inside cells. Viruses are engulfed by those vesicles and are often destroyed by the cell before an infection can take hold.

Elledge suspects that differences in the amount of IFITM among people might explain differences in their susceptibility to getting sick with the flu. "Natural variation [in this gene] could easily translate into resistance or sensitivity," he said. "We know some people get the flu and are knocked out while others just sniffle a bit."

Drugs aimed at IFITM might fight the flu without an increase in interferon, he said. Interferons are used to combat some viruses such as hepatitis C, but they come with nasty side effects. "You might be able to turn up resistance without making people feel bad," Elledge said. It might even be possible to deliver the protein directly to cells via liposomes. Such a strategy might help in the fight against many infectious diseases, as Elledge showed that IFITM has a similar influence on other viruses, including those responsible for the prevalent mosquito-borne diseases, dengue and West Nile. Likewise, it should be possible to make transgenic animals such as swine or fowl that make more IFITM3 to prevent these animals from becoming reservoirs for flu, where it can recombine with human flu to generate more virulent strains for humans.

The many other proteins they found to be required for the replication of H1N1 could each also yield new ways to fight flu. For its part, IFITM proteins may additionally hold the key to faster production of the weakened virus, as is needed for the making of vaccine.

"If IFITM proteins are also rate-limiting for influenza A virus infection in other organisms such as chickens, whose embryos are employed to passage attenuated viruses for vaccine production, the inhibition of IFITM protein expression could reduce the amount of time it takes to produce vaccine and thereby boost yields," Elledge's team wrote. "This has been a critical issue confounding vaccine production in the current [H1N1] influenza pandemic."