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RNAi Screen Uncovers Host Genes Needed for Flu Virus Replication

NEW YORK (GenomeWeb News) – Researchers may be a step closer to finding new ways to combat influenza, after an RNA interference study in fruit fly cells uncovered more than a hundred host genes that are exploited by flu viruses during replication.
In a paper appearing online today in Nature, an international team of researchers described how they used a genome-wide RNAi screen in Drosophila melanogaster cells to identify host genes required for flu virus replication and infection. Because fruit flies share sequence homology with humans and other mammals, the results have implications for understanding mammalian influenza infections as well — something the team has begun verifying in human tissue culture cells.
“All of the evidence is that this is a high-value set of genes for testing in mammalian cells,” co-senior author Paul Ahlquist, an oncologist and molecular virologist at the University of Wisconsin at Madison, said in a statement. “It has implications for virology and control.”
Influenza kills hundreds of thousands of people around the world in a typical year. In the event of a flu pandemic, that can jump to millions. The pandemic scenario has been a concern for both health officials and the public, particularly since sporadic cases of humans infected by bird flu have been documented in recent years. Together, the burden of annual influenza cases and the specter of larger outbreaks are spurring efforts to develop better influenza preventions and treatments.
In an effort to lay the groundwork for improved influenza treatment and control, Ahlquist and his colleagues looked for previously unknown interactions between the virus and a host cell. First, they modified a human H1N1 influenza virus so that it could infect Drosophila cells. They then screened the infected Drosophila cells twice with an Ambion RNAi library containing double-stranded RNA targeted against 13,071 genes — some 90 percent of the Drosophila genome — to look for genes that were required for flu virus function.
Although some viral proteins weren’t expressed in the cell (including those required for virion assembly and infectivity), the virus did enter and replicate in Drosophila cells, providing the researchers with an opportunity to identify host genes involved in processes such as viral protein expression.
Using this approach, the researchers found 110 genes that were required for efficient viral replication in both trials. Six were excluded from further consideration because the researchers suspected that their knockdown may have had cytotoxic effects independent of the viral effect.
Next, the researchers tested the human homologs of three of the 100 or so candidate genes — ATP6V0D1, a gene involved in endocytosis, COX6A1, a gene involved in mitochondrial function, and NXF1, a gene involved in mRNA nuclear export — in HEK 293 cells. By knocking each of the genes down with short interfering RNAs and then infecting the cells with flu virus, the researchers were able to determine whether the genes had a role in the influenza infection of human cells.
Indeed, the team found that ATP6V0D1 or COX6A1 knockdown decreased the replication of mutated flu virus while NXF1 knockdown decreased viral activity by almost five times. When they targeted ATP6V0D1 or COX6A1 with siRNA in the presence of the wild type H1N1 or H5N1 viruses, the researchers also found that viral yields dropped by about ten times. Targeting NXF1, meanwhile, slashed the H5N1 viral titer by some 20 times.
In contrast, knocking down the same genes did not seem to affect the replication or activity of other viruses such as the vaccinia virus or the vesicular stomatitis virus. That suggests that the RNAi screen specifically picks up genes required for influenza virus function — increasing the potential of this method for finding new therapeutic targets.
“The genes we identified in mammalian cells are very specific for influenza viruses,” co-senior author Yoshihiro Kawaoka, a virologist affiliated with the University of Wisconsin at Madison, the University of Tokyo, and Kobe University, said in a statement. “Now we know influenza viruses are using specific host proteins so those become targets for drug development.”
And, the team added, the strategy employed in this study holds promise for understanding other viral-host relationships, too. “We suggest that the same strategy could be applied to identify previously unknown host factors involved in the replication of other viruses, whenever at least a portion of their replication is supported by Drosophila cells,” they wrote.

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