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German Team Finds Host Factors Needed For Flu Virus Replication

NEW YORK (GenomeWeb News) – German researchers have tracked down hundreds of candidate host genes that seem to influence influenza A virus replication, validating dozens against both a non-pandemic H1N1 strain and an H1N1 swine flu strain from the 2009 pandemic.

The team used a genome-wide RNA interference screen combined with a luciferase reporter assay to pinpoint host factors influencing the replication of a non-pandemic H1N1 flu virus and/or the pandemic H1N1 swine flu virus. Their search yielded genes involved in everything from viral trafficking within the cell to viral messenger RNA splicing. And, researchers say, by narrowing in on promising candidate genes it may be possible to develop influenza treatments that are effective against many flu viruses.

"[O]ur results highlight the potency of genome-wide RNAi screening for the dissection of virus-host interactions and the identification of drug targets for a broad range of influenza viruses," senior author Thomas Meyer, a molecular biologist at the Max Planck Institute for Infection Biology, and his colleagues wrote in the journal Nature.

Although a great deal of effort has gone into developing drugs and vaccines targeting influenza A viruses themselves, the researchers explained, viruses still tend to escape such measures because of their high mutation rates. By focusing on host rather than viral factors alone, they argue, it may be possible to come up with treatments that are effective against numerous flu viruses, regardless of their source and mutations.

First, the team did a genome-wide RNAi screen in a human lung epithelial cell line called A549, transfecting the cells with roughly 62,000 siRNAs targeting about 17,000 annotated and 6,000 predicted genes. Two days later, the same cells were infected with influenza A H1N1 virus. The researchers then checked infection rates in the cells a day later with an antibody targeting the virus.

They also transferred some of the supernatant from these lung cells into a culture of 293Y human embryonic kidney cells containing a flu virus-luciferase reporter to further test for viral replication.

Using this approach, the team found 287 human genes that seemed to affect flu virus replication, including several genes that have been previously implicated in influenza replication, such as the nuclear export factors NXF1 and XPO1.

The set of suspect genes contained an unusually high representation of pre-messenger RNA splicing, spliceosome, small ribosomal subunit, eukaryotic translation initiation factor 3, and transport-related genes.

When the researchers independently tested siRNAs targeting these 287 candidate genes in a dog kidney cell line infected with either the seasonal H1N1 flu virus tested originally or a 2009 swine flu H1N1 virus, they were able to validate nearly 60 percent of the genes.

In particular, they noted, siRNAs targeting 119 of the genes led to a five times or greater decrease in the number of non-pandemic H1N1 flu viruses in A549 cells. Meanwhile, 121 of the genes curbed pandemic H1N1 swine flu virus numbers by at least five times.

In their subsequent experiments, the team showed that some of the same siRNAs that effectively decreased both types of H1N1 virus replication also decreased H5N1 bird flu replication by at least two orders of magnitude.

"The observation that a subset of common factors blocked replication of both swine- and avian-origin virus variants corroborates that these proteins constitute crucial sub-type-independent host-cell checkpoints," Meyer and his co-workers wrote.

Using immunofluorescence staining, the researchers also illustrated how knocking down some of the host genes identified in their screen affected viral localization and/or viral mRNA synthesis. Meanwhile, a series of experiments in a human bronchial epithelial cell line narrowed in on genes whose knockdown led to the most dramatic decreases in viral growth.

Based on their findings so far, the team suggests it may eventually be feasible to come up with flu treatments that temporarily target host factors and are effective against a range of influenza viruses.

"Importantly, most of the validated hits are essential for a broad spectrum of influenza viruses, including the pandemic swine-origin H1N1 influenza virus and even a highly pathogenic avian H5N1 strain," they concluded. "This holds promise for the therapeutic potential of these targets against novel emerging influenza viruses with minimized likelihood of developing drug-resistant variants."

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