Drosophila RNAi screen identifies host genes important for influenza virus replication.

Linhui Hao, Akira Sakurai, Tokiko Watanabe, Ericka Sorensen, Chairul A. Nidom, Michael A. Newton, Paul Ahlquist & Yoshihiro Kawaoka 

Nature 2008 454;890-893 doi:10.1038/nature07151

[Full Text – subscription required]; [pdf – subscription required]; [abstract]

A couple of years ago, I attended a meeting in Barcelona that concerned the use of Drosophila as a model for human disease.  At the time I was impressedby the ingenuity of Drosophila researchers in utilising Drosophila as a platform for investigating disease.  This paper describes the use of Drosophila cells in culture to identify cellular factors required for influenza virus replication

With the possible advent of novel influenza variants with the potential to trigger a global pandemic, new approaches in identifying therapeutic targets are important. The iInfluenza virus, like all viruses, requires a panoply of host encoded factors to support its own replication.  Unfortunately for those seeking to model influenza virus replication, it cannot replicate in Drosophila cells, principally because the cell surface receptor to which the virion attach is absent.

To enable viral infection of Drosophila cells and detection of virus, the investigators engineered a virus in which the HA and NA genes were replaced with genes encoding Vesicular stomatitis virus glycoprotein G (VSV-G) and Green Fuorescent Protein (GFP) respectively.  For convenience, this engineered virus is referred to as FVG-G, and can be shown to infect Drosophila cells by visualising GFP expression.  Interstingly, while the cells permit infection and replication of FVG-G, no virions are released from the cells.

A second viral variant, FVG-R, was engineered , in which the HA and NA genes were replaced with VSV-G and a gene encoding luciferase (an enzyme that produces light).  Cells were infected with this virus, and the effects on luciferase activity of each of 13,071 dsRNAs directed against Drosophila genes were tested. (This collection of dsRNA covers approximately 90% of known and predicted Drosophila genes.)  From this screen 110 Drosophila genes were identified, which, when knocked down by RNAi, reduced or prevented viral replication as measured by luciferase activity. Among this set, several candidates were discarded as they seemed to have cytotoxic effects when suppressed. 

The authors selected three candidates for further examination on the basis that (a) there were human homologues, and (b) that these genes encoded components of pathways pathways known to be involved in the life cycle of influenza virus.  This actually surprised me, as it seemed a curious way to validate the strategy – presumably one could have taken a short cut and selected these genes directly…

 Candidate genes
gene encoded protein  function
ATP6V0D1  subunit D of vacuolar (H+}-ATPase  Involved in endocytosis pathway
COX6A1  subunit of cytochrome oxidase  mitochondrial electron transport chain
 NXF1  nuclear export factor  export of mRNAs containing exon-exon junctions

The human homologues of the three genes were then investigated in human cells.  In the first round, FVG-R was used to infect the cells, and luciferase activity assayed following various siRNA treatment, demonstrating that knockdown of these three genes did indeed suppress expression of the luciferase (and therefore viral replication).

A second round of analyses used two different influenza virus strains, an H1N1 and an H5N1 strain, comparing the efficacy of siRNA knockdown of the three target genes in suppressing these two viruses.  They also tested the efficacy against two unrelated viruses, VSV and vaccinia.  As a positive control, siRNAs directed against a viral gene (corresponding to the virus being investigated) was used.

The upshot is that not only does knockdown of the three candidate gens suppress influenza virus, but the effect appears to be influenza specific, as neither VSV or vaccinia viruses were affected.

Because one of the candidates encoded a component of the mitochondrial ETC, various drugs targeting the ETC were evaluated for effects on influenza virus replication – some did indeed suppress the virsu, and what’s more did so to a greater degree than they suppressed cell viability or another virus (an MLV derivative).

In summary, this paper presents a further Drosophila-based approach to fundamental biomedical studies of disease, and one that should prove widely applicable.  

Linhui Hao, Akira Sakurai, Tokiko Watanabe, Ericka Sorensen, Chairul A. Nidom, Michael A. Newton, Paul Ahlquist, Yoshihiro Kawaoka (2008). Drosophila RNAi screen identifies host genes important for influenza virus replication Nature, 454 (7206), 890-893 DOI: 10.1038/nature07151