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were included in the process of primary hit identification: luciferase expression,
percentage of infected cells (as determined by immuno fl uorescence microscopy),
and total number of infected cells. Results from each of the three parameters were
combined, and from a total of over 22,000 human genes, 287 were designated pri-
mary hits, affecting replication of WSN virus. Among the high-confidence candi-
dates pivotal in IAV replication were genes encoding for nuclear export factors
NXF1
and
XPO1
, as well as the vacuolar ATPase
ATP6V0D1
. Furthermore, our data
set was markedly enriched in genes belonging to the spliceosome, the small ribo-
somal subunit, and those involved in eukaryotic translation initiation and coated
vesicle transport. One hundred and sixty-eight hits were confirmed in subsequent
analysis, using A/WSN/33 and the pandemic H1N1 virus A/Hamburg/04/2009
influenza strains. A gene was considered a hit if two or more individual siRNAs
showed clear reduction of virus replication, thus also selecting against off-target
effects by siRNA-sequence-independent immunostimulation. In order to avoid false-
positive hits caused by RNAi-mediated toxicity, we also performed WST-1 assays to
monitor viability of cells. Also, H5N1 virus (A/Vietnam/1203/2004) replication was
found to be reduced by two orders of magnitude by a subset of siRNAs, suggesting
that influenza A viruses of different subtypes share a dependency on a specific group
of host factors.
Shapira et al. used an integrative genomics approach to investigate the IAV-host
cell relationship. They used a combination of yeast two-hybrid analysis, genome-
wide expression profiling, and RNAi screen to determine genes transcriptionally
regulated by IAV infection [
27
]. First, they performed a systematic yeast two-hybrid
screen to build a physical interaction map for viral proteins and human cellular
genes using computational analysis. They subsequently examined differential gene
expression in primary human bronchial epithelial cells (HBECs) exposed to either
IAV, or to viral RNA, IAV lacking the NS1 gene (DNS1), or to interferon beta
(IFN-b). These different approaches yielded 1,745 candidate genes, 1,056 of which
were shown to be transcriptionally regulated by influenza virus infection upon fur-
ther analysis. Further validation using siRNA pools to mediate knockdown resulted
in a list of 616 human genes affecting virus replication and/or IFN production
(Table
12.1
).
12.2.3
Results and Interpretation
The genome-wide siRNA screens have provided an overabundance of likely
influenza host factors required for steps throughout all stages of the infection cycle;
however, only a limited number of factors actually overlap among the individual
screens when analyzed at gene level. Only ~8% of genes identified are common to
at least two of the five siRNA screens discussed above, with no gene shared by all
five. If the respective data is analyzed at the level of cellular function instead of that
of gene name, however, more matches appear, suggesting that the screens agree at
the level of functional pathway, just not by individual gene name. Also, while
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