Genome-Wide RNAi Screening to Identify Human Host Factors Crucial for In fl uenza Virus Replication - RNA Interference from Biology to Therapeutics - page 244

The first genome-wide screen to identify host factors involved in IAV infection
was by Hao et al. using Drosophila RNAi technology [ 23 ] . Hao and coworkers fi rst
modified IAV to allow for infection of Drosophila cells and for expression of a
reporter gene product in infected cells. They then tested an RNAi library against
13,071 genes (90% of the Drosophila genome), identifying over 100 genes whose
suppression in Drosophila cells significantly inhibited or stimulated reporter gene
( Renilla luciferase) expression from an IAV-derived vector. Among the over 100
candidate genes found to be crucial to IAV replication in Drosophila , they selected
the human homologues of several encoding components in host pathways/machin-
eries known to be involved in the life cycle of IAV, such as ATP6V0D1 (an ATPase
involved in the endocytosis pathway), COX6A1 (a cytochrome c oxidase subunit
with mitochondrial function), and NXF1 (a nuclear RNA export factor) for further
analysis in mammalian cells. The validated factors were found to play key roles in
the replication of H5N1 and H1N1 influenza A viruses, but not in vesicular stoma-
titis virus or vaccinia virus, in human HEK293 cells [ 23 ] .
With the rapid development of more advanced RNAi-based screening technol-
ogy including more sophisticated, commercially available siRNA libraries as well
as improved robotics, image acquisition, and analysis instruments and software,
comprehensive analysis of mammalian host cell functions was made possible.
Recently, three different groups have performed genome-wide RNAi screens for
influenza virus host factors in human cells [ 4, 24, 25 ] .
Experiments by Brass and coworkers used A/PR/8/34 virus-infected human osteo-
sarcoma cells (U2OS) and replication efficiency was detected by microscopy-based
quantification of the HA signal. This system allowed for the identification of host gene
products involved in the early and mid-stages of IAV infection only since readout of the
assay was the measurement of a viral protein. The primary screen identified 312 human
genes regulating susceptibility or resistance to influenza PR8 virus infection and
confirmed 129 required host factors for IAV infection. Notably, they discovered that
the interferon-inducible transmembrane proteins (IFITM) 1, 2, and 3 restrict an early
step in influenza A viral replication [ 24 ]. The IFITM proteins were shown to be induced
by interferons type I and II, subsequently leading to basal resistance against influenza
A virus. In addition, it was shown that IFITM proteins inhibit the early replication of
flaviviruses, including Dengue virus and West Nile virus [ 24 ] .
König et al. performed a genome-wide siRNA screen with human lung epithelial
(A549) cells [ 25 ] . The coding region for the in fl uenza A/WSN/33 virus hemagglu-
tinin (HA) protein was replaced with that of Renilla luciferase to facilitate the read-
out of the screen. Since no HA was produced, the recombinant virus could not
complete its replication cycle. Similar to the readout system employed by Brass
et al., this RNAi screen also focused on the cellular factors required for viral entry,
uncoating, vRNP nuclear import, genome transcription, and viral protein transla-
tion, excluding late events such as virus assembly, budding, and release. König et al.
identified 295 cellular cofactors required for early-stage influenza virus replication,
219 of which were confirmed to be required for efficient wild-type influenza virus
growth. Further analysis of a subset of genes showed 23 factors necessary for viral
entry, including members of the vacuolar ATPase (vATPase) and COPI-protein
families, fibroblast growth factor receptor (FGFR) proteins, and glycogen synthase