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In-Depth Information
retroviral proteins. Having said that, this suffers an inherent drawback in that
some steps are dependent on retroviral proteins and therefore limits the scope of
the screen. One limitation to the siRNA screen is specificity and cell toxicity,
which can be overcome by the use of pseudotypes. Indeed, a siRNA screen can be
done with different pseudotypes in parallel. If, contrary to the pseudotypes of
interest, some pseudotypes are not affected by siRNA, this indicates that the
siRNA specifically inhibits the virus of interest and, moreover, that the siRNA is
not toxic. To better characterize the entry pathway of the hepatitis C virus, a
small interfering RNA library dedicated to membrane trafficking and remodeling
was screened in the context of the Huh-7.5.1 liver cell line cells infected by
HCV pseudoparticles (HCVpp) (Trotard
., 2009). Results showed that the
downregulation of different factors implicated in clathrin-mediated endocytosis
inhibit HCVpp cell infection. In addition, knockdown of the phosphatidylino-
sitol 4-kinase type III-alpha (PI4KIIIalpha) prevented infection by HCVpp, and
the presence of PI4KIIIbeta in the host cells influenced their susceptibility to
HCVpp infection. This library screening using pseudoparticles identified two
kinases, PI4KIIIalpha and beta, as being involved in the HCV life cycle. These
results have been confirmed in published works of the identification of cellular
factors required for the HCV life cycle using siRNA libraries screening either
over 4500 drugable genes (Ng
et al
et al
., 2007; Vaillancourt
et al
., 2009), 140 cellular
membrane-trafficking genes (Berger
et al
., 2009; Coller
et al
., 2009), kinome
(Supekova
., 2009).
These works are much more time- and cost-consuming, and even though pseu-
doparticles may appear to be an overly simple model to study and identify host
factors necessary for viral infection, they are also powerful and flexible tools that
have led to major discoveries, as these examples have shown.
Recently, however, in order to identify host factors necessary for viral
infections, researchers are turning to genome-wide genetic screens. Indeed, the
sequencing of the human genome and the emergence of technologies that allow
the silencing of individual genes in cells one by one using siRNA, when
combined with the use of genome-wide siRNA libraries and automated HTS
methodology, allows molecular information to be gained about virtually every
critical intracellular event occurring during virus infection. Within the last few
years, there have been several publications describing such genome-wide siRNA
screens that have been applied to virus infections in tissue-cultured cells. RNAi
screens have now been preformed for several viruses, including HIV (Brass
et al
., 2008), or the entire genome (Li
et al
., 2009; Tai
et al
et al
.,
2008; Konig
et al
., 2008; Yeung
et al
., 2009; Zhou
et al
., 2008), West Nile Virus
(Krishnan
et al
., 2008), Influenza (Karlas
et al
., 2010; Konig
et al
., 2008), and, in
drosophila cells, Dengue (Sessions
.,
2010). From the hundreds of cellular factors that have been implicated in the
outcome of infection, some were already known from other studies, but the
majority are entirely novel, and the validation and analysis of data is ongoing
et al
., 2009) and vaccinia virus (Moser
et al
