Genome-Wide RNAi Screening to Identify Human Host Factors Crucial for In fl uenza Virus Replication - RNA Interference from Biology to Therapeutics - page 242

Whereas functions of the viral proteins have been studied extensively during the
last decade, relatively little is known about the cellular factors involved and neces-
sary for viral infection. A missing cellular function would be more difficult for the
virus to adapt to, so targeting a host factor essential for influenza infection should
affect replication, independent of the type, strain, and antigenic properties of the
invading influenza virus, since viruses may share dependence on certain cellular
pathways. The obvious caveat with such an approach is the necessity to insure that
inhibition of the cellular protein is not detrimental to the host. In an acute disease
like influenza, drug administration is only short term however, thereby offering
more flexibility in the choice of chemical compound to be administered.

12.2 Genome-Wide Screens for Human Host Factors Crucial

for In fl uenza A Virus Replication

12.2.1 Background on RNAi

Several laboratories have followed this rationale of targeting host factors required
for viral replication by expending great effort into performing genome-wide RNA
interference (RNAi) screens to identify these genes.
RNA interference (RNAi) is a process of sequence-specific, posttranscriptional
gene silencing in animals and plants by degradation of mRNA. The RNA interfer-
ence pathway was first described in 1998 by Fire and Mellow, who injected double-
stranded (dsRNA) into the nematode Caenorhabditis elegans , initiating
sequence-specific degradation of cytoplasmic mRNAs containing the same sequence
as the dsRNA trigger [ 11 ]. Early applications using long dsRNA were not effective
in most mammalian cells, however, because they induced an antiviral interferon
(IFN) response hereby functioning as the host's first line of defense against viral
infection but ultimately leading to cell death [ 12 ] . Further investigations regarding
the RNA silencing mechanism in different organisms revealed that the mediators of
sequence-specific messenger RNA degradation were 21- and 22-nucleotide small
interfering RNAs (siRNAs) generated by ribonuclease III cleavage from longer
dsRNAs by the enzyme termed Dicer [ 13 ]. Components of the RNAi machinery
specifically recognize these small interfering RNA (siRNA) duplexes and incorpo-
rate a single siRNA strand into the so-called RISC complex (RNA-induced silenc-
ing complex) [ 14 ]. After integration into the RISC, the antisense “guide” strand
base-pairs to the cognate site of the mRNA and induces cleavage of the mRNA,
thereby preventing it from being used as a translation template. This mechanism,
therefore, enables strong reduction of de novo protein synthesis for the correspond-
ing mRNA in a sequence-specific manner [ 14 ]. The natural function of RNAi
appears to be the protection of the genome against invasion of either mobile genetic
elements such as transposons or of viruses, which exploit the RNA-producing
machinery of the host cell upon infection to produce aberrant RNA or dsRNA.