RNAi as Antiviral Therapy: The HIV-1 Case - RNA Interference from Biology to Therapeutics - page 218

mammalian cells [ 1- 7 ]. RNAi also holds promise for antiviral therapy against
pathogenic viruses, either the ones that cause acute infections like respiratory syn-
cytial virus (RSV) and influenza virus or the ones that cause chronic infections such
as hepatitis B virus (HBV), hepatitis C virus (HCV), and HIV-1. We will focus on
the chronic infections that require an RNAi-based gene therapy approach.
In the following, we will briefly describe the natural microRNA (miRNA) path-
way. It is estimated that human cells express more than 500 miRNAs to control the
process of cell differentiation and development by regulating gene expression at the
posttranscriptional level [ 8- 13 ]. The natural miRNA pathway uses RNA polymerase
II or III to produce primary transcripts or pri-miRNAs that encode a single or mul-
tiple miRNAs as a polycistronic transcript [ 14 ]. The pri-miRNA is processed by the
microprocessor complex, consisting of the Drosha enzyme and the dsRNA-binding
protein DGCR8/Pasha [ 15- 19 ], into a pre-miRNA with a 5¢ -monophosphate and
3¢-hydroxyl 2-nucleotide (nt) overhang [ 20 ]. The pre-miRNA is processed in the
nucleus and subsequently exported by Exportin-5 (Exp-5) to the cytoplasm [ 21- 23 ] .
The cytoplasmic Dicer endonuclease cleaves the base-paired stem approximately
22 base pairs (bp) away from its base, generating a 2-nt overhang at the 3¢ -end [ 24 ] .
Dicer is associated with TAR RNA-binding protein (TRBP) that recruits Argonaute
2 (Ago2) [ 25 ]. The Ago2-RNA complex forms the minimal core of the RNA-
induced silencing complex (RISC) [ 26, 27 ]. RISC unwinds the miRNA duplex and
cleaves the passenger strand, such that only the guide strand remains in the complex
to execute the subsequent mRNA silencing step [ 28- 30 ] .
Mammalian miRNA-mediated gene silencing occurs by translational repression
of the target mRNA [ 9 ]. However, a recent study suggested mRNA cleavage as the
favorite mode of miRNA action [ 31 ]. Degradation of the targeted mRNA is initiated
by endonucleolytic mRNA cleavage opposite nucleotides 10 and 11 of the miRNA.
The level of base-pairing complementarity in the miRNA-mRNA duplex is a major
determinant, leading to mRNA cleavage with a perfect complementarity and trans-
lational repression with suboptimal complementarity [ 32- 36 ] . The “seed” region of
the miRNA (5¢ terminal nucleotides 2-8) typically finds multiple partially comple-
mentary target sequences in the 3¢ untranslated region (3¢UTR) of the cellular
mRNA target, and the silencing efficiency is determined by the overall arrangement
of these 3¢UTR targets, including their number and intermotif distance [ 37 ] . At least
one human miRNA-mRNA set with perfect base-pairing complementarity has been
reported [ 38 ] .
In plants and insects, small interfering RNAs (siRNAs) arise from extended
virus-derived double-stranded (ds) RNA molecules upon cleavage by Dicer. These
siRNAs have antiviral activity and belong to an ancient antiviral mechanism of the
host cell, which is less apparent in mammalian cells because the siRNAs are not
readily detectable in infected human cells [ 39 ]. However, recent studies reported the
accumulation of small virus-derived RNAs in cells infected with HIV-1, poliovirus,
HCV, flock house virus (FHV), Drosophila C virus (DCV), Sindbis virus, and vesic-
ular stomatitis virus (VSV) [ 40- 42 ] .
The most popular strategy for activation of RNAi-mediated gene silencing in
mammalian cells is transfection of siRNAs either introduced directly into cells as