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Inhibition of viral replication
One of the main steps in the process of viral infection
is DNA and RNA genome replication of the virus. The
genome of RNA viruses, especially with plus polarity,
serves both as mRNA and as a replication template.
Many research groups applied the siRNA method to
inhibit replication of viruses in vitro and in vivo. In the
absence of an efficient cell culture system for growing
HCV, the sensitivity of HCV to RNAi was shown in the
replicon-based system. An HCV replicon is derived from an
HCV consensus genome that was cloned from a viral RNA
isolated from an infected human and used to construct
subgenomic selectable replicons. Upon transfection of
these subgenomic selectable constructs into a cell line,
these RNAs were found to replicate to high levels. In
several studies, siRNAs were directed against different
targets in the virus genome [18–27]. For example, siRNA
specific for the 5′ untranslated region (UTR) of the HCV
genome, introduced into Huh7 cells carrying the replicon
system, inhibits HCV replication by up to 90% [20], as
measured by the expression level of the replicon luciferase
reporter gene. siRNAs targeting the viral polymerase NS5B
region reduced expression of NS5B-Luc chimera in mice
[27] or in the replicon system in vitro. Other studies
that target other regions of the HCV genome reported a
significant decline in the level of HCV proteins and the
level of both the sense and antisense RNA strands [25].
The siRNA effect shown for HCV is IFN- and cell-cycle-
independent [23].
In the hepatitis A virus (HAV) replicon-based system,
it has been reported that siRNAs targeting the regions
coding for the non-structural proteins of the virus give
rise to partial inhibition of HAV replication [28]. In that
study, two siRNAs specific for HAV sequences increased
rather than inhibited HAV replication. This could be due to
complex secondary structures of the target region that can
limit and reduce the efficiency of the RNAi process [29]. In
another study, siRNA targeted to various domains of the
HAV internal ribosomal entry site (IRES) induced efficient
and sustained suppression of viral genome translation and
replication [30].
Poliovirus is a highly cytopathic RNA virus. siRNAs
specific to the poliovirus genome inhibited viral replica-
tion, as was demonstrated in a poliovirus replicon system.
The siRNA effect led to viral genome clearance from the
infected cells, without destruction of the cells harboring
the virus [31].
Additional examples of inhibition of viral replication by
siRNA originated from the study of positive RNA viruses
such as dengue (DENV), West Nile (WNV), and severe
acute respiratory syndrome (SARS) [32–36]. siRNAs
targeting the 3′-UTR sequence of DENV, in a region
that is conserved in all the dengue serotypes, reduced
viral replication and infection in dendritic cells [32].
siRNAs targeting the SARS-CoV RNA polymerase gene
inhibited viral RNA replication, protein synthesis and
reduced the viral cytopathic effects on Vero cells [36].
Likewise, expression vectors of siRNAs specific for two
different regions of the WNV genome protected 293T
cells from WNV infection, and significantly reduced viral
RNA replication and virus production [35]. Coxackievirus
B3 (CVB3), a member of the Picornaviridae family,
is a major cause of many human diseases, such as
meningioencephalitis and myocarditis. Synthetic siRNA
targeted to the VP1 or to the viral polymerase showed
antiviral effects in infected HeLa cells by inducing a
significant reduction of viral replication [37]. The foot-
and-mouth disease virus (FMDV) replication was inhibited
in BHK-21 cells by siRNAs targeting various conserved
regions of the FMDV genome [38]. Multiple siRNAs have
been used to target multiple conserved viral genes that
are essential for virus replication, including a long 5′-
non-coding region, a short 3′-non-coding region, the viral
protein VPg, the viral polymerase, and the viral capsid
protein VP4. The combination of those siRNAs gave rise
to a 10–1,000-fold inhibition in virus yield by specific
inhibition of viral replication [38]. The antiviral properties
of RNAi have not been assessed in comparison for their
effectiveness upon targeting the different intracellular
stages of the viral life cycle. However, from current
reports, we could surmise that targeting viral replication,
similar to what has been described in several other types
of antiviral methods, would probably be the suggested
approach to suppress viral infection. Replication of DNA
viruses can be inhibited by targeting their viral mRNA,
whereas replication of RNA viruses can be inhibited by
targeting either their mRNAs or their viral RNA, as was
elegantly demonstrated for HIV [39].