Viruses and RNA interference in mammalian cells

UNIVERSITY OF TARTU
FACULTY OF SCIENCE AND TECHNOLOGY
INSTITUTE OF MOLECULAR AND CELL BIOLOGY
Jekaterina Gusseva
Viruses and RNA interference in mammalian cells
Bachelor’s Thesis
Supervisor MD, PhD, Eva Žusinaite
TARTU 2008

TABLE OF CONTENTS
TABLE OF CONTENTS.......................................................................................................2
ACRONYMS AND ABBREVIATIONS..............................................................................3
INTRODUCTION..................................................................................................................4
LITERAUTRE OVERVIEW................................................................................................5
1. Intracellular anti-viral responses......................................................................................5
Interferone response.......................................................................................................5
RNAi………….............................................................................................................7
2. RNA interference................................................................................................................7
History of discovery.....................................................................................................7
General RNAi mechanism............................................................................................8
RNAi functional elements............................................................................................9
Molecular mechanism of RNAi in mammalian cell...................................................12
Link between miRNA, siRNA and RNAi. (siRNA versus miRNA).........................13
3. RNAi as an antiviral defense in plants, insects and intervertebrates..........................15
Plants...........................................................................................................................15
Insects.........................................................................................................................15
Intervertebrates...........................................................................................................16
4. RNA interference in mammals........................................................................................17
Artificially triggered RNAi........................................................................................17
5. Suppression of cellular anti-viral RNAi by viruses.......................................................19
RNA silencing suppressors.........................................................................................20
Virus-encoded miRNAs..............................................................................................21
Further directions of investigations............................................................................22
CONCLUSIONS..................................................................................................................23
KOKKUVÕTE.....................................................................................................................24
REFERENCES.....................................................................................................................25
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ACRONYMS AND ABBREVIATIONS
CrPV - Cricket Paralysis virus
CVB3 - Coxsackievirus B3
DCV - Drosophila C virus
ds - double-stranded
EBV - Epstein–Barr virus
EGFP - enhanced green fluorescent protein
eIF2 – Eukaryotic Initiation Factor
FHV - Flock House virus
FMDV - Foot and Mouth Disease Virus
HCC - hepatocellular carcinoma
HCV - hepatitis C virus
HIV - Human Immunodeficiency virus
IFN - interferon (InterFeroN)
miRNA - micro RNA
mRNA - Messenger RNA
NoV - Nodamura virus
nt- nucleotides
ONNV - O’nyong–nyong virus
PTGS - Post-Transcriptional Gene Silencing
PVX - potato virus X
RdRp – RNA-dependent RNA polymerase
RNAi - RNA interference
RSS - RNA Silencing Suppressor
RT - reverse transcriptase
SARS - severe acute respiratory syndrome
SARS-CoV - SARS- associated coronavirus
SINV - Sindbis virus
siRNA - small or short interfering RNA
ss - single stranded
TMV - tobacco mosaic virus
VA – virus-associated
VSV - vesicular stomatitis virus
WNV - West Nile virus
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INTRODUCTION
Epochal discoveries are happening not very often, but when they are, such findings take
their niche in the history of humankind. During multiple investigations scientists try to apply
the disclosures in as many fields, as possible. All this makes sense in the case of outstanding
discovery of RNA interference (RNAi). The 2006 year’s Nobel Prize for achievement in
Physiology or Medicine was awarded to professor A. Fire and professor C. Mello for
discovering RNA interference. This fact already proves the importance of RNAi.
One decade has passed from its discovery, but it has already become known in the scientific
world as one of the most exploited and perspective defense mechanisms of the cell against
invaders, including viruses. It exists as an widely spread (from single-cell organisms to
humans) ancient nucleic acid-based immune system. It is clearly established, that in plants
and insects RNAi functions as an antiviral defense, however it still being under
investigation, whether RNAi has a similar function in mammals. Firstly RNAi was discribed
as a natural cell response to the introduction of the foreign double stranded RNA (dsRNA),
but recent researches highlighted new opportunities of RNAi - therapeutic modality. To
apply it in safe medical therapies, more detailed basics of this process should be concerned.
This refers to actuality of the current thesis.
The objectives of this bachelor's thesis were:
- on the basis of literature to make an overview of intracellular anti-viral responses;
- to introduce principal RNA interference mechanisms;
- to explore evidences of the existence of RNA interference in mammalian cells as an
effective antiviral response;
- to find out, whether viruses have specific mechanisms against anti-viral RNAi.
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LITERATURE OVERVIEW
1. Intracellular anti-viral responses
Refering to Newton's third law of motion: For every action, there is an equal and
opposite reaction. Even to the viral influence, there is an intracellular reaction in the form
of antiviral responses, such as an interferon (IFN) and RNA interference. These processes
are induced in respond to viral replication and are aimed to control viral replication inside
the infected cell, and through intercellular signaling, in the neighbor cells.
Interferon system
The history of IFN started when, Isaacs and Lindenmann discovered in 1957 that chick cells
infected with influenza virus produced a factor that mediated the transfer of a virus-resistant
state against both homologous and heterologous viruses. Similar findings for vaccinia virus
infection were published by Nagano and Kojima in 1958. These facts have led in future to
the elucidation of the IFN system in exquisite detail. Now this antiviral response with nonspecific
mechanism is known as IFN response (Samuel, 2001).
Interferons are proteins and glycoproteins, inducible cytokines (little peptide information
molecules, that modulate interactions between cells). IFN actions are pleiotropic and affect
many biological processes: antiviral response, regulation of cell growth, cell differentiation
and apoptosis. In general, interferons are divided into two types. Interferons induced by viral
infection - the viral IFNs, which include IFN-α (leukocyte), IFN- β (fibroblast), and IFN- ω,
pertain to type I. Type II - IFN-γ, also known as immune IFN, can be induced by mitogenic
(cause cell transformation or mitosis) or antigenic (cause immune response) stimuli.
Almost all cell types produce type I interferons. Type II IFN-s are produced only by the
cells of immune system.
Production of IFN-s by cells is induced, when abnormally large quantity of dsRNA-s
(double-stranded RNA) is found in a cytoplasm. One of the genes induced by type I IFN-s is
RNA-dependent protein kinase (PKR) gene, which is translated into PKR protein (future
enzyme). However, it is catalytically inactive and requires RNA to be converted in
catalytically active form. The PKR protein changes its conformation, affecting the kinase
catalytic subdomains. Then PKR kinase inhibits initiation of translation by phosphorylating
eIF-2a - the eukaryotic initiation factor of translation. PKR activation and subsequent
phosphorylation of eIF-2a change the translational pattern of the host cell (Figure 1). PKR
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interferes with the replication of an invading virus and also suppresses normal cell ribosome
functioning, killing both the virus and the host cell. It happens, if response lasts for
sufficient long time.
Figure 1 Mechanism of IFN action. Functions of selected IFN-inducible protein - PKR. It takes place among
the IFN-induced proteins believed to affect virus multiplication within a single cell. It inhibits initiation of
translation by phosphorylating eIF-2a (Samuel, 2001).
Other IFN-induced proteins, which affect virus replication within the cell, except PKR
kinase, are the the 2’,5’-oligoadenylate synthetase (OAS) family (catalyze the synthesis of
oligoadenylates) and RNase L nuclease (a latent endoribonuclease) - mediate RNA
degradation; the family of Mx protein GTPases (Mx proteins are GTPases that belong to the
superfamily of dynamin-like GTPases) – target viral nucleocapsids and inhibit RNA
synthesis; and RNA-specific adenosine deaminase (ADAR) – edits double-stranded RNA by
deamination of adenosine to yield inosine. Still they are not completely investigated.
(Samuel, 2001)
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RNAi
The development of biotechnological methods of research in the last years made possible
RNA interference to be discovered. RNAi is a regulatory mechanism, which silences genes
degrading mRNA in a sequence-specific manner with help of small pieces of doublestranded
RNAs: small or short interfering RNAs (siRNAs) and micro RNAs (miRNAs). It
suppresses gene expression at the translation stage or by interfering the transcription of
specific genes. RNAi has been observed in organisms as diverse as plants, protozoa,
nematodes, fungi, insects, and mammals (Hu et al., 2004). This process takes an integral part
in various eukaryotic functions such as viral defense, chromatin remodeling, genome
rearrangement, developmental timing, brain morphogenesis and stem cell maintenance
(MacRae et al., 2006).
2. RNA interference
History of discovery
The history of RNAi discovery started when scientists tried to compare the activity of
silencing of antisense or sense single-stranded RNAs (ssRNAs) with their double-stranded
RNA (dsRNA) hybrid in two types of experiments in 1990. First group of experiments was
made to estimate antisense suppression for selective silencing of plant gene expression. In
theory, antisense RNA encoded by a transgene should prevent translation of complementary
mRNA of a plant gene into protein by basepairing to it. But the control “sense” transgene
RNAs are unable to do it, because they can not basepair to mRNA. Hence, the silencing
should not been observed, but surprisingly it was (Smith et al., 1990). The other type of
experiments, has led to the conclusion that the expression of both introduced and
homologous endogenous genes in petunia leaves was suppressed by introduction of a
pigment-producing gene that was under a control of a powerful promoter. This phenomenon
scientist called “co-suppression”. It was discovered while scientists made the efforts to
enhance coloration of petunia flowers. They tried to overexpress a transgene that encoded a
protein involved in a synthesis of pigments. However, it led to a partial or complete color
loss. That was the result of “co-suppression” or coordinate silencing (Napoli et al., 1990;
Van der Krol et al., 1990). “Co-suppression” also occurs at the post-transcriptional level.
Such post-transcriptional gene silencing (PTGS) in plants is equivalent of RNAi. Posttranscriptional
gene silencing is a mechanism involving dsRNA, nucleases, small RNAs and
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RNA synthesis. It destructs the RNA transcripts (De Carvalho et al., 1992 ; Van Blokland et
al., 1994).
RNAi was first described in 1998 when the researchers found that gene interference
produced by dsRNAs was considerably more effective than were sense or antisense strand
separately. Moreover, the introduction of specific dsRNA sequences in minimal quantities
into the nematode Caenorhabditis elegans could effectively silence the expression of a
target gene in the injected animals, as well as in their progeny (Fire et al., 1998).
General RNAi mechanism
DsRNA (more than 30 bp) is produced from the invading genes, either during viral
replication or by aberrant transcription from promoters located near the transgene insertion
site. Initiative dsRNA can be either exogenic or endogenic. RNAi is carried out in two steps
(Figure 3). Frst, long dsRNA homologous in sequence to the silenced gene (Elbashir et al.,
2001) is converted to 21–23-nucleotide small interfering RNA (siRNA) duplexes by
ribonuclease III (RNase III)-like enzyme Dicer in the cytoplasm (Meister and Tuschl, 2004).
In the second step, the small dsRNA products of the cleavage are assembled into a
multiprotein RNA-induced silencing complex (RISC) with the help of the Dicer in the
cytoplasm. Then the duplexes of siRNA unwound and one single (Nykanen et al., 2001;
Martinez et al., 2002) strand with lowest stability at its 5’-end (Schwarz et al., 2003), guides
RISC to its complementary mRNA. Further, mRNA is degraded by RISC nuclease activity.
As a result, the translation stops (Elbashir et al., 2001). The other anti-guide strand is
degraded during RISC activation (Gregory et al., 2005). Below, more detailed description of
RNAi functional elements is given.
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Figure 3. RNAi mechanism. RNAi is triggered by siRNAs, which are located in cytoplasm. Either dsRNA or
small hairpin RNA (shRNA) are their precursors, cleaved by Dicer in ATP-depending manner. The siRNA
sequences associated with RISC unwind also in ATP-depending manner. This complex recognizes target
mRNA and degrades it, releasing RISC. (Rutz and Scheffold, 2004)
RNAi functional elements
siRNAs are small RNAs formed through cleavage of long dsRNA molecules, with
complementary nucleotide sequences to the targeted RNA strand. They are 20–25 (21–25 nt
in plants) nucleotides in length and they usually have a two-base overhang on the 3' end,
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which allows them to be recognized by the enzymatic machinery of RNAi that eventually
leads to homology-dependent degradation of the target mRNA. siRNAs lead to a strong
reduction of either cellular or viral gene expression (Elbashir et al., 2001; Aagaard and
Rossi, 2007). Precursors of siRNAs - dsRNA form when complementary DNA strands are
transcribed into RNA sequences. Viral infection of a cell can also supply dsRNAs. Almost
all eukaryotic organisms have siRNAs or cellular mechanisms to produce them, except
budding yeast Saccharomyces cerevisiae. Also they have started the new era of small RNAs
in medical area, as synthetic siRNAs can be artificially expressed for many therapeutical
purposes, hence a promising approach for treating medical conditions in humans (Grosshans
and Filipowicz, 2008).
Dicer is a ribonuclease (the enzyme which catalyses the hydrolysis of RNA into
oligonucleotides and smaller molecules of the RNase III family, it cleaves dsRNA and premicroRNA
into siRNA It contains an amino-terminal DEXH-box RNA helicase/ATPase
domain: the role of ATP-dependent RNA helicase domain remains to be elucidated (Meister
and Tuschl, 2004); a carboxy-terminal dsRNA-binding motif; two catalytic ribonuclease III
(RNase III) domains and one Piwi-Argo-Zwille/Pinhead (PAZ) domain (Figure 4). The last
mentioned is a module that binds the end of dsRNA. A flat, positively charged surface
separates these two regions. The distance of 65 angstrom between the PAZ and RNase III
domains conforms to the length spanned by 25 base pairs of RNA (Jaronczyk et al., 2005).
In addition, Dicer helps to load its small RNA products into large multiprotein complexes –
RNA-induced silencing complexes (RISC). Mammalian Dicer enzymes are ATPindependent
(Zhang et al., 2002). However Dicer of D. melanogaster is not (Ketting et al.,
2001).
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Figure 4. Dicer components. 2 helicase domains, Domian of Unknown Function – DUF, PAZ domain, 2
ribonuclease III domains, double-stranded RNA binding domain (motif). (Jaronczyk et al., 2005)
RISC is a multiprotein complex in RNAi, into which a siRNA strand is assembled to become
effective in gene silencing (Hong, 2008). This complex uses the siRNA guide to identify
mRNAs with a complementary sequence to the siRNA. It also cleaves the mRNA in the
middle of the mRNA–siRNA duplex. RISC is formed in an ATP-dependent manner.
(Meister and Tuschl, 2004). It consists of subunits: helicase, exonuclease, endonuclease, and
homology searching domains (Nykanen et al., 2001). Endonuclease domains are proteins,
which belong to the argonaute family, which cleave the target mRNA strictly
complementary to their siRNA. Argonaute-2 (AGO2) has been identified as the catalytic
center of RISC, which cleaves the target mRNA strand complementary to their bound
siRNA, as they have endonuclease activity (Gregory et al., 2005) (Figure 5). AGO 2 protein
performs the siRNA anti-guide strand cleavage, using one strand of the siRNA duplex as a
guide to find mRNAs containing complementary sequences. Then at a specific site
measured from the guide strand’s 5’ end, cleaves the phosphodiester backbone (Rand et al.,
2005).
MicroRNA (miRNA) is another important class of small RNAs in cells 20–25 nucleotides in
length. They regulate gene expression during development and defense against viruses.
They are processed from long ssRNA (Grosshans and Filipowicz, 2008). miRNAs are
generated from primary transcripts (pri-miRNAs). Their length ranges from several
hundreds to several thousands bases. Pre-miRNAs are produced from pri-miRNAs by
RNase III-like enzyme –Drosha (van Rij and Andino, 2006). They are approximately 75 nt
long and contain the active, mature miRNA component (Hammond, 2006). Then, pre-
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miRNAs are taken from the nucleus to the cytoplasm by Ran-GTP and the export receptor
Exportin-5 (Yi et al., 2003; Lund et al., 2004). Pre-miRNAs are processed by Dicer into
miRNAs. Hence, miRNAs are produced endogenously (Carmell and Hannon, 2004).
Drosha - RNase III, nuclease that executes the initiation step of miRNA processing in the
nucleus: processes pri-miRNA to pre-miRNA (Lee et al., 2003) (Figure 5). This nuclear
RNase III enzyme, which was first discovered in humans and subsequently in Drosophila
and C. elegans, is implicated in initiation of the miRNA pathway (Tang, 2005).
Molecular mechanism of RNAi in mammalian cells
In mammalian cells, siRNAs are also produced with the help of Dicer from dsRNA.
Mammals encode only one Dicer gene, while for instance the plant Arabidopsis thaliana,
encodes four different Dicer-like (DCL) genes (van Rij and Andino, 2006). Dicer is
complexed with the TAR-RNA binding protein (TRBP), which binds to the transactivation
response element (TAR) of RNAs through different sites (Dorin et al., 2002), and delivers
the siRNAs to the RISC. Its core components are the Argonaute family proteins. Only Ago-
2 possesses an active catalytic domain for cleavage activity in humans. The Ago-2 protein
cleaves the target mRNAs between 10 and 11 bases, relative to the 5′ end of the antisense
siRNA strand (Aagaard and Rossi, 2007)
Figure 5. Human AGO2 and Drosha. Ago proteins contain PAZ domains. Ago proteins carry an extra PIWI
domain. The PIWI domain is a protein domain homologous to PIWI proteins. RNAse III domains (RIIIa,
RIIIb) are the parts of Drosha. Drosha also contains double-standed RNA binding domains (dsRBDs). (Meister
and Tuschl, 2004).
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Link between miRNA, siRNA and RNAi (siRNA versus miRNA)
Both miRNAs and siRNAs trigger RNA silencing, the repression of specific sequences of
mRNA. The siRNAs are equivalent to miRNAs (Montanucci et al., 2005). MiRNAs as well
as siRNAs belong to the small RNA class. It is supposed, that they differ only in their
biogenesis: miRNAs originate from short hairpin RNA (shRNA, also called micro RNA
precursors, pre-miRNA), produced by Drosha. SiRNAs are cleaved from dsRNAs by Dicer
(Carmell and Hannon, 2004). Mature miRNAs are ssRNA molecules, 21 or 22 nt in length,
that are endogenously produced. However, siRNAs are dsRNA molecules 21-25 nt long and
can be endogenous as well as exogenous (Aagaard and Rossi, 2007; Carmell and Hannon,
2004; Elbashir et al., 2001). The perfect match recognition (complementary with targeted
RNA) sequences for miRNA can be 7-8 nt and the complete sequence permit mismatches,
not as siRNAs (Zeng et al, 2003). Endogenous siRNAs are rarely conserved in related
organisms that is not the case for miRNA sequences (Lee et al., 2003). Endogenous siRNA
specify “auto-silencing” in that they silence the same locus or a very similar loci from which
they originate, whereas miRNA specify “hetero-silencing”, they are directed for silencing
different genes (He and Hannon, 2004).
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A
B
Figure 6. RNA interference pathways. The processing of dsRNAs proceeds in two steps, and is catalyzed by
the enzymes Drosha (in the nucleus) and Dicer (in the cytoplasm). A. Antiviral defence. The double-stranded
RNA silencing pathway is involved in two different modes of post-transcriptional gene silencing. One is
triggered after cleaving dsRNAs to siRNAs by Dicer. Then siRNAs in the consistent of RISC guide cleavage
of their target RNA. siRNAs must perfectly match its target. Then, the catalytic domain of the RISC cleaves
the target RNA. B. Regulation of gene expression. The repression of protein synthesis is initiated by
microRNA genes. Pri-miRNA transcripts are cleaved by the nuclear RNase III-like enzyme – Drosha in the
nucleus into pre-miRNA. Pre-miRNAs are exported to the cytoplasm and cleaved by Dicer into miRNAs,
which are loaded into RISC complex. RISC complex guides inhibition of translation without destroying the
target mRNAs (imperfect mach with target mRNA). (van Rij and Andino, 2006)
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3. RNAi as an antiviral defense in plants, insects, and intervertebrates
Plants
RNA silencing, as an antiviral response, has been studied widely in plants. It provides a
nucleic acid-based defense against viruses that can induce systemic immunity:
RNA and DNA viruses produce virus-derived siRNAs, during their infection.
(Hamilton and Baulcombe, 1999)
Increased viral replication and spread was observed, if RNAi components in plants
are impaired. It was shown that plants, deficient in component of RNAi, exhibited markedly
enhanced susceptibility to tobacco mosaic virus (TMV) and potato virus X (PVX) (Xie et
al., 2001).
A local and systemic silencing signal is initiated upon virus infection. (Baulcombe,
2004)
As a contra-defense against RNAi pathway, viruses have evolved proteins capable of
suppressing RNA silencing. The first viral suppressors of RNA silencing reported were HC-
Pro and the 2b protein encoded by potyviruses and cucumoviruses (Anandalakshmi et al.,
2000; Li et al., 1999). This all is a circumstantial evidence, that RNA silencing serves as an
antiviral defense mechanism in plants (Ding et al., 2004; van Rij and Andino, 2006).
Insects
RNA silencing also provides a natural antiviral defense in insects. It was demonstrated that:
silencing RNAi components in Aedes aegypti result in transient increase of Sindbis virus
(SINV; family Togaviridae, genus Alphavirus) replication; the production of virus-specific
siRNAs indicates that RNAi in Aedes aegypti is active during SINV infection; the RNAi
response varies in a virus-dependent manner. These data define important features of RNAi
anti-viral defense in Aedes aegypti (Campbell et al., 2008).
In addition, it was demonstrated that RNAi acts as an antagonist to alphavirus replication in
mosquitoes (Anopheles gambiae). An increase in titer and spread of O’nyong–nyong virus
(ONNV) supports an antiviral role of RNAi in suppression of alphavirus replication in its
natural vector, after depletion of RISC components: Argonaute proteins 2 and 3. These
observations prove that RNAi prohibits ONNV replication in Anopheles gambiae, and also
suggest that the innate immune response conditions vector competence. The presented data
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supported the idea that RNAi is a mechanism, which protects mosquitoes from viral
infection (Keene et al., 2004).
RNA interference pathway protects adult flies from infection by two evolutionarily diverse
viruses. It was also shown that the central catalytic component of the RISC, the nuclease
Ago-2, is necessary for antiviral defense in adult Drosophila melanogaster. Increased
mortality in ago-2–defective flies, which were infected with Drosophila C virus (DCV), and
with Cricket Paralysis virus (CrPV) was associated with a dramatic increase in viral RNA
accumulation and virus titers. The physiological significance of RNAi as antiviral
mechanism is emphasized by finding that DCV encodes the RNAi potent suppressor. This
suppressor binds long dsRNA and inhibits processing of dsRNA into siRNA, but does not
bind short siRNAs or disrupt the miRNA pathway. Based on these results it was suggested
that RNAi is a major antiviral immune defense mechanism in Drosophila. Whether RNAi
has a similar function in mammals is under intense investigation (van Rij and Andino,
2006).
Invertebrates
Along with insect studies, successful researches of Caenorhabditis elegans have been made.
C. elegans contains a single Dicer gene. Similar to plants, C. elegans also supports transient
RNAi and systemic RNAi (Voinnet, 2005). It was shown that multiple genes of C. elegans
required for RNAi are also required for resistance against vesicular stomatitis virus (VSV).
VSV was engineered to encode a EGFP (enhanced green fluorescent protein) fusion protein;
two types of cells were infected: wild type and cells lacking components of the RNAi. The
RNAi-defective cells produced more EGFP and infectious particles, establishing that RNAi
of invertebrates is a part of an antiviral defense (Wilkins et al., 2005; Schott, et al., 2005).
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4. RNA interference in mammals
The problem, whether there is mammalian antiviral RNAi counterpart, is the subject of
many studies. It was supposed, that RNA silencing machinery is not typical for mammalian
cells. This skepticism was derived from the fact that mammalian cells respond to the
presence of dsRNA with activation of IFN system. Plants and invertebrates do not possess
this system; hence it might functionally replace the RNA interference (Gitlin and Andino,
2003). Then scientists discovered that siRNAs themselves are not able to induce IFN
system. (Elbashir et al., 2001). This finding entailed other question whether siRNAs could
target mammalian virus and whether viruses would have means to avoid such an onset
(Schütz and Sarnow, 2006).
It was considered, that mammalian cells might not need to interfere with the RNAi pathway,
because they have an effective IFN, which responds to the accumulation of viral dsRNAs by
inducing the synthesis of a large group of genes which exert inhibitory effects on viral gene
expression (Gale et al., 2000; Katze et al., 2002).
Viruses have evolved highly sophisticated mechanisms for interacting with the host cell
machinery, and recent evidence indicates that these interactions also involve RNAi
pathways. Last years the evidences that RNAi functions as an antiviral defense mechanism
in mammalian cells are accumulating. To the present time, there are only few papers
describing the mechanism of antiviral action of RNAi in mammalian cells.
Artificially triggered RNAi
The mammalian cellular RNAi machinery can inhibit virus replication, when the formation
of siRNAs is experimentally induced (Li et al., 2004). To attest this supposition, the
experiment with poliovirus replication, sense ssRNA virus, in the presence of siRNAs was
performed. The poliovirus’ genome is identical to mRNA, therefore can immediately initiate
translation of virus-encoded proteins. In one-step growth curves artificial siRNAs resulted in
a 100-fold reduction of the progeny titer (Gitlin et al., 2002). But the first RNAi-mediated
suppression of mammalian virus replication in tissue culture has been shown on
Coxsackievirus B3 (CVB3), which is the most common causal agent of viral myocarditis.
CVB3 belongs to the Picornaviridae family and has a ss(+)RNA genome (Yuan et al.,
2004).
Also the down regulating viral gene expression and replication of negative- and positivestrand
RNA viruses were demonstrated by the examples of influenza A and West Nile virus
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(WNV) (McCown et al., 2003). An enveloped, segmented, negative-strand RNA virus -
Influenza A virus belongs to the Orthomyxoviridae virus family (Chen et al., 2001) WNV is
an enveloped arbovirus with ss(+)RNA genome, belongs to the Flaviviridae family of
viruses (Chambers et al., 1990). siRNAs were shown to block expression of viral genes in a
specific manner and thus interfering the replication of the mentioned viruses (McCown et
al., 2003).
The acknowledgement of hypothesis, that the mammalian cellular RNAi machinery can
inhibit virus replication with the help of artificial siRNAs, was obtained by investigation of
foot-and-mouth disease virus (FMDV) replication. The VP1-specific siRNAs showed an
inhibitory effect on VP1 of FMDV in BHK-21 cells (baby hamster kidney cells), as well as
in and suckling mice, a commonly used small animal model. VP1 plays a key role in virus
attachment to susceptible cells and is essential during the life cycle of the virus.
Approximately 90% reduction in the expression of FMDV VP1 in BHK-21 cells was
observed with transfection of siRNAs. Suckling mice became significantly less susceptible
to FMDV (Chen et al., 2004).
In 2003 the investigations of SARS-CoV virus with ss(+)RNA genome has led to the
observation, that the infection and replication can be inhibited by siRNAs. They targeted the
replicase 1A region of the virus genome; this appeared to be effective in vitro in different
strains of SARS-CoV. This coronavirus has been identified as a cause of severe acute
respiratory syndrome (SARS). But clinical usefulness of such method of viral replication
inhibition remained to be undemonstrated (He et al., 2003).
Interest in RNAi as an alternative antiviral for human immunodeficiency virus type 1 (HIV-
1) is strong because of the problems of drug resistance, the cost and toxicity of modern
antiviral therapy against this virus (Manjunath et al., 2008). Several reports using siRNA
target sequences have shown potent RNAi activity against HIV-1 replication. Targeting the
region encoding the HIV-1 reverse transcriptase (RT) by siRNAs reduced viral replication
by 90% (Gimenez-Barcons et al., 2007). The siRNAs targeting tat/rev (Novina et al., 2002;
Lee et al., 2002; Coburn et al., 2002 ), pol (Surabhi and Gaynor, 2002),TAR, vif , 3’UTR
region (Jacque et al., 2002), as well as gag and the HIV-1 receptor CD4 (Novina et al.,
2002) and co-receptor CCR5 has led to the suppression of HIV-1.These results suggest that
RNAi represents an important new therapeutic approach for treating HIV-1 infection (Qin et
al., 2002).
It was observed that siRNAs directed against NS4B and core regions of hepatitis C virus
(HCV) specifically decreased quantity of HCV RNA. HCV is a main cause of chronic liver
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diseases, including liver cirrhosis and hepatocellular carcinoma (HCC). It has a ss(+)RNA
genome, and belongs to the Flaviviridae family. Other regions of the HCV’s RNA genome,
including 5'UTR and the coding sequences of core (Yokota et al., 2003; Seo et al., 2003;
Randall et al., 2003; Kapadia et al., 2003; Wilson et al., 2003), NS3 and NS5B (Takigawa et
al., 2004) are sensitive to the action of siRNA.
Similar results with the replication inhibition of RNA viruses and others like Coxsackievirus
B3 (Yuan et al., 2005), human rhinovirus 16 (Phipps et al., 2004), poliovirus (Gitlin et al.,
2002), human parainfluenza virus-3, vesicular stomatitis virus (Barik, 2004), hepatitis delta
virus (Chang and Taylor, 2003), and rotavirus (Dector et al., 2002) with the help of artificial
siRNAs and mammalian cell enzymes serve as an evidence, that the mammalian cellular
RNAi machinery can inhibit virus replication. These findings open a wide range of
enormous therapeutical potential of RNAi.
In summary, these in vitro studies is the first step to demonstrate that siRNA technology is a
very promising approach to antiviral gene therapy. RNAi based therapies for some viruses
have already reached clinical trials. However, there are still many concerns of the
widespread application of RNAi in treatment of human diseases (Kim and Rossi, 2007).
5. Suppression of cellular anti-viral RNAi by viruses
During infection dsRNA molecules are produced, which can trigger RNAi. To counteract
this and avoid recognition by the RNAi machinery of the host, viruses have evolved special
strategies (Berkhout and Haasnoot, 2006). Many mammalian viruses can encode proteins or
RNA molecules that suppress existing RNAi pathways or trigger the silencing of specific
host genes (Chen et al., 2005). Like the plant viruses, which encode proteins that interfere
with one or more aspects of Dicer action and/or siRNA targeting (Vance and Vaucheret,
2001; Voinnet et al., 1999), mammalian viruses also encode such molecules. The fact, that
animal RNA viruses have RNA silencing suppressors (RSSs) might serve as evidence that
RNAi acts as an antiviral defense also in mammalian cells. These suppressors block induced
RNAi against reporter gene constructs. However, functional significance of RNAi for viral
pathogenesis must be investigated (van Rij and Andino, 2006). Recently, several human
pathogenic viruses have been shown to encode RSSs, suggesting that RNAi serves as an
innate defense response in mammals (Li and Ding, 2001).
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RNA silencing suppressors
The fact that B2 of both the Nodamura virus (NoV) and the Flock House virus (FHV) can
inhibit RNAi triggered artificially in human cells was proved by scientists in 2005 (Sullivan
and Ganem, 2005). The NoV is not enveloped ss(+)RNA virus. It occurs in nature as an
unapparent infection of mosquitoes in Japan, also asymptomatically infects pigs (Bailey et
al., 1974). Many viral RSS identification in mammalian cells include the use of shRNAs.
Scientist developed a highly sensitive assay of RNAi in mammalian cells, which shows that
RNAi triggered by either shRNAs or siRNAs, can be inhibited by the NoV B2 protein. In
the cell, NoV B2 binds to pre-Dicer substrate RNA and RISC-processed RNAs, inhibiting
the Dicer cleavage reaction. Both endogenous genes and transgenes encoding EGFP or
luciferase have been used as the reporter target gene in RNAi suppression assays. The assay
based on RNAi of a destabilized EGFP (dEGFP) reporter gene was developed to identify
inhibitors of RNAi in mammalian cells (Sullivan and Ganem, 2005). Less EGFP was
detected, when cotransfected with the anti-EGFP shRNA than, when co-transfected with the
control anti-luciferase (anti-Luc) irrelevant shRNA. The facts, that transfected siRNAs could
suppress viral replication by reducing viral RNA accumulation and that viral RSSs function
in mammalian cells, could enhance the evidence, that mammalian RNAi machinery
possesses an antiviral response (Gitlin et al, 2002).
VP35 protein of Ebola virus has RSS activity, which is functionally equivalent to that of the
HIV-1 Tat protein. VP35 can replace HIV-1 Tat and thereby support the replication of a Tatminus
HIV-1 variant. It shows that viruses need to counteract RNAi in order to replicate.
Also VP35 protein is known as IFN antagonist. The VP35 dsRNA-binding domain is
required for this RSS activity. (Haasnoot et al., 2007)
Influenza A, B, C viruses NS1 protein and vaccinia virus E3L protein were demonstrated to
be essential proteins that suppress RNA silencing in Drosophila. The major fact is, that
these mammalian virus proteins, are also distinct dsRNA-binding proteins essential for
pathogenesis by inhibiting IFN antiviral response. It was also demonstrated that the dsRNAbinding
domain of NS1 is essential and sufficient for RNAi suppression (Li et al., 2004).
Influenza A virus NS1 protein and vaccinia virus E3L protein are also capable to replace the
HIV-1 Tat RSS function (Haasnoot et al., 2007).
The adenovirus virus-associated RNAs I and II (VA-RNAI and II) are also capable of
inhibiting RNAi antivaral response. VA RNA are highly structured RNAs, about 160 nt in
length, encoded by adenoviral genome and expressed by host RNA polymerase III.
(Mathews and Shenk, 1991). At late infection human adenovirus inhibits RNAi antiviral
20

esponse by suppressing the activity of Dicer and RISC. The VA RNAs bind Dicer and
function as competitive substrates squelching Dicer. The siRNAs, received from VA RNAI
and VA RNAII are incorporated into active RISC. A panel of mutant adenoviruses defective
in virus-associated (VA) RNA expression was used to detect the mechanism by which
adenovirus blocks RNAi. The investigation results suggested that the adenovirus VA RNAs
antagonize the cellular defense pathways directed against both long (interferon-induced) and
short (RNAi-induced) dsRNA by binding and inactivating PKR and Dicer enzymes VA
RNAI potently inhibited RNAi induced by endogenously transcribed pre-miRNAs and
shRNAs and weakly inhibited RNAi induced by in vitro transcribed, transfected shRNAs.
But it did not affect RNAi induced by artificial siRNA-miRNA duplexes. Also it was
observed, that VA RNAI expression inhibited the production of mature miRNAs from premiRNA
precursors, because VA RNAI is a potent inhibitor of pre-miRNA or shRNA
nuclear export, competing for a limited pool of the cellular Exp5 nuclear export factor. To
conclude, adenovirus VA RNAI is a gene product, which is able to specifically block RNAi
in human cells (Lu and Cullen, 2004)
These findings support the hypothesis that mammalian virus proteins can inhibit RNA
silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in
mammalian cells. Moreover, the results indicate that RSSs play a critical role in mammalian
virus replication (Haasnoot et al., 2007).
Virus-encoded miRNAs
The fact, that viruses use the RNAi machinery serves as another evidence of its existence.
Viruses encode miRNA-like molecules, that can potentially affect cellular miRNA
processing and functioning (Berkhout and Haasnoot, 2006) Virus-encoded miRNAs were
identified while cloning the small RNA profile in cells infected with Epstein–Barr virus
(EBV). This is a large DNA virus, which belongs to the herpesvirus family (Pfeffer et al.,
2004). Other members of the herpesvirus family and other viruses with large DNA genomes
could encode miRNAs to exploit RNAi for the regulation of host and viral expression.
(Berkhout and Haasnoot, 2006). Processing of virus-encoded miRNAs is going through the
same pathway as host miRNAs. The early miRNAs are cleaved by the late miRNA at the
predicted position in the replication cycle, thus reducing early gene expression, and possibly
evading immune recognition of the infected cell. The viral miRNA targets the early
transcript for degradation. Viruses exploits the RNAi machinery to regulate their gene
21

expression, thereby reducing susceptibility to the adaptive immune response (Berkhout and
Haasnoot, 2006; van Rij and Andino, 2006)
Further directions of investigations
Advances in our understanding of the mechanisms of RNAi indicate that RNAi-based
therapies might soon provide a powerful new arsenal against viral diseases for which
treatment options are currently limited. Recent findings have highlighted both promise and
challenges in using RNAi for therapeutic applications. There are still a number of major
concerns and possible impediments to the widespread application of RNAi for the treatment
of human disease. For instance, chronic diseases such as hepatitis C virus or HIV infections
will require lifelong treatments with RNAi. So the further effetcs of RNAi, design and
delivery strategies for RNAi effector molecules must be carefully considered to address
safety concerns and to ensure effective, successful treatment of viral diseases. The coming
years are likely to see the detailed investigation of this process and an increasing range of
applications for RNAi-based treatments (Berkhout and Haasnoot,2006; Kim and Rossi,
2007).
22

CONCLUSIONS
In the present bachelor’s thesis on the basis of literature:
1. an overview of intracellular antiviral responses are made;
2. RNAi mechanism are described;
3. evidences, that RNA interference in mammalian cells acts as a viral antagonist, are
given;
4. evidences, that mammalian viruses have potent anti-RNAi mechanisms, are collected
and analyzed.
RNAi is established to be an adaptive nucleic acid-based antiviral response. Thorough
investigations on RNAi have been made in plants and insects. Nevertheless, till the recent
time it was debated, whether RNAi acts as an antiviral response in mammalian cells. Some
investigation achievements to support this opinion are described in this bachelor’s thesis.
Recent researches in this field showed that RNAi is a part of antiviral response in
mammalian cells. Viruses are used to exploit RNAi machinery in order to replicate, could be
the first confirmation of this. Second evidence was found to be RSSs, which are encoded by
animal viruses, as counteracting viral elements to RNAi. Hence, the benefits of the fact,
RNAi acts as an antiviral response in mammalian cells, are that RNAi has a potential of an
antiviral treatment or diagnostic tool. Still many aspects and possibilities of this process
remained unknown, but many thorough investigations and clinical trials are yet to come.
23

Viirused ja RNA interferents imetajarakkudes
Jekaterina Gusseva
KOKKUVÕTE
Biotehnoloogiliste uurimismeetodite areng viimastel aastatel tegi võimalikuks RNA
inteferensi (RNAi) avastamist. RNA interferents on regulatoorne mehhanism, mis viib võõra
mRNA degradeerimiseks, dsRNA indutseerimisega järjestus-spetsiifilisel viisil. Ta surub
maha geenide ekspressiooni translatsiooni staadiumil või sekkumisega spetsiifiliste geenide
transkriptsiooni.
RNAi oli eelnevalt hästi kirjeldatud taimedel ja putukatel, eeskätt kui osa antiviiruslikust
rakulisest vastusest. Mis puutub imetajatesse, siis arvati, et nende rakud ei oma RNAi
viiruse-vastast toimet, kuna seal on olemas võimas interferooni süsteem.
Käesolev bakalaureusetöö on kirjanduse põhjal koostatud uurimistöö. Kirjanduse analüüsi
põhjal:
- tehakse ülevaadet rakusisestest viiruse-vastastest mehhanismidest (interferooni
süsteem ja RNAi);
- antakse RNAi mehhanismi detailne kirjeldus;
- esitatakse hulgalisi tõestusi selle kohta, et RNAi on äärmiselt oluline viiruse-vastane
mehhanism mitte ainult taimedes ja putukates, vaid ka imetajarakkudes;
- tuuakse fakte selle kohta, et ka loomsetel viirustel on olemas spetsiifilised
mehhanismid rakusisese RNAi mehhanismi mahasurumiseks.
Tänapäevases maailmas on olemas suur hulk haigusi, mis on esile kutsutud viiruste poolt
ning mille vastu ei ole efektiivset ravi. Viiruse-vastase RNA interferentsi uurimine on
paljulubav ja lootust andev teaduse suund, mis tulevikus võiks palju kaasa aidata uute
viiruste-vastaste ravimeetodite väljatöötamisele. Selle saavutamiseks oleks praegusel hetkel
vaja arendada teadustööd edasi sellistel suundadel nagu: RNAi kõrvaltoimed, RNAi efektor
molekulite moodustumine ja nende kohaletoimetamine.
24

REFERENCES
Aagaard, L. and Rossi, J. J. (2007) RNAi Therapeutics: Principles, Prospects and Challenges. Adv Drug
Deliv Rev. 2007 Mar 30;59(2-3):75-86. Epub 2007 Mar 16.
Anandalakshmi R, Marathe R, Ge X, Herr JM Jr, Mau C, Mallory A, Pruss G, Bowman L, Vance VB.
(2000) A calmodulin-related protein that suppresses posttranscriptional gene silencing in plants. Science. 2000
Oct 6;290(5489):142-4.
Andersson, M.G., Haasnoot, P.C.J., Xu, N., Berenjian, S., Berkhout, B. and Akusjarvi, G. (2005)
Suppression of RNA interference by adenovirus virus-associated RNA. J. Virol. 79, 9556–9565.
Bailey, L., Newman, J.F. and Porterfield, J.S. (1974) The multiplication of Nodamura virus in insect and
mammalian cell cultures. J Gen Virol. 1975 Jan;26(1):15-20.
Barik, S. (2004) Control of nonsegmented negative-strand RNA virus replication by siRNA. Virus Res. 102,
27–35.
Baulcombe D. (2004) RNA silencing in plants. Nature. 2004 Sep 16;431(7006):356-63.
Berkhout B, Haasnoot J. (2006) The interplay between virus infection and the cellular RNA interference
machinery. FEBS Lett. 2006 May 22;580(12):2896-902.
Bitko, V. and Barik, S. (2001) Phenotypic silencing of cytoplasmic genes using sequence-specific doublestranded
short interfering RNA and its application in the reverse genetics of wild type negative-strand RNA
viruses.BMC Microbiol. 2001;1:34.
Campbell, C.L., Keene, K.M., Brackney, D.E., Olson, K.E., Blair, C.D., Wilusz, J., Foy, B.D. (2008)
Aedes aegypti uses RNA interference in defense against Sindbis virus infection. BMC Microbiol. 2008 Mar
17;8:47.
Carmell, M. A. and Hannon, G. J. (2004) RNase III enzymes and the initiation of gene silencing. Nat Struct
Mol Biol. 2004 Mar;11(3):214-8.
Chambers, T.J., Hahn, C.S., Galler, R., Rice, C.M., (1990) Flavivirus genome organization, expression, and
replication. Annu. Rev. Microbiol. 44, 649–688.
Chang, J. and Taylor, J.M. (2003) Susceptibility of human hepatitis delta virus RNAs to small interfering
RNA action. J. Virol. 77, 9728–9731.
25

Chen, W., Liu, M., Cheng, G., Yan, W., Fei, L. and Zheng, Z. (2005) RNA silencing: A remarkable parallel
to protein-based immune systems in vertebrates? FEBS Lett. 2005 Apr 25;579(11):2267-72.
Chen, W., Yan, W., Du, Q., Fei, L., Liu, M., Ni, Z., Sheng, Z. and Zheng, Z. (2004) RNA Interference
Targeting VP1 Inhibits Foot-and-Mouth Disease Virus Replication in BHK-21 Cells and Suckling Mice. J
Virol. 2004 Jul;78(13):6900-7.
Chen, W., Calvo, P.A., Malide, D., Gibbs, J., Schubert, U., Bacik, I., Basta, S., O’Neill, R., Schickli, J.,
Palese, P., Henklein, P., Bennink, J.R., Yewdell, J.W., (2001) A novel influenza A virus mitochondrial
protein that induces cell death. Nat. Med. 7, 1306–1312.
Coburn, G.A. and Cullen, B.R. (2002) Potent and specific inhibition of human immunodeficiency virus type
1 replication by RNA interference. J Virol 2002; 76:9225-31.
De Carvalho, F., Gheyson, G., Kushnir, S., van Montagu, M., Inzй, D. (1992) Suppressionof β-1,3-
glucanase transgene expression in homozygous plants. EMBO J 11: 2595–2602.
Dector,M.A. , Romero, P., López, S., Arias, C.F. (2002) Rotavirus gene silencing by small
interferingRNAs. EMBO Rep. 3, 1175–1180.
Ding, S.W., Li, H., Lu, R., Li, F., Li, W.X. (2004) RNA silencing: a conserved antiviral immunity of plants
and animals. Virus Res. 2004 Jun 1;102(1):109-15.
Dorin, D., Bonnet, M.C., Bannwarth, S., Gatignol, A., Meurs, E.F. and Vaquero, C. (2003) The TAR-
RNA binding protein, TRBP, stimulates the expression of TAR-containing RNAs in vitro and in vivo
independently of its ability to inhibit the dsRNA dependent kinase PKR. J Biol Chem. 2003 Feb
14;278(7):4440-8.
Elbashir, S.M., Lendeckel, W. and Tuschl, T. (2001) RNA interference is mediated by 21- and 22-
nucleotide RNAs. Genes and Development 15 188–200.
Fire, A.Z. (1998) Gene silencing by double-stranded RNA. Cell Death Differ. 2007 Dec;14(12):1998-2012.
Gale, M. Jr., Tan, S.L., Katze, M.G. (2000) Translational control of viral gene expression in eukaryotes.
Microbiol Mol Biol Rev. 2000 Jun;64(2):239-80.
Gimenez-Barcons, M., Clotet, B., Martinez, M.A. (2007) Endoribonuclease-prepared short interfering RNAs
induce effective and specific inhibition of human immunodeficiency virus type 1 replication. J Virol. 2007
Oct;81(19):10680-6.
26

Gitlin, L., Karelsky, S. and Andino, R. (2002) Short interfering RNA confers intracellular antiviral immunity
in human cells. Nature 418, 430–434.
Gitlin, L. and Andino, R. (2003) Nucleic acid-based immune system: the antiviral potential of mammalian
RNA silencing. J Virol. 2003 Jul;77(13):7159-65.
Gregory, R. L., Chendrimada, T. P., Cooch, N. and Shiekhattar, R. (2005) Human RISC Couples
MicroRNA Biogenesis and Posttranscriptional Gene Silencing. Cell. 2005 Nov 18;123(4):631-40.
Grosshans, H. and Filipowicz W. (2008) Nature. 2008 Jan 24;451(7177):414-6
Haasnoot, J., Berkhout, B. (2006) RNA interference: its use as antiviral therapy. Handb Exp Pharmacol.
2006;(173):117-50.
Haasnoot, J., de Vries, W., Geutjes, E.J., Prins, M., de Haan, P. and Berkhout B. (2007) The Ebola Virus
VP35 Protein Is a Suppressor of RNA Silencing. PLoS Pathog. 2007 Jun;3(6):e86.
Hamilton, A.J., Baulcombe, D.C. (1999) A species of small antisense RNA in posttranscriptional gene
silencing in plants. Science. 1999 Oct 29;286(5441):950-2.
Hammond, S.M. (2006) RNAi, microRNAs, and human disease. Cancer Chemother Pharmacol. 2006 Nov;58
Suppl 1:s63-8.
He, L., Hannon, G.J. (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet.
2004 Jul;5(7):522-31.
He, M.L., Zheng, B., Peng, Y., Peiris, J.S., Poon, L.L., Yuen, K.Y., Lin, M.C., Kung, H.F., and Guan, Y.
(2003) Inhibition of SARS-Associated Coronavirus Infection and Replication by RNA Interference. JAMA.
2003 Nov 26;290(20):2665-6.
Hong, J., Wei, N., Chalk, A., Wang, J., Song, Y., Yi, .F, Qiao, R.P., Sonnhammer, E.L., Wahlestedt, C.,
Liang, Z., Du, Q. (2008) Focusing on RISC assembly in mammalian cells. Biochem Biophys Res Commun.
2008 Apr 11;368(3):703-8.
Hu, W.Y., Bushman,
Res;102(1):59-64.
F.D., and Siva, A.C. (2004) RNA interference against retroviruses. Virus
Iannello, A., Debbeche, O., Martin, E., Attalah, L. H., Samarani, S. and Ali Ahmad. (2005) Viral
strategies for evading antiviral cellular immune responses of the host. J Leukoc Biol. 2006 Jan;79(1):16-35.
27

Jacque, J.M., Triques, K. and Stevenson, M. (2006) Modulation of HIV-1 replication by RNA interference.
Nature. 2002 Jul 25;418(6896):435-8.
Jaronczyk, K., Carmichael, J. B. and Hobman T. C. (2005) Exploring the functions of RNA interference
pathway proteins: some functions are more RISCy than others? Biochem J. 2005 May 1;387(Pt 3):561-71
Kapadia, S.B., Brideau-Andersen, A. and Chisari, F.V. (2003) Interference of hepatitis C virus RNA
replication by short interfering RNAs. Proc Natl Acad Sci USA 2003; 100: 2014-2018.
Katze, M.G., He, Y. and Gale, M. Jr. (2002) Viruses and interferon: a fight for supremacy. Nat Rev
Immunol. 2002 Sep;2(9):675-87.
Ketting, R.F., Fischer, S.E., Bernstein, E., Sijen, T., Hannon, G.J. and Plasterk, R.H. (2001) Dicer
functions in RNA interference and in synthesis of small RNA involved in developmental timing in C. elegans.
Genes Dev. 2001 Oct 15;15(20):2654-9.
Keene, K.M., Foy, B.D., Sanchez-Vargas, I., Beaty, B.J., Blair, C.D. and Olson, K.E. (2004) RNA
interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of
Anopheles gambiae. Proc Natl Acad Sci U S A. 2004 Dec 7;101(49):17240-5.
Kim, D.H. and Rossi, J.J. (2007) Strategies for silencing human diseases using RNA interference. Nat Rev
Genet. 2007 Mar;8(3):173-84.
Lu, S. and Cullen, B.R. (2004) Adenovirus VA1 Noncoding RNA Can Inhibit Small Interfering RNA and
MicroRNA. J Virol. 2004 Dec;78(23):12868-76.
Lee, N.S., Dohjima, T., Bauer, G., Li, H., Li, M., Ehsani, A., et al. (2002) Expression of small interfering
RNAs targeted against HIV-1 rev transcripts in human cells. Nat Biotechnol 2002; 19:500-5.
Lee, Y., Ahnm, C., Han, J., Choi, H., Kim, J., Yim, J., Lee, J., Provost, P., Rådmark, O., Kim, S. and
Kim, V.N. (2003) The nuclear RNase III Drosha initiates microRNA processing. Nature. 2003 Sep
25;425(6956):415-9.
Li, W.X. and Ding, S.W. (2001) Viral suppressors of RNA silencing. Curr Opin Biotechnol. 2001
Apr;12(2):150-4.
Li, H.W., Lucy, A.P., Guo, H.S., Li, W.X., Ji, L.H., Wong, S.M. and Ding, S.W. (1999) Strong host
resistance targeted against a viral suppressor of the plant gene silencing defence mechanism. EMBO J. 1999
May 17;18(10):2683-91.
28

Li, W.X. and Ding, S.W., (2001) Viral suppressors of RNA silencing. Curr. Opin. Biotechnol. 12, 150–154.
Li, H.W., Li, W.X., Ding, S.W., (2002) Induction and suppression of RNA silencing by an animal virus.
Science 296, 1319–1321.
Li, W.X., Li, H., Lu, R., Li, F., Dus, M., Atkinson, P., Brydon, E.W., Johnson, K.L., Garcia-Sastre, A.,
Ball, L.A., Palese, P. And Ding, S.W. (2004) Interferon antagonist proteins of influenza and vaccinia viruses
are suppressors of RNA silencing. Proc. Natl. Acad. Sci. USA 101, 1350–1355.
Li, X., X. Zhao, Y. Fang, X. Jiang, T. Duong, C. Fan, C. C. Huang, and S. R.
Kain. (1998) Generation of destabilized green fluorescent protein as a transcription
reporter J. Biol. Chem. 273:34970–34975.
Li, H.W. and Ding ,S.W. (2005) Antiviral silencing in animals. FEBS Lett. 2005 Oct 31;579(26):5965-73.
Lu, S. and Cullen, B.R. (2004) Adenovirus VA1 noncoding RNA can inhibit small interfering RNA and
microRNA biogenesis. J.Virol. 78, 12868–12876.
Lund, E., Güttinger, S., Calado, A., Dahlberg, J.E. and Kutay, U. (2004) Nuclear export of microRNA
precursors. Science. 2004 Jan 2;303(5654):95-8.
Macrae, I..J., Li, F., Zhou, K., Cande, W.Z. and Doudna, J.A. (2006) Structure of Dicer and mechanistic
implications for RNAi. Cold Spring Harb Symp Quant Biol. 2006;71:73-80.
Martinez, J., Patkaniowska, A., Urlaub, H., Lührmann, R. and Tuschl, T. (2002) Single-stranded
antisense siRNAs guide target RNA cleavage in RNAi. Cell. 2002 Sep 6;110(5):563-74.
Manjunath, N., Kumar, P., Lee, S.K. and Shankar, P. (2006) Interfering antiviral immunity: application,
subversion, hope? Trends Immunol. 2006 Jul;27(7):328-35.
Mathews, M.B. and Shenk, T. (1991) Adenovirus virus-associated RNA and translation control. J Virol.
1991 Nov;65(11):5657-62.
McCown, M., Diamond, M.S., Pekosz, A. (2003) The utility of siRNA transcripts produced by RNA
polymerase i in down regulating viral gene expression and replication of negative- and positive-strand RNA
viruses. Virology. 2003 Sep 1;313(2):514-24.
Meister, G. and Tuschl, T. (2004) Mechanisms of gene silencing by double-stranded RNA. Nature. 2004 Sep
16;431(7006):343-9.
29

Montanucci, L., Piero Fariselli, P., Martelli P. L., Rossi, I. and Casadio, R. (2007) In silico evidence of the
relationship between miRNAs and siRNAs.
Napoli, C., Lemieux, C. and Jorgensen, R. (1990) lntroduction of a Chimeric Chalcone Synthase Gene into
Petunia Results in Reversible Co-Suppression of Homologous Genes Ín trans. Plant Cell. 1990 April; 2(4):
279–289.
Novina, C.D., Murray, M.F., Dykxhoorn, D.M., Beresford, P.J., Riess, J., Lee, S., Collman, R.G.,
Lieberman, J., Shankar, P. and Sharp, P.A. (2002) siRNA-directed inhibition of HIV-1 infection. Nat Med
2002; 8:681-6.
Nykanen, A., Haley, B. and Zamore, P.D. (2001) ATP requirements and small interfering RNA structure in
the RNA interference pathway. Cell 107 309–321.
Pfeffer, S., Zavolan, M., Grasser, F.A., Chien, M., Russo, J.J., Ju, J., John, B., Enright, A.J., Marks, D.,
Sander, C. and Tuschl, T. (2004) Identification of virus-encoded microRNAs. Science 304, 734–736.
Phipps, K.M. Martinez, A., Lu, J., Heinz, B.A. and Zhao, G. (2004) Small interfering RNA molecules as
potential anti-human rhinovirus agents: in vitro potency, specificity, and mechanism. Antiviral Res. 61, 49–55
Qin, X., An, D., Chen, I.S.Y. and Baltimore, D. (2002) Inhibiting HIV-1 infection in human T cells by
lentiviral mediated delivery of small interfering RNA against CCR5. PNAS 2002; 100:183-8.
Rand, T.A., Petersen, S., Du, F. and Wang, X. (2005) Argonaute2 Cleaves the Anti-Guide Strand of siRNA
during RISC Activation. Cell. 2005 Nov 18;123(4):621-9.
Randall, G., Grakoui, A. and Rice, C.M. (2003) Clearance of replicating hepatitis C virus replicon RNAs in
cell culture by small interfering RNAs. Proc Natl Acad Sci USA 2003; 100: 235-240.
Rutz, S. and Scheffold, A. (2004) Towards in vivo application of RNA interference – new toys, old problems.
Arthritis Res Ther. 2004;6(2):78-85.
Samuel, C.E. (2001) Antiviral actions of interferons. Clin Microbiol Rev. 2001 Oct;14(4):778-809
Schott, D.H., Cureton, D.K., Whelan, S.P. and Hunter, C.P. (2005) An antiviral role for the RNA
interference machinery in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 102, 18420–18424
Schwarz, D.S., Hutvágner, G., Du, T., Xu, Z., Aronin, N., Zamore, P.D. (2003) Asymmetry in the assembly
of the RNAi enzyme complex. Cell. 2003 Oct 17;115(2):199-208.
30

Schütz, S. and Sarnow, P. (2006) Interaction of viruses with the mammalian RNA interference pathway.
Virology. 2006 Jan 5;344(1):151-7. Review.
Seo, M.Y., Abrignani, S., Houghton, M. and Han, J.H. (2003) Small interfering RNA-mediated inhibition
of hepatitis C virus replication in the human hepatoma cell line Huh-7. J Virol 2003; 77: 810-812.
Smith, C.J., Watson, C.F., Bird, C.R., Ray, J., Schuch, W. and Grierson, D. (1990) Expression of a
truncated tomato polygalacturonase gene inhibits expression of the endogenous gene in transgenic plants. Mol
Gen Genet. 1990 Dec;224(3):477-81.
Sullivan, C.S. and Ganem, D. (2005) A Virus-Encoded Inhibitor That Blocks RNA Interference in
Mammalian Cells. J Virol. 2005 Jun;79(12):7371-9.
Surabhi, R.M. and Gaynor, R.B. (2002) RNA interference directed against viral and cellular targets inhibits
human immunodeficiency Virus Type 1 replication. J Virol. 2002 Dec;76(24):12963-73.
Takigawa, Y., Nagano-Fujii, M., Deng, L., Hidajat, R., Tanaka, M., Mizuta, H. and Hotta, H. (2004)
Suppression of hepatitis C virus replicon by RNA interference directed against the NS3 and NS5B regions of
the viral genome. Microbiol Immunol 2004; 48: 591-598.
Tang, G. (2005) siRNA and miRNA: an insight into RISCs. Trends Biochem Sci. 2005 Feb;30(2):106-14.
Van Blokland, R., van der Geest ,N., Mol, J. and Kooter, J. (1994) Transgene-mediated suppression of
chalcone synthase expression in Petunia hybrida results from an increase in RNA turnover. Plant J 6: 861–877.
van der Krol, A.R., Mur, L.A., Beld, M., Mol, J.N.M. and Stutje, A.R. (1990) Flavonoid genes in petunia:
addition of a limited number of gene copies may lead to a suppression of gene expression. Plant Cell 4: 291–
299.
van Rij, R.P. and Andino, R. (2006) The silent treatment: RNAi as a defense against virus infection in
mammals. Trends Biotechnol. 2006 Apr;24(4):186-93.
Vance, V. and Vaucheret, H. (2001) RNA silencing in plantsdefense and counterdefense. Science 292, 2277–
2280.
Voinnet, O., Pinto, Y.M. and Baulcombe, D.C. (1999) Suppression of gene silencing: a general strategy used
by diverseDNA and RNA viruses of plants. Proc Natl Acad Sci U S A. 1999 Nov 23;96(24):14147-52.
Voinnet, O. (2005) Non-cell autonomous RNA silencing. FEBS Lett. 579, 5858–5871.
31

Wilkins, C., Dishongh, R., Moore, S.C., Whitt, M.A., Chow, M. and Machaca, K. (2005) RNA
interference is an antiviral defence mechanism in Caenorhabditis elegans. Nature 436, 1044–1047.
Wilson, J.A., Jayasena, S., Khvorova, A., Sabatinos, S., Rodrigue-Gervais, I.G., Arya, S., Sarangi, F.,
Harris-Brandts, M., Beaulieu, S. and Richardson, C.D. (2003) RNA interference blocks gene expression
and RNA synthesis from hepatitis C replicons propagated in human liver cells. Proc Natl Acad Sci USA 2003;
100: 2783-2788.
Xie, Z., Fan, B., Chen, C., Chen, Z., (2001) An important role of an inducible RNA-dependent RNA
polymerase in plant antiviral defense. Proc. Natl. Acad. Sci. U.S.A. 98, 6516–6521.
Yi, R., Qin, Y., Macara, I.G. and Cullen, B.R. (2003) Exportin-5 mediates the nuclear export of premicroRNAs
and short hairpin RNAs. Genes Dev. 2003 Dec 15;17(24):3011-6.
Yokota, T., Sakamoto, N., Enomoto, N., Tanabe, Y., Miyagishi, M., Maekawa, S., Yi, L., Kurosaki, M.,
Taira, K., Watanabe, M. and Mizusawa, H. (2003) Inhibition of intracellular hepatitis C virus replication by
synthetic and vector-derived small interfering RNAs. EMBO Rep 2003; 4: 602-608.
Yuan, J., Cheung, P.K., Zhang, H.M., Chau, D. and Yang, D. (2005) Inhibition of Coxsackievirus B3
Replication by Small Interfering RNAs Requires Perfect Sequence Match in the Central Region of the Viral
Positive Strand. J Virol. 2005 Feb;79(4):2151-9.
Zeng, Y., Yi, R. and Cullen, B.R. (2003) MicroRNAs and small interfering RNAs can inhibit mRNA
expression by similar mechanisms. Proc Natl Acad Sci U S A. 2003 Aug 19;100(17):9779-84.
Zhang, H., Kolb, F.A., Brondani, V., Billy, E. and Filipowicz, W. (2002) Human Dicer preferentially
cleaves dsRNAs at their termini without a requirement for ATP. EMBO J. 2002 Nov 1;21(21):5875-85.
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