Perspectives on Contemporary Virology
- Volume 1, 2007
Vaccine and Infectious Disease Organization and
Dept. of Veterinary Microbiology
University of Saskatchewan
for: Advanced Virology (VTMC 833)
RNA interference (RNAi) is a natural response to the presence of double
stranded RNA (dsRNA) which results in the sequence-specific silencing
of gene expression. RNAi is a nucleic-acid based immune defense against
viruses, transgenes and transposons. In eukaryotic cells, RNAi is
triggered either by short interfering RNAs (siRNA) or by micro RNAs
(miRNA) molecules. Recent findings reveal that certain viruses encode
their own miRNAs that are processed by cellular RNAi machinery.
However, it was unclear what the roles of these virus-encoded miRNAs
play and whether some cellular miRNAs play a role in viral replication
and phatogenicity. Here I have reviewed the current findings on
virus-encoded miRNAs, and their roles in viral replication. I have also
examined the role of cellular miRNA in the virus replicative cycle,
mechanisms of virus countradefense as well as roles of viral miRNAs in
cancer development and latest achievements in antiviral therapy using
The mi/siRNA induced
RNA interference pathway
RNAi was first discovered in the nematode Caeonrhabditis elegans in
1998 , but previous studies in plants and
fungi had exposed homology-dependent gene silencing mechanism which can
be triggered by viral replication or transgene expression . The RNAi
is a conserved sequence-specific gene silencing mechanism present in
most eukaryotes that is induced by dsRNA. This natural response was
initially shown to be posttranscriptional gene silencing (PTGS)
resulting in sequence-specific degradation of targeting mRNA . Beside PTGS, further research revealed that
RNAi pathway could cause transcriptional gene silencing (TGS) and
translational inhibition . Presently, it has been shown that RNAi
can be triggered by two similar mechanisms:
The first one is the regulation of cellular gene expression by miRNAs (Figure 1a.). Mammalian miRNA genes are found as
single or clustered transcription units [4, 5]
which are expressed either from intron regions of protein-coding or
exon non protein-coding genes . miRNAs
originate from a wide variety of monocistronic, dicistronic or
polycistronic primary transcripts (pri-miRNAs) [7, 8] that are generated by RNA polymerase II
(pol II) in all eukaryotes or by RNA polymerase III (pol III) in some
viruses . Within the pri-miRNA, 21~23 nt
mature miRNA forms part of one arm of ~70 nt imperfect stem-loop
sequence . Processing of pri-miRNAs
requires the recognition and nuclear cleavage of the RNA stem-loop
structures by nuclear RNase III enzyme Drosha. Drosha activity is
supported by Di George-syndrome critical protein 8 (DGCR 8), which
directly interacts with pri-miRNAs and stabilizes the junction between
the helical RNA and single-stranded RNA in the pri-miRNA hairpin . Cleavage of pri-miRNAs liberates a RNA
hairpin intermediate (~70nt) containing a characteristic 2 nt 3’
overhang, named a premature miRNA (pre-miRNA) .
The next step in miRNA biogenesis requires nuclear export of the
pre-miRNA. Exportin5 (Exp5) together with the GTP-bound form of
cofactor Ran, recognize and bind the 2 nt 3’overhang and carry out the
export of pre-miRNA from nucleus to cytoplasm 
. Once in the cytoplasm, GTP hydrolyses
and provides release of the pre-miRNAs, which become targets for a
second cellular RNase III enzyme, Dicer. Dicer rapidly cleaves
pre-miRNAs and yield to the generation of mature miRNAs duplexes
(~21-23nt). Dicer activity is aided by transactivating (TAR) RNA
binding protein (TRBP) and PACT (interferon-inducible dsRNA dependent
protein kinase (PKR) activator) which are both co-factors for strand
selection [15, 16]. Dicer binds to the 2nt
3’overhang at the base of the pre-miRNA hairpin and cuts the terminal
loop, leaving a second 2nt 3’overhang structure in the miRNA duplex
intermediate. Further, Dicer facilitates assembly of the miRNA strand
of the duplex into the RNA-induced silencing complex (RISC). The strand
with the lower thermodynamic stability at the 5’end of miRNA is named
“guide strand” and it is incorporated in RISC while the “passenger”
strand is released and degraded . The
composition of RISC is not fully defined, but it is known that key
component presents Argonaute protein (Ago). Besides Ago enzyme, eight
different enzymes (FMRP, FXR1, FXR2, Dicer, Gemin-3, Gemin-4, MOV10,
TNRC6B and PRMT5) have been identified as components of RISC involved
in the recognition of target mRNA by the miRNA [18,
19]. The specific steps in how these proteins interact with each
other and associate in RISC complex as well as their roles in the
recognition and targeting mRNA is not fully understood. The
incorporated miRNA strand act as a guide to direct RISC to the
complementary mRNA. The most common scenario of target recognition by a
miRNA in mammalian cells involves imperfect complementarity, which
leads either to TGS or degradation of the target mRNA.
The second mechanism that triggers RNAi is related to the presence of
viral genomes or transponsable elements and generation of siRNAs
molecules (~19-21nt) (Figure 1b.). The
processing of secondary RNA structures in the viral RNA genome or dsRNA
replication intermediates by RNase III enzyme Dicer generates these
siRNAs. After Dicer processing, siRNAs molecules follow the same
pathway as miRNAs described previously. The only difference is that
upon loading into the RISC complex, siRNAs trigger cleavage of the
target mRNA, since they are perfectly complementary to the target mRNA.
verification of virus encoded miRNAs
Since miRNAs have been discovered and their role in gene regulation
established, it has been theorized that viruses could generate miRNAs
as well and that these viral encoded miRNAs could regulate cellular
mechanisms and viral replication. There are several lines of evidence
to support this theory:
The first being that miRNAs unlike viral proteins are not antigenic.
Their small size is enough to avoid INF/PKR induced pathway triggered
by long dsRNAs. Secondly, miRNAs are able to down-regulate the
expression of host gene products, which might interfere with stages in
viral life cycle. Finally, miRNAs are able to occupy ~200 nt or ~200bp
of viral genome which is significant advantage given the tight
constraints on viral genome size . A
comparison of the crucial stages in the miRNA biogenesis and viral
replication has shown that there is no unique theory for the synthesis
of viral miRNA. Most of the RNA viruses replicate in the cytoplasm
while the replication site for the majority of DNA viruses is nucleus . Initial processing of cellular miRNA
occurs in the nucleus suggesting that only viruses that replicate in
nucleus are able to generate miRNA. This theory is supported by recent
reports [22-24]. However, there are some
indications that Pox viruses, DNA viruses that replicate in the
cytoplasm are also able to synthesis miRNA, what is inconsistent with
the previous theory.
More than 60 viral miRNAs have been identified within four different
virus families. Most of the reported virus encoded miRNAs are reported
from Herpesvirues and small number within Adenovirus (1), Retrovirus
(1) and Polyomavirus (1) families (Table1.)
Methods for identification
and purification of viral miRNA
In all reports where the presence of viral encoded miRNA is
confirmed, two methods appear to be the most reliable and efficient .
(i) Small RNA cloning method is based on the
isolation of size-fractionated small RNAs and their cloning from
virus-infected cells. Briefly, 18 to 25 nt long RNAs are first purified
by excision from acrylamide gel and individual linkers are ligated to
the 3’ and 5’ ends of purified RNAs. Using the linkers, the small RNAs
are reverse transcribed and amplified by PCR. Presence of the
restriction sites within linkers allows the cloning of the small RNAs
into plasmids. Sequencing of the cloned plasmids identifies small RNAs
including miRNAs in the cells .
(ii) Computer-driven predictions are the second way
to identify virally encoded miRNAs. Currently, all miRNA prediction
programs scan viral genomes for the presence of stem-loop structures
present in the pri- and pre- miRNAs. The advantage of this approach is
that the computer program can scan a large number of sequences and
identify potential candidate stretches able to form stable secondary
stem-loop structures. Further, predicted miRNAs-candidates are selected
and validated by Northern blotting.
The internet and several RNA companies offer a variety of different
miRNA prediction software (Promega, Ambion, ViTA, siVirus) which are
usually connected with virus sequence databases (PubMed). These
programs are quite reliable and efficient and most importantly, the
majority are free. Although initially some viral miRNA were found using
these methods, a number of problems can also be encountered. Small RNA
cloning is highly dependent on the abundance of miRNA in virus-infected
cells and the timing and expression pattern of viral miRNAs are
unpredictable. Small RNA cloning depends on stage, robustness of
infection and cell type as well. In addition, computer-driven
predictions are not always accurate and at times misleading.
miRNA with known functions
The role of most reported viral
miRNAs (vi-miRNA) in the viral life cycle and cellular processing are
still unknown. At present only four vi-miRNAs has been reported with,
more or less established roles and functions (Table
Simian polyomavirus 40
(SV40) encodes a set of miRNAs whose in vivo targets have been defined . Computer-based predictions identified one
vi- pre-miRNA encoded by sequences located within 3’ of the late poly
(A) cleavage site in the viral mRNA. Subsequent Northern blot analyses
verified the presence of ~ 60nt pre-miRNAs processed from late SV40
transcripts . The SV40 miRNAs were
excised from both arms of pre-miRNAs and both found to be perfectly
complementary to early SV40 transcripts, which encode the large “T” and
small “t” SV40 tumor antigens. This finding was unusual because it is
predicted to be energetically favorable for only one pre-miRNA strand
to enter the RISC . Further experiments
confirmed that target mRNA (large “T” and small “t” early transcripts)
were cleaved in cells infected with the wild type SV40 (wtSV40). Cells
infected with mutant SV40, which contained mutations to disrupt the
predicted pre-miRNA hairpin structure did not produce the cleaved early
mRNA fragments and contained increased amounts of early viral mRNA
transcripts. These observations insinuate that SV40 miRNAs have an
autoregulatory function that regulates the amount of early viral
transcripts. Interestingly, downregulation of the large “T” and small
“t” antigens did not affect the final viral yield in infected cells.
Cells infected with wtSV40 showed a lower susceptibility to lysis by
T-antigen specific cytotoxic T lymphocytes and released less
interferon-γ than cells infected with the mutant SV40 . This indicates that vi-miRNAs induced
downregulation of SV40 tumor antigens resulting in the reduced
susceptibility to the activation of responsive cytotoxic T lymphocites .
Epstein-Barr virus (EBV)
– Eighteen potential miRNAs were detected in small RNA cloned from a B
cell line latently infected with EBV. [25,
27]. The function of the EBV miRNAs has not been established, but
the use of algorithms to predict possible targets of EBV miRNAs
revealed cellular genes that are involved in cell proliferation and
apoptosis as well as genes that encode B-cell specific chemokines and
cytokines. In additon, one of the EBV miRNAs apparently has a viral
target. miR-BART2 is antisense to a region in a lytic mRNA, BALF5
(BamHI L fragment 5) which encodes the EBV polymerase. Since it is
fully complementary to that mRNA, it would be predicted to cleave the
transcript. This hypothesis is supported by previous studies that
detected a shortened version of BALF5 mRNA that showed a relationship
in size with the predicted miR-BART2 mediated cleavage product . At this point, what is unclear is the role
of this cleavage. A reasonable explanation could be use of cleaved
BART2 mRNA to downregulate the expression of the EBV DNA
polymerase, suggesting the possibility that viruses not only use miRNAs
to downregulate host genes but also as autoregulatory tool.
Human Immunodeficiency virus
(HIV-1) - Although early attempts to clone virally encoded
miRNA from cells infected with HIV-1 failed after the screening of more
than 1500 clones , Omoto and colleagues,
reported evidence for a miRNA within nef region of HIV-1 genome and
proposed a role for it in the downregulation of viral transcription . In addition, it was recently reported that
HIV-1 encodes a siRNA precursor derived from hairpin structures
composed of a 19 bp perfectly complementary stem and small loop . Dicer processes this structure into
functional siRNAs that target the HIV-1 genome. HIV nef derived miRNA
(miR-N367) downregulates nef expression and suppress transcription by
reducing HIV-promoter activity, which helps in maintaining low viremia
in long-term non progressors. However, further studies will be needed
to resolve the following questions (i) why these HIV siRNAs have not
been cloned in previous studies and (ii) whether or not these hairpins
occur endogenously during the natural infection. At present, it is
unclear whether experimental designs influenced the outcomes of
previous studies [23, 29, 30]
Herpes simplex virus-1
(HSV-1 ) - The most recently discovered miRNA with a known function is
encoded by HSV-1 latency associated transcript (LAT) gene. miR-LAT
targets genes of apoptotic pathway including those involved in TGF-
pathway, thus protecting the cells from apoptosis 
(see vi-miRNA and
Cellular miRNA with a
It is well established that
cellular miRNAs play important roles in the regulation of cellular
genes. Recent evidence indicates that cellular miRNAs can also target
the genetic material of invading viruses. A perfect example comes from
analysis of the replication of the retrovirus primate foamy virus type
1 (PFV-1) and hepatits C virus (HCV) [31, 32].
mir-32 and PFV-1
A comparison of PFV-1 genome and
human miRNAs revealed several host cell miRNAs that could potentially
shut down gene expression and viral replication of this virus . Only cellular miR-32 showed
complementarity to the 3’ UTR shared by five mRNA of PFV-1 indicating
ability to down-regulate virus replication (Figure
2.). Mammalian miR-32 binds to ORF 2 of PFV-1 and restricts the
viral RNA abundance in cultured human embryonic kidney cells (293T).
When this miR-32 is knocked out, the virus doubles its replication rate
. Since the miR-32 is highly conserved in
all vertebrates, its antiviral activity against PFV-1 is not due to
evolutionary selection of miR-32 which confers an antiviral phenotype . The target sequence for miR-32 is not
highly conserved within other primate foamy viruses and it does not
exist in non-primate foamy viruses. This is the reason for unrestricted
replication of other primate/non-primate foamy viruses in different
miR-122 and hepatitis C virus (HCV)
A different mode of viral genome
regulation by cellular miRNA occurs in HCV. Cellular miRNAs usually
bind to the 3’UTR of mRNA, thereby repressing mRNA translation.
Examination of the HCV RNA sequence led to the identification of two
potential binding sites for liver specific miR-122; one in the 3’UTR
and one in the 5’UTR. A study by  showed
that miR-122 upregulates the expression of the HCV in cultured liver
cells Huh7, by binding to 5’UTR of HCV genome. The evidence for this
comes from the observation that sequestration of miR-122 by methylated
oligonucleotides result in reduction of HCV RNA  (Figure 3.). In addition, mutation of the 3’UTR
did not have an effect on virus replication whereas point mutation in
the 5’UTR abolished viral RNA accumulation. The level of HCV
replication was re-established by ectopic expression of miR-122
molecules that contained mutation to restore predicted base
complementarity. Experiments with replication-deficient genomes of HCV
indicated that miR-122-HCV interactions affected viral replication but
not the mRNA translation .
Overall, these findings provide an illustration of how cellular miRNAs
can be used in virus life cycle, either to downregulate or upregulate
viral gene expression. Even though these results have enormous
scientific weight, some questions remain. For instance, what pathway or
RNAi mechanism does PFV-1 employ, to evade the cellular defense
mechanism (see Viral vs Cellular
Defense). In addition, what are the natural targets for cellular
miR-122 in the liver and does the high concentration of virus in
hepatic cells suppress miR-122 targeting?
Viral vs cellular defense
From the aspect of cell virus interaction it is worth while to refresh
our knowledge about the important mechanisms involved in cellular
defense against viruses as well as the virus countradefense.
Reflecting the intimate relationship between viruses and their host,
infected cells have several signaling mechanisms to sense and respond
to virus infection. These mechanisms involve cross-talk between
different cellular pathways to modulate the expression and antiviral
function of interferons (IFNs) and specific gene products. IFNs – an
cytokines that are important regulators of innate and adaptive immune
responses. Besides their antiviral role, which is well established,
they are potent regulators of cell growth and have immunomodulatory
activity. INFs mediate their effects through interactions with type
specific receptors that are different and not redundant for the INF
type I and INF type II (Figure 4.). Virus
infection triggers the activation of INFs, probably through production
of viral dsRNA and other virus specific signals. The most intensely
studied pathway for INF production is the dsRNA-activated
serine/threonine protein kinase (PKR). Activation of PKR occurs by the
presence of cytoplasmic dsRNA, and leads to the rapid phosphorylation
of eukaryotic initiation factor eIF2 and subsequent inhibition of both
host and viral mRNA .
The discovery that the expression of small viral mi/siRNAs does not
induce the mammalian interferon machinery, raises a number of questions
(i) Can mammalian viruses be a target for cellular miRNAs in RNAi
machinery and (ii) What mechanisms mammalian viruses employ to avoid
and suppress such a sophisticated defense mechanism. A partial answer
to the first question has been given through the example of miR-32 and
PFV-1 virus, as well recent computational analysis conducted by
Brahmachari and colleagues  who
discovered two human miRNAs (miR-507 and miR-136) with potential
binding sites in polymerase B2 (PB2) and hemagglutinin (HA) genes
of influenza H5N1 virus. These two influenza genes encode proteins
crucial for viral pathogenicity and replication. Interestingly, miR-507
and mi-136 were not found in the chicken genome implicating the
difference in infectivity and lethality of the H5N1 virus in chicken
and humans. In an attempt to answer the second question, one should
look into different virus families and their mechanisms to suppress
RNAi. Currently, it has been reported that five mammalian viruses have
mechanisms that suppress RNAi and cellular defenses (INF/PKR) in
Adenoviruses use two countradefense mechanisms to shut down cellular
innate immunity. VA1 is adenovirus encoded ~160nt non-coding RNA that
is expressed at high levels during the late stage of lytic replication . VA1 is transcribed by RNA polymerase III
(polIII) in the nucleus and transported to cytoplasm by Exp5 and Ran.
In the cytoplasm, VA1 binds to PKR preventing its activation and
phosphorylation of eIF2, which results in no INF synthesis (Figure4. B). Another scenario is: VA1 like
miRNAs is transported by Exp5 from nucleus and it shares the same
sequence recognized by Exp5. This sequence is located in the 3’
overhang at the short RNA stem and it is recognized by Dicer-TRBP
complex [37, 38] suggesting that VA1 act as a
repressor of RNA silencing and cellular miRNA processing . Dicer processes only 1% of VA1 into viral
miRNAs but this percentage could give rise to up 10 VA1 encoded miRNAs
with a possible role in the viral life cycle in virus-infected cells
 (Figure 5/1).
Recent in vitro studies on HIV-1 have shown that HIV-1 uses two
pathways to repress RNA silencing. One pathway involve transactivation
responsive RNA (TAR), a 59 nt secondary stem-loop structure located at
the 5’end of all HIV-1 transcripts, which is essential for efficient
viral replication. Because TAR is a high-affinity ligand for TRBP, it
competes with Dicer for TRBP preventing TRBP incorporation in RISC.
Elimination of TRBP results in destabilization of Dicer and loss in
miRNA biogenesis  (Figure5/2a).
Another pathway utilizes the HIV-1 protein Tat. Tat is a regulatory
protein that binds viral RNA to facilitate initiation and elongation of
viral transcripts. In the study conducted by Bennasser and colleagues
it is shown that, Tat protein partially repress the processing activity
of Dicer [30, 41] (Figure5/2b).
Since TAR posses stem-loop structure which can be processed by Dicer
into miRNA most likely HIV-1 use both mechanisms (Tat along with TAR)
to increase suppression activity and provide safe virus assembling.
PFV-1 also employs the viral encoded protein as a suppressor of RNAi as
mentioned earlier in chapter 4. miR-32 down-regulates virus gene
expression and in order to counteract this RNAi pathway, PFV-1 uses Tas
protein synthesized early in infection. Lecellier and their group
reported that ectopic expression of Tas suppress miR-32 activity  (Figure 5/3).
Influenza NS 1 and vaccinia virus E3L proteins are known PKR
inhibitors, but recent findings revealed that they are also potent
inhibitors of the host RNAi pathway 
Virus encoded miRNA
& apoptosis /latency
The latency-associated transcript (LAT) of the HSV-1 is involved in
maintenance of the latent infection in neurons .
However, no protein product has been identified within the LAT gene and
therefore the mechanism by which the LAT confers resistance to
apoptosis was unknown until recently. Gupta et al. 2006  reported that LAT codes for miRNA, named
mir-LAT that is responsible for the observed antiapoptotic activity of
LAT in infected cells. Deletion of miR-LAT resulted in more extensive
apoptosis in infected cells upon stress. miR-LAT itself was able to
suppress apoptosis significantly in stressed cells. In order to gain an
insight into the mechanism how miR-LAT is able to suppress apoptosis,
the authors searched the human genome for possible target genes by
computational analysis. Using miRANDA computer prediction program, they
identified TGF-ß and SMAD3 as a potential targets. Moreover, it
is known that TGF-ß is a potent inhibitor of cell growth and
inducer of apoptosis . In further
investigation into the correlation of miR-LAT, SMAD3 and TGF-ß,
Gupta and colleagues observed that mRNA levels as well as the protein
levels of these two genes were lower in LAT expressing cells.
Furthermore, the activity of luciferase reporter gene of either
TGF-ß or SMAD3 was significantly reduced upon cotransfection with
LAT or a double stranded RNA oligonucleotide encoding miR-LAT itself.
These findings lead to the conclusion that miR-LAT miRNA helps the
virus to establish latency by allowing avoidance of TGF-B induced
apoptosis following infection .
Another interesting study was published by Bagsara and colleagues  suggesting the involment of cellular miRNA
in lentiviral latency. They hypothesized that in the case of lentivirus
infection, cellular miRNAs are involved in the inhibition of these
retroviruses by the formation of the intramolecular triplex formation
between the polypurine track sequences in the viral genome and miRNAs.
In addition, these triplex formations should block the viral
replication at the preintegration stage, placing the viruses into a
suspended latency. Using several latently and chronically infected cell
lines and human PBMCs from HIV-1 infected individuals, they confirmed
the presence of triplexes in lentivirus-infected cells. In addition,
the number of triplexes decreased in cells with productive replication
of lentivirus . This correlation was
further confirmed by stimulation of PBMCs and lentivirus-infected cell
line with the appropriate mitogenes .
However, additional research has to be done to answer what cellular
miRNAs are involved in triplex formation; could triplexes be formed in
vivo in CD4+ T cells, and finally, what molecular interactions
initiate/trigger their formation?
Cancer development has long been an enigma for researchers. Today we
know that viruses represent one of the main factors that cause
uncontrolled cell proliferation leading to the malignant
transformation. The discovery that viruses are able to produce high
level of viral miRNAs, currently with no determined specific roles in
viral life cycle, indicated that vi-miRNAs might be involved in cancer
development. Reports on precise roles of number identified vi-miRNAs
have not been published as of yet. However, according to the current
observations, we can hypothesize what roles vir-miRNAs might have in
(i) One possibility could be direct oncogene
function. Vi-miRNAs could directly stimulate oncogenesis by inhibition
of the major cellular tumor suppressor p53. Computational analysis,
suggests that p53 could be a possible target for EBV encoded miR-BHRF1 . Another EBV encoded miRNAs, miR-BART5-12,
found in EBV associated gastric carcinoma cells but not in the latently
infected B cell lymphomas, suggested that BART miRNAs may play
important roles in EBV induced epithelia cell transformation but not in
B-cell transformation 
(ii) A second scenario might involve viral
miRNAs in evasion of innate and adaptive immune responses during viral
infection, which can result in chronic or latent infection, an
environment conducive for cancer development. This scenario appears to
be the most likely; there are examples of SV40 and MHV68, which use
vi-miRNAs to evade cellular immune respond [22, 48].
(iii) The third theory could suggest that viral
miRNAs are involved in oncogenesis through the facilitation of tumor
progression in tissues that are already prone to develop cancer in the
absence of an infection. This hypothesis is based on research which has
observed that the expression of the mir17-92 significantly accelerated
lymphoma development in the mouse B-cell lymphoma model [49, 50]. There are some indications that some
EBV-derived miRNAs could act in a similar manner to miR17-92 based on
studies conducted by Taub et al.1982.
(iv) Finally, it is possible that viral miRNAs
can be expressed abundantly in infected cells resulting in alteration
of miRNA processing, export or RISC function leading to oncogenesis.
(example of adenovirus VA1). In addition, cellular miRNAs found in
cancer tissue could be involved in maintenance of chronic viral
infection and play role in viral affinity for specific organs (HCV and
Since the discovery that miRNAs can silence gene expression in
mammalian cells, their potential role as a therapeutic tool has been
considered. Currently, application of miRNAs based therapy is focused
on genetic inflammatory disorders, certain cancers as well as viral
diseases. The advantage of this approach is that miRNAs have the
potential to be more selective, more effective and less toxic than
current drugs. The concept of miRNA therapy is based on computational
prediction and design of artificial short hairpin RNAs molecules with a
sequence complementarity to the target mRNA. These synthetic miRNAs are
subsequently processed into active si/miRNAs following RNAi pathway in
targeted cells. Administration of artificially miRNAs can be via
different routs, such as direct injection, intravenous, intraperitoneal
or by expression from polII/polIII or viral vectors. Recent reports
showed that artificially designed miRNAs expressed from U6 or
lentivirus vectors are able to induce RNAi and shutdown viral
replication in vitro by targeting highly conserved regions or vital
genes required for viral replication. (Table 3.)
However, despite some encouraging results, the miRNA-based therapy has
some pitfalls. One of the drawbacks of synthetic miRNAs is transient
nature of the inhibition in mammalian cells. Their activity is mainly
dependent on the rate of cell growth and the turnover of targeted
proteins (3 to 6 days). Perhaps a more serious problem is the
unanticipated off-target effects that occur by synthetic miRNAs
recognition of other mRNAs bearing partial homology. As well,
considering the size of the human/animal genome and the number of known
genes, it is difficult to imagine that artificially design miRNAs will
be able of targeting a single gene of interest in vivo model. Hopefully
ongoing and future preclinical studies in animal models will facilitate
the development of highly efficient RNAi based therapies with minimal
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Figure 1. mi/siRNA induced RNA interference
A comparison of the miRNA and siRNA pathways. 1a) miRNAs are deriverd
from highly-structured precursors pri-miRNAs transcribed by RNA Pol
II/Pol III in the nucleus. Drosha and DGCR 8 processed pri-miRNAs into
~ 70nt hairpin structure with 2nt at the 3’ overhang (pre-miRNAs).
pre-miRNAs are exported by Exp5 into the cytoplasm where are further
processed by RNase III Dicer. Dicer trimmed loop structrures of
pre-miRNAs leaving mature ~21nt long miRNAs . One strand of miRNAs is
incorporated into RISC complex and acts as a guide for RNA silencing.
The remaining passenger strand is degradated. 1b) siRNAs are
derived from viral or artificial dsRNAs and share the common processing
pathway which also involves Dicer/TRBP and RISC complex. mRNAs with
perfect complementary to the si RNAs are targeted to the degradation
while transcripts captured by miRNAs with incomplete complementary
undergo translation repression. Figure created by using references [51,
Figure 2. miR-32 and PFV-1
Binding to the ORF2 of PFV-1, miR-32 restricts viral RNA
synthesis and accumulation
Figure 3. miR-122 and HCV
Upregulation of HCV replication induced by binding of miR-122 to 5’UTR
of HCV genome
Figure 4. INF/PKR pathway and inhibition
A. viral dsRNA activate PKR and phosphorilation of eIF2α which results
in translation inhibition and INF synthesis. B. Influenza NS1, Vaccinia
E3L, and Adenovirus VA1 bind to PKR and block phosphorilation of eIF2α,
which results in blocking INF synthesis and translation inhibition.
Figure 5. Viral mechanisms to suppress RNAi
5/1- Adenovirus VA1 is exported from nucleus by Exp5 to the cytoplasm
where it binds to Dicer/TRBP and suppresses RNA silencing and cellular
processing. 5/2a- HIV-1 Tar sequence is abundantly expressed; it
competes with Dicer for TRBP and suppress RNAi. 5/2b HIV-TAT protein
binds to Dicer and blocks its incorporation into RISC complex.
5/3-PFV-1 TAT protein binds to RISC complex with incorporated miR-32
and suppresses miR-32 activity
Figure 6. Overview