Review Article Silencing Viral MicroRNA as a Novel Antiviral Therapy? - Hindawi

19 Pages·2014·1.29 MB·English

HindawiPublishingCorporation
JournalofBiomedicineandBiotechnology
Volume2009,ArticleID419539,18pages
doi:10.1155/2009/419539
Review Article
Silencing Viral MicroRNA as a Novel Antiviral Therapy?
UgoMoens
DepartmentofMicrobiologyandVirology,UniversityofTromsø,N-9037Tromsø,Norway
CorrespondenceshouldbeaddressedtoUgoMoens,[email protected]
Received28December2008;Accepted20March2009
RecommendedbyBibekanandMallick
Virusesareintracellularparasitesthatensuretheirexistencebyconvertinghostcellsintoviralparticleproducingentitiesorinto
hidingplacesrenderingthevirusinvisibletothehostimmunesystem.Somevirusesmayalsosurvivebytransformingtheinfected
cellintoanimmortaltumourcell.MicroRNAsaresmallnon-codingtranscriptsthatfunctionasposttranscriptionalregulators
of gene expression. Viruses encode miRNAs that regulate expression of both cellular and viral genes, and contribute to the
pathogenicpropertiesofviruses.Hence,neutralizingtheactionofviralmiRNAsexpressionbycomplementarysingle-stranded
oligonucleotidesorso-calledanti-miRNAsmayrepresentastrategytocombatviralinfectionsandviral-inducedpathogenesis.
ThisreviewdescribesthemiRNAsencodedbyhumanviruses,anddiscussesthepossibletherapeuticapplicationsofanti-miRNAs
againstviraldiseases.
Copyright©2009UgoMoens.ThisisanopenaccessarticledistributedundertheCreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalworkisproperlycited.
1.Introduction [10]. Thus the pathogenic properties of viruses necessitate
thedevelopmentofefficientantiviraltherapies.
Viruses are common habitants of the human population, Viruses attempt to create a favorable cellular environ-
where they establish different forms of infection, including ment allowing viral replication or survival by establishing
anacute,achronic,orapersistentinfectionwithproduction a lifelong latent infection through evading the immune
of low levels of virions. Some viruses can exist in a true system of their hosts. Viruses can hide within a cell by
latent state in which infectious particles are only produced restricting their activity to a minimum so as not to conceal
upon reactivation stimuli. Viruses that reside harmlessly in their presence to the immune system and at the same time
theirhostcanundercertainconditionsorinimmunocom- they will also try to avoid apoptosis. For these purposes,
promised persons be responsible for malignant and non- viruses have developed different strategies, one of which
malignant diseases, which may even lead to the death of includes the posttranscriptional regulation of both cellular
the host. A causal role for human polyomaviruses (HPyV), and viral gene expressions through modulating the host’s
papillomaviruses (HPV), herpesviruses (HHV), hepatitis B RNA-interference (RNAi) machinery. Viruses can suppress
virus (HBV), hepatitis C virus (HCV), and human T-cell the RNAi pathway by viral microRNA (vmiRNA) targeting
lymphotropic virus type-I (HTLV-I) and cancer is accepted cellular or viral transcripts, or by viral proteins (e.g.,
(forrecentreviewssee[1–7]).Itisestimatedthatoncoviruses humanimmunodeficiencyvirusTatprotein,influenzavirus
areassociatedwith15%ofthehumancancers[8],whilenon- NS1/NS2 protein, Ebola VP35 protein, and vaccinia virus
malignant infections from human immunodeficiency virus E3L protein) or viral RNA (Adenovirus VA transcripts)
(HIV), HBV and HCV alone cause more than 3 million that counteract the host’s RNAi machinery (for recent
deaths annually worldwide [9]. Other viral infections (HIV reviews see [11–17]). This review summarizes the recent
no included) were responsible for the death of more than findings on virus-encoded miRNAs and their described
6000 patients in Japan in 2006, ∼7000 individuals in the functions and briefly discusses the potential of antiviral
USA in 2005, and 555 people in United Kingdom in 2006 miRNA as a novel therapeutic strategy in combating virus
according the statistics of the World Health Organization infections.
2 JournalofBiomedicineandBiotechnology
2.MicroRNA(miRNA) duplex formation with their target sequences and they
display excellent mismatch discrimination, hence avoiding
MiRNAs are noncoding small RNA molecules that act as off-target effects (for recent reviews see [28] and [29]).
posttranscriptionalregulators.Theyseemtobeaninherent LNAinjectionsagainstmiR-122,acellularmiRNAinvolved
part of the genomes of most living organisms as they have in lipid metabolism, resulted in efficient and long-lasting
been described in plants, unicellular and lower inverte- decrease in plasma cholesterol in African green monkeys
brates, all vertebrates, and in viruses. Their exact functions without any evidence for toxicities [30]. A second type
start to emerge and include control of cellular processes of modification is the phosphorothioate backbone which
such as differentiation, morphogenesis, organogenesis, and reduces the affinity to the target somewhat, but it confers
metabolism [18–22]. MiRNAs are typically generated by significantstabilitytonucleasedegradation(Figure1(b)).A
RNA polymerase II. The primary transcript (pri-miRNA) thirdgenerationofantisenseoligonucleotidesarephospho-
is processed by the RNase III enzyme Drosha, in concert diamidatemorpholinooligomers(PMO)inwhichtheribose
with double-stranded (ds) RNA-binding protein DGCR8 ringisreplacedwithamorpholinering.Addinganarginine-
into a ∼60 pre-miRNA hairpin. This nuclear pre-miRNA richpeptide(RXR) furtherincreasedthestabilityandtissue
4
isthentransported into thecytoplasma byexportin 5/Ran- retention of the PMO (reviewed in [31]). An additional
GTP and cleaved by the cytoplasmic RNase III Dicer to modificationcanbemadetoimprovethecellularuptakeof
generate an imperfect ds RNA of 21–25 nucleotides. One theAMO.Kru¨tzfeldtetal.linkedacholesterolmoietytotheir
of the strands, the mature miRNA strand or guide strand, AMOs and referred to these anti-miRNAs as antagomirs.
is loaded in the RNA induced silencing complex (RISC), Antagomirs should be >19 nucleotides in length to provide
and directs RISC to the target mRNA, where the complex highest efficiency in silencing target miRNA [32–34]. The
hybridizesto(partially)complementarysequencesresulting putative therapeutic potentials of antagomirs were recently
in cleavage or translational inhibition of the target mRNA. demonstrated in treatment of lipid metabolic disease in
The unincorporated strand, called the passenger strand, is animals[35].AnalternativeclassofAMOsispeptidenucleic
degraded. The seed region, which encompasses nucleotides acids (PNA), which are synthetic oligonucleotides with N-
2 to 8 of the 5’ ends of miRNA, plays a pivotal role in (2-aminoethyl)-glycine replacing the deoxyribose or ribose
target selection by RISC-bound miRNA (for recent reviews backbone[36].Astudypublishedin2008reportedthatPNA
see [23–25]). In animals, mature miRNAs do not require can efficiently block the action of cellular miRNAs [37].
completecomplementaritytotheirtargetmRNAs,enabling Finally, another approach in silencing miRNA is the use of
them to bind to and prevent translation of several mRNAs. so-calledmicroRNAsponge,asyntheticmRNAthatcontains
Experimental evidence suggests that a single miRNA can multiple binding sites for a particular miRNA and that is
potentiallytargetasmanyas200differentmRNAs[26–28]. transcribed from a plasmid containing a strong promoter
Assuch,miRNAshavemergedaspivotalposttranscriptional (reviewed in [28]). In conclusion, different classes of AMO
regulatorsofgeneexpressioninmulticellulareukaryotesand havebeenshowntobeefficientinsilencingmiRNAandmay
aberrant expression can contribute to diseases ([28] and beusefultherapeutictools(reviewedin[29,32–34,38]).
referencestherein).
3.SilencingofmiRNAby 4.ViralmiRNAsEncodedbyHumanViruses
Anti-miRNAOligonucleotides
4.1. Human polyomaviruses. (HPyV) are nonenveloped
Anti-miRNAoligonucleotides(AMOs)arechemicallymod- viruses with a circular ds DNA genome of approximately
ified synthetic oligonucleotides that are complementary to 5000 base-pairs. The members BK virus and JC virus were
their target sequence and this will silence the action of first isolated in 1971 from the urine of a renal transplant
the target. AMOs are modified with the dual purpose patient with the initials B.K., and from the brain of a
to stabilize them and to improve their affinity for their Hodgkin’slymphomapatientwithinitialsJ.C.whosuffered
targets. One modification is the 2’ sugar modification from progressive multifocal leukoencephalopathy (PML),
which implies a chemical modification of the 2’-O of respectively. Human infections with the rhesus macaque
the ribose residue (Figure1(a)). The 2’-O -methyl AMOs (Macaca mulatta) Simian vacuolating virus 40 (SV40) were
have a methyl group linked to the 2’-O of the ribose considered as accidental transmission of the virus from
residue, while the 2’-O - methoxyethyl AMOs contain a monkeystopeoplelivinginclosecontactwiththeseanimals
methoxygroup.ThismodificationprovidesimprovedRNase or through vaccination with the contaminated poliovirus
resistanceandbindingaffinitytoRNAcomparedtounmod- vaccines.However,recentobservationssupportthepossibil-
ified antioligonucleotides. However, 2’-O -methoxyethyl itythatSV40canspreadinhumansbyhorizontalinfection
AMOspossessahigheraffinityandspecificitytoRNAthan andevenverticaltransmission,suggestingthatmanmaybe
their 2’-O -methyl AMOs. Other 2’ sugar modifications a natural host for this virus. The human polyomaviruses
that have been used include 2’-fluor and locked nucleic seem to be implicated in tumours of the brain, bone,
acid (LNA). In LNA-modified oligonucleotides, the 2’-O - colon,mesothelium,pancreas,stomach,urogenitaltract,and
oxygen is bridged to the 4’-position via a methylene linker lymphomasandleukaemias(reviewedin[39,40]).In2007,
to form a rigid bicycle, locked into a C3’-end (RNA) two independent research groups reported the isolation
sugar conformation (Figure1(a)). LNAs give very strong of two new human polyomaviruses from nasopharyngeal
JournalofBiomedicineandBiotechnology 3
O O O O
O
-O P = O -O P = O -O P = O -O P = O -S P = O
O O O O
O O O O O
Base Base Base Base O
Base
O OH O O CH3 O O O O O O OH
-O P = O -O P = O -O P = O CH3 -O P = O -S P = O
O O O O
O O O O O
Base Base Base Base O
Base
O OH O O CH O O O O
3 O O OH
CH3
RNA 2’-O-methyl-RNA 2’-O-methoxyethyl-RNA LNA Phosphorothioate
(a) (b)
Figure1:Commonchemicalmodificationsusedforanti-miRNAoligonucleotides(AMO).(A)Modificationsofthe2’-Oresidueinribose.
(B)Modificationofthephosphate-ribosebackbone.LNA=lockednucleicacid.Seetextfordetails.
samples and they are referred to as KIPyV WUPyV [41, therapeutic effect [47]. Expression of a corresponding or
42]. This year a novel human polyomavirus, Merkel cell otherviral-encodedmiRNAfortheotherHPyVWUandKI
polyomavirus (MCPyV), was identified that is associated islackingsofar.
withMerkelcellcarcinoma[43].
TheHPyVgenomecanbedividedintothreefunctional
regions. The early region encodes the early proteins large 4.2. Human Papillomaviruses. Human papillomaviruses
T-antigen (LT-ag) and small t-antigen (st-ag), while the (HPV) are nonenveloped viruses with a circular dsDNA
late region encodes the capsid proteins VP1-VP3 and the genomeofapproximately8000base-pairs.Thesevirusesare
regulatory protein agnoprotein. Both regions are separated associatedwithbenignandmalignantlesionsoftheskinand
bythenoncodingcontrolregionthatencompassestheorigin the genital tract. More than 100 different HPV genotypes
ofreplicationandthepromoter/enhancersequencesforthe have been identified and based on their association with
early and late genes (reviewed in [44]). The SV40 genome benign warts or cancer, they are classified as low-risk and
encodes a viral miRNA (vmiRNA) of which both arms high-riskvariants,respectively[1].
are complementary to the early viral mRNAs and reduces One study with HPV type 31 failed to clone vmiRNA
expression of the early proteins (Figure2(a); Table1). Cells fromvirus-infectedcells[58].However,thisdoesnotexclude
infectedwithmutantSV40lackingthismiRNAorwithwild- that other strains may encode vmiRNA. Moreover, the
type SV40 yielded comparable levels of infectious viruses, expression of vmiRNA may be regulated in a temporal
but the latter were less sensitive to lysis by cytotoxic T cells and spatial manner, so that the experimental conditions
andproducedlessinterferon-γ.ThusSV40-encodedmiRNA for capturing vmiRNA may be tricky. In addition, the high
allowsthevirustoevadetheimmunesystem[45].TheSV40 mutation rate of HPV genome sequences may impede the
miRNA is conserved in BKV and JCV and both miRNAs predictionofthepresenceofputativevmiRNA.
generatedfromtheprecursorhairpinbindtothesametarget,
thatis,theearlytranscripts.TheBKVandJCVmiRNAsserve
the same role as SV40 miRNA, that is, downregulation of 4.3. Human Adenovirus. Adenoviruses are naked dsDNA
early expression. JCV miRNA, miR-J1, was readily detected viruses that can cause mild respiratory, gastrointestinal,
in brain samples of PML patients, suggesting a biological urogenital,andoculardisease.Morethan50serotypeshave
role of this miRNA [46]. The group of Sullivan has also been described in human. Although there is no proof for
identifiedaMCPyV-encodedmiRNA,miR-M1,whichdoes a causative role in malignancies, adenoviruses can induce
not share sequence identity with the known miRNAs of cancerinanimalmodelsandhavebeenextensivelystudiedto
the other polyomaviruses. MCPyV miR-M1 is located in scrutinizeviralmechanismsforcellulartransformation[59].
the early region (Figure2(a)) and can downregulate early Adenovirus encodes small noncoding RNAs, known as
gene expression. In accordance with SV40, this may allow virus-associated RNA or VAI and VAII RNA, which are
thevirustoevadetheimmunesystem.However,MCPyVis generated by RNA polymerase III. This noncoding RNA
associatedwithMerkelcellcarcinomaandtheviralgenome playsanimportantroleforviralreplicationandneutralizes
is integrated in these tumours [43]. Blocking miR-M1 by, the antiviral action of interferon by blocking the dsRNA-
for example, antagomirs will increase the expression of the induced protein kinase (PKR), which phosphorylates and
viraloncoproteinLT-antigenandassuchhavelittlebeneficial thereby inactivates the eukaryotic translational initiation
4 JournalofBiomedicineandBiotechnology
HPyV
ORI
HSV-1
miR-M1
UL US
TRL IRL IRS TRS
Late region Early region
miR-H1 miR-H3
miR-H6 miR-H2 miR-H4 miR-H5
miR-S1
miR-J1 LAT locus
miR-B1 ~118300 ~127000
(a) (b)
HCMV
miR miR
US5-2 US25-2
miR miR miR miR miR miR miR miR
UL22A-1 UL36-1 UL70-1 UL112-1 UL148D-1 US4-1 miR US25-1 US33-1
US5-1
miR UL71 US6 miR-US24
TRL UL23 IRL/IRS TRS
UL21 UL23 UL36 UL37 UL70 UL112/UL113 UL114 UL150 US4US5 US7 US24 US26 US28US29
UL US
(c)
EBV
EBERs EBNA-LP EBNA2BHRF1 EBNA3 EBNA1 BARTs LMP2A,B
BHRF1 LMP1
miRNAs
BART miRNAs
(d)
KSHV
117600 124000
K12 ORF 71 ORF 72
12 10 9 8 7 11 6 5 4 3 2 1
miR-K
(e)
Figure2:Continued.
JournalofBiomedicineandBiotechnology 5
HIV
tat
miR-N367
gag vif nef
vpu
U3 R U5 pol vpr env U3 R U5
mi-TAR #1 #2 #3 rev #4 #1 #5
HAAmiR
(f)
Figure2:Schematicrepresentationofthegenomesofdifferenthumanvirusesandlocationofviral-encodedmiRNAs.(A)Thehuman
polyomavirus(HPyV)BKvirus,JCvirus,SV40,andMerkelCellpolyomavirusencodetheviralmiRNAsmiR-B1,miR-J1,miR-S1,andmiR-
M1,respectively[45–47].(B)Herpessimplexvirus-1genomewithdetailoftheLATlocus.Thenumbersrefertotheapproximatesequence
coordinatesoftheLATlocus.L=long,S=short,U=unique,TR=terminalrepeat,andIR=internalrepeat.Thefigureismodifiedafter[48].
(C)GenomicmapofHCMVwithrelativepositionofsomeofthegenes.Theopenreadingframesaredepictedbythickarrows,whilethe
positionoftheviralmiRNAsareindicatedbynarrowarrows.L=long,S=short,U=unique,TR=terminalrepeat,andIR=internalrepeat.
Modifiedafter[49]and[50].(D)LocationofthemiRNAsintheEBVgenome.Thelatencygenesareshownasgreyboxes,whilethemiRNAs
areindicatedbyverticallines.(E)Kaposi’ssarcoma-associatedherpesvirus(KSHVorHHV-8).MostKSHVmiRNAsareclusteredbetween
thekaposin(K12)geneandthev-FLIP(ORF71)gene,whiletwoarelocatedwithintheK12openreadingframe(ORF).Adaptedfrom[51].
(F)TheHIV-1genomeandlocationoftheviral-encodedmiRNAs.Thefigureisbasedonacompilationofthestudiesby[52–57].
factor 2 [60]. A recent study showed that a minor frac- but it can also cause a spectrum of diseases from sight-
tion of VAI RNA is processed by Dicer into functional threateningocularinfectionsinimmunocompetentadultsto
RISC-associated ssRNA which can act as miRNA (Table1). moresevereinfectionsinnewbornsandimmunosuppressed
Blocking of VAI-derived small RNA by 2’-O -methyl AMO patients(reviewedin[89]).
complementarytoVAIdecreasedvirionproduction[61–63]. An important gene in HSV latency is the LAT gene.
In an attempt to identify potential targets for VAI-derived This gene encodes the latency-associated transcript (LAT),
miRNA, computational analysis was used. Putative targets which does not code for a protein. LAT seems to promote
include genes encoding apoptosis-related protein NAPOR cell survival of the infected cells [90]. Gupta and cowork-
and PKR-activating protein PACT. Therefore VAI RNA- ers proposed that the antiapoptotic activity of LAT was
derived miRNA may help adenovirus to escape the actions achieved by an miRNA-entrapped in the LAT (miR-LAT),
ofthehostdefensemechanisms[64]. whichdownregulatestheexpressionoftransforminggrowth
factor-β (TGF-β) and SMAD3. The latter is a mediator
of the signalling pathway induced by TGF-β, while TGF-
4.4. Human Herpesviruses. Herpesviruses are enveloped
β can prevent cell proliferation and induce cell death [67].
dsDNAviruseswithagenomesizerangingbetween∼130to
However,alaterstudyrevealedthatthedescribedmiR-LAT
∼250kilobase-pairs.Theyaredividedintothreesubfamilies
wasnotviralencoded,butinfactacellularmiRNAexpressed
denoted α, β, and γ. Approximately 130 different her-
in SH-SY-5Y cells [67] and the report describing miR-LAT
pesviruseshavebeenidentifiedtodate,includingthehuman
was retracted [91]. Work by Umbach et al. could also not
α-herpesvirusesherpessimplexvirus1(HSV-1orHHV-1),
confirmtheexistenceofthismiRNAinHSV-1infectedSH-
herpes simplex virus 2 (HSV-2 or HHV-2), Varicella-zoster
SY-5Ycells[48].Later,itwasshownthattheHSV-1LATexon
virus(VZVorHHV-3),theβ-herpesvirusescytomegalovirus
encodesHSV-1miR-LAT-ICP34.5,whichcanbedetectedin
(HCMV or HHV-5), HHV-6A, HHV-6B, and HHV-7, the
HSV-1infectedcells[89].Circa120base-pairsupstreamin
γ-herpesviruses Epstein-Barr virus (EBV or HHV-4), and
this region, a sequence with 77% homology to the HSV-2
Kaposi’s sarcoma-associated virus (KSHV or HHV-8). All
miRNAmiR-I(seefurther)ispresent,butnomaturemiRNA
human herpesviruses are able to establish latent infections
wasdetectedinHSV-1infectedcells.However,theexistence
with only a small subset of viral genes expressed (reviewed
of this miRNA during HSV-1 latency in vivo remains to be
in[64]).Aswillbediscussedinthenextsection,amongthe
confirmed[68].
viraltranscriptsthatcanbedetectedinlatentlyinfectedcells
Computationalanalysispredicted24-miRNAcandidates
are viral-encoded miRNAs, which seem to be required to
in the HSV-1 genome, 8 of which were conserved in HSV-
maintain a latent state of infecton, but may also contribute
2, suggesting they may be functional miRNAs [69]. The
tothepathogenicpropertiesofthevirus.
authors confirmed the expression of one mature miRNA,
designated miR-H1, in HSV-1 infected Vero cells, where it
4.4.1. HSV-1. Herpes simplex virus-1 (HSV-1 or human wasexpressedlateinproductivereplication.ThismiRNAis
herpes virus 1; HHV1) infects the majority of the human encodedapproximately450bpupstreamofthetranscription
population,butremainsalatentcohabitantinmostpeople. startsiteoftheLATtranscript,butacorrespondingsequence
Reactivation of the viruses usually results in cold sores, is not conserved in the HSV-2. The function of miR-H1
6 JournalofBiomedicineandBiotechnology
Table1:MicroRNAsencodedbyhumanvirusesandtheirtargets.Seetextfordetails.
Virus miRNA Target/function Reference
Humanpolyomaviruses
Downregulationearly
SV40 miR-S1 [45]
expression;Immunomodulating
BKV miR-B1 Downregulationearlyexpression [46]
JCV miR-J1 Downregulationearlyexpression [46]
MCPyV miR-M1 Downregulationearlyexpression [47]
Humanpapillomavirus notpredicted [65]
notdetected [66]
Humanadenovirus unnamed [61–63]
Humanparvovirus notpredicted [65]
Preventsapoptosisbytargeting
miR-LAT [67](retracted),[48,68,
HSV-1(HHV1) translationofthegenesencoding
(withinexon1) 69],[70]
TGF-β1andSMAD3
miR-LAT-
ICP34.5
Repressionoftheexpressionof
miR-H1 theviralproteinICP0,which
promotesviralreplication
DownregulationofICP34.5,a
miR-H2
keyviralneurovirulencefactor
DownregulationofICP34.5,a
miR-H3
keyviralneurovirulencefactor
miR-H4
DownregulationofICP4,aviral
miR-H5
transcriptionalactivator
ReducedexpressionofICP34.5,a
miR-H6
keyviralneurovirulencefactor
HSV-2(HHV2) miR-I
miR-II
miR-III
VZV(HHV3) notpredicted [65,71]
HCMV(HHV5) miR-UL23 Immunomodulating [12,49,65,72–74]
miR-UL36-1
miR-UL54-1
miR-UL70-1
miR-UL22A-1
Downregulatestheexpressionof
CMVgenesinvolvedinitsown
replicationprocess,forexample,
transactivatorsIE72andIE86;
miR-UL112-1 UL120/121;UL114MHCclass
I-relatedchainB(MICB),a
cellularligandfortheactivating
receptorNKG2D;
downregulationofIE-1
miR-UL148D-1
miR-US4-1
miR-US5-1
miR-US5-2
miR-US24
JournalofBiomedicineandBiotechnology 7
Table1:Continued.
Virus miRNA Target/function Reference
miR-US25-1
miR-US25-2
miR-US33-1
Bndscomponentsofthe
mitochondrialrespiratorychain
RNAβ2.7
complexIandthuspreventing
apoptosis
miR-BART1-1
EBV(HHV4) InhibitionLMP1expression [24],[51,75–79]
to-3
InhibitEBVDNApolymerase
BALF5
miR-BART2 InhibitionLMP1expression
miR-BART3
Antiapoptoticby
miR-BART4
downregulationofPUMA
miR-BART5
miR-BART6 InhibitionLMP1expression
miR-BART7
miR-BART8
miR-BART9
miR-BART10
miR-BART11
miR-BART12
miR-BART13
miR-BART14 InhibitionLMP1expression
miR-BART15 InhibitionLMP1expression
miR-BART16
miR-BART17
miR-BART18
miR-BART19
Downregulationchemokine
miR-BART20
CXCL-11
miR-BHFR-1
miR-BHFR-2
miR-BHFR-3
Downregulationof
KSHV(HHV-8) miR-K12-1 [51,66,75,80–82]
thrombospondin1andBACH
Downregulationof
miR-K12-2
thrombospondin1
miR-K12-3
miR-K12-4
Downregulationof
miR-K12-5
thrombospondin1andBACH
miR-K12-6
miR-K12-7
miR-K12-8
miR-K12-9
miR-K12-10a
Downregulationof
miR-K12-10b
thrombospondin1andBACH-1;
miR-K12-11 IdenticaltomiR-155
8 JournalofBiomedicineandBiotechnology
Table1:Continued.
Virus miRNA Target/function Reference
miR-K12-12
Poxvirusvacciniavirus 3predicted [65]
Poxvirusvariolavirus 1predicted [65]
HepatitisBvirus [54]
HepatitisCvirus
Humanimmunodeficiencyvirus miR-H1
HDAC-1-mediatedrepressionof
Type1(HIV-1) miR-TAR [83]
viralgeneexpression
miR-TAR-5p [84]
miR-TAR-p
DownregulationIL-15,IL-2
HAAmiRNA receptorγchain,IRAK1,and [56]
FMRP
Proteinsinvolvedin,for
example,signaltransduction,
VmiRNA#1-5 [85]
proteinsynthesis,and
degradation,DNAmethylation
miR-N367 HIVpromoterinterference [86]
HIV-2 miR-TAR2-5p [87]
miR-TAR2-3p
HTLV-I notdetected [88]
Paramyxoviridae(measlesvirus) 1predicted [65]
remains to be established and no cellular target mRNAs HSV-2 infection varies between 10–60%. Reactivation can
were identified [69]. Umbach and coworkers detected in causeoro-facialandgenitalherpes,butHSV-2infectioncan
addition to miR-H1 five novel viral miRNAs in trigeminal alsocauseencephalitisandneonatalherpes,andformsarisk
ganglia of mice latently infected by HSV-1, as well as in factor for HIV acquisition [89]. The only detectable viral
HSV-1-infected Vero cells. These miRNAs, that is, miR-H1 transcriptduringHSV-2latencyistheLAT,butthemolecular
tomiR-H6,areencompassedintheLATlocus(Figure2(b)). function of this transcript remains largely unknown [92].
By quantitative RT-PCR, the authors were able to roughly HSV-2 LAT exon encodes an miRNA, referred to as miR-
estimate the number of copies of each miRNA during
I, which is expressed during latent, as well as during acute
productiveinfectionofVerocells.MiR-H1andmiR-H6were
infection. Remarkably, several promoters regulate miR-I
expressed at ∼1200 and 300 copies, respectively, while the expression in different stages of the viral life cycle. This
othermiR-Hswerepresentatlessthan40copiesperinfected HSV-2 miRNA efficiently diminishes the expression of
cell. In latently infected trigeminal ganglia, much higher
the viral neurovirulence factor ICP34.5, a multifunctional
levels were monitored with 63000 copies of miR-H2, 8000
protein required for viral replication in neuronal cells in
copiesofmiR-H3,800000copiesofmiR-H4,80000copiesof
miR-H5,and40000copiesofmiR-H6.Thelargedifference vivo,andwithintrinsicneurovirulentproperties[93].Thus,
miR-I may affect the outcome of infection (latent versus
in numbers of miRNA transcripts in latently infected cells
productive) by modulating the protein levels of ICP34.5.
comparedtocellswithproductiveHSV-1infectionindicates
WhethermiR-Ihasothertargetsremainstobeinvestigated,
that miR-Hs play an important role in establishing latent
HSV-1infection.Indeed,miR-H2expressiondiminishedthe butanmiR-IanalogueisalsoexpressedbyHSV-1,indicating
protein levels of ICP0, a viral transcriptional activator that the importance of this miRNA for these viruses [68]. Tang
promotesviralreplication,whilemiR-H6inhibitsexpression and colleagues identified two new HSV-2 miRNAs, miR-
of ICP4, which is required for expression of most HSV-1 II, which includes miR-II-5p and miR-II-3p, and miR-III,
genesduringreproductiveinfection[48]. both encoded byexon 2 of LAT. The expression of miR-I, -
II, and –III increased during infection of cells, but miR-III
4.4.2.HSV-2. HSV-2typicallyinfectsthegenitalregionand displayedslowerkineticsthanthetwoothermiRNAs.Similar
establishesalifelonglatentinfection.Theprevalenceoflatent to miR-I, miR-II silences the expression of ICP34.5,while
JournalofBiomedicineandBiotechnology 9
miR-IIIfunctionallyresemblesHSV-ImiR-H2inthatitcan of them is the viral transactivator protein called immediate
downregulatetheexpressionofICP0[70]. early72(IE72)thatregulatesthetranscriptionofviralgenes
requiredforacutereplication[50].IE72playsapivotalrolein
controllinglatencyandreactivation.miR-UL-112-1canthus
4.4.3.Varicella-ZosterVirus(VZV). VZVisacommonvirus
restrictreactivationofthevirusthroughnegativeregulation
thatcauseschickenpoxorvaricelladuringprimaryinfection.
ofIE72expression[50,95].Twoseparatestudiesshowedthat
Thevirusestablishesalatentinfection,butreactivationleads
miR-UL112-1 also inhibited expression of the immediate
to herpes zoster, commonly referred to as shingles. Acute
earlyproteinIE1.Murphyetal.detectedincreasedIE1levels
VZV reactivation may lead to post-herpetic neuralgia [94].
in cells infected with either a virus lacking miR-UL112-
NoputativemiRNAscouldbepredictedintheVZVgenome
1 or with mutations in the seed sequence of the ie1 gene
[65, 71], but experimental studies to unambiguously proof
comparedtocellsinfectedwithwild-typeHCMV[73].Grey
theexistenceofVZVmiRNAarelacking.
and coworkers demonstrated that addition of miR-UL112-
1 RNA prior to infection reduced IE1 protein levels and
4.4.4. Human Cytomegalovirus (HCMV). HCMV causes blocked viral replication [74]. IE1 is a crucial protein to
mild or subclinical diseases in immunocompetent adults, ascertainlyticreplicationofHCMV,thusdownregulationof
but can lead to life-threatening complications in immuno- IE1mayhelpthevirustoestablishlatency.Thisstrategymay
compromised patients such as organ transplant recipients beacommonfeatureforherpesvirusesbecauseimmediate-
and AIDS patients. In addition, HCMV is one of the early genes may be putative targets for HSV-1 miR-LAT,
leadingviralcausesofbirthdefectsandhasbeenimplicated EBV miR-BART15 and miR-BHRF1-3, and KSHV miR-
in the acceleration of long-term vascular diseases such K12-6-3p [73]. Yet another target for miR-UL112-1 is the
as atherosclerosis. Depending on the tissue type and the viral protein UL114, a homologue of the mammalian DNA
host’s immune state,HCMV canestablish acute,persistent, repair enzyme uracyl-DNA glycosylase. UL114 is required
or latent infections characterized by different viral gene for efficient viral DNA replication. Hence, miR-UL112-1-
expression [95, 96]. Because other herpesviruses encode mediateddownregulationofUL114maypreventviralDNA
miRNAs, it was assumed that HCMV may encode miRNAs replication and favor a latent infection state [65]. Taken
whichcouldbeinvolvedindeterminingthetypeofinfection. together,theactionsofmiR-UL112-1seemtobeassociated
Todate,15HCMVvmiRNAs,scatteredthroughouttheviral with latent viral infection, a state which allows the virus to
genome, have been identified (see Table1 and Figure2(c)). hide from the immune system. Ablating expression of this
ThreeviralmiRNAs,designatedasUL23-5p,miR-UL23-3p, viral-encoded miRNAs by AMOs may therefore force the
and miR-US24, were identified that are expressed during virus into a lytic cycle and provide the immune system the
productiveHCMVinfectionofpermissivecells(humanfore- opportunitytogetrideoftheviralinfection.
skinfibroblasts,astrocytomaU373MGcells,retinalpigment
epithelialcells,andhumanmicrovascularendothelialcells).
Their putative cellular target genes include genes encoding 4.4.5. EBV. Epstein-Barr virus (EBV) or human herpses-
transcriptionfactors(e.g.,HNF3andTGIF2),receptors(e.g., virus-4(HHV-4)isaγ-herpesvirusthatestablishesalifelong
IL-18 receptor 1 precursor; CD206), proteins implicated in latent infection in B-lymphocytes in more than 90% of
T-cell activation (AHNAK1), in signal transduction (e.g., the human population. EBV is associated with infectious
RAB2L), and in biosynthesis of leukotrienes that sustain mononucleosisandhasbeenimplicatedinthepathogenesis
inflammatory reactions (coactosin-like protein). Whether of several malignancies including Burkitt’s and Hodgkin’s
thesegenesrepresentbonafidetargetsaswellasthebiological lymphomas,posttransplantandT-celllymphomas,X-linked
relevance of HCMV miRNA-mediated silencing of these lymphoproliferative syndrome, nasopharyngeal, and gastric
genesremainselusive[49]. carcinomas [97]. Latently EBV-infected cells are classified
HCMV usually establishes a lifelong persistent or latent in stage I, II, or III, each of them characterized by distinct
state in healthy individuals by ensuring that infected cells EBVgeneexpression[98].Amongthelatencystage-specific
avoid immune recognition. The HCMV-encoded miRNA EBV transcripts are miRNAs. More than 20 different EBV
miR-UL112-1seemstoplayacentralroleinhelpingthevirus miRNAs have been identified that are transcribed during
to hide from the host’s immune system. This viral miRNA latentinfection(Table1;[25,51,75–79]).TheEBVmiRNAs
targetsmRNAforMHCclassI-relatedchainB(MICB),and areorganizedintwomajorclusterswithintheEBVgenome
toalesserextendMICA.Theseproteinsarecellularligands (Figure2(d)).OneclusterresidesintheBART(abbreviation
for the activating receptor NKG2D, which is expressed on for BamHI-A rightward transcripts) region. The BART
some natural killer (NK) cells, γ/δ T cells, and CD8+ T region gives rise to multispliced transcripts and is highly
cells.Duringcellularstress(suchasviralinfection)MICBis expressed in EBV-positive cancers and in epithelial tissues,
induced,thusactivatingNK-cellsandTcellsthatcanleadto while there is low BART expression in B lymphocytes. The
thekillingofinfectedcells.Cellsinfectedwithmutantvirus exact function of BART mRNAs remains obscure [76]. The
lackingthismiRNAweremoresusceptibletobeingkilledin intronic region of BART also encodes the miRNAs miR-
anNKG2D-dependentmannerbyNKcells[72]. BART1 to miR-BART20. The second region that encodes
The miR-UL112-1 also represses the expression of multiple miRNAs is the untranslated region of the gene
HCMVgenesinvolvedinitsownreplicationprocess,inpart encoding BHRF1 (BamHI fragment H rightward open
bytargetingmRNAencodingimmediateearlyproteins.One reading frame 1), a viral Bcl-2 homologue that prevents
10 JournalofBiomedicineandBiotechnology
apoptosis. BHRF1 encompasses the miRNAs miR-BHRF-1- inhibited with an anti-miR-BART5 oligonucleotide, PUMA
1tomiR-BHRF-1-3[51,75]. protein levels decreased and apoptosis was triggered. Thus,
The expressions of EBV-encoded miRNAs in clinical EBV may promote survival of infected epithelial cells by
samples and computational analysis to predict putative modulating the expression of an apoptotic protein through
targets were applied to unravel the biological functions of an miRNA-mediated mechanism. This finding may have
EBVmiRNAs.TheseapproachesshowedthatthemiR-BARTs important implications in the development of anti-EBV
areabundantlyexpressedinlatentlyinfectedepithelialcells, agentssuchasAMOsdirectedagainstmiR-BART5.
nasopharyngeal carcinomas, EBV-associated gastric carci- Fewerstudieshavebeendirectedtodeterminethetargets
nomacelllinesandtissues,Burkitt’slymphomaslatencytype ofmiR-BHRF1s.miR-BHRF1-2isinvolvedinthecleavageof
I, EBV positive primary effusion lymphomas, and diffuse BHRF1 RNA in the cytoplasm, but the biological relevance
large B-cell lymphomas, but at a significantly lower level in remains to be determined [98]. In another study, Xia et al.
B cells. This corresponds well with the expression pattern observed that high levels of miR-BHRF1-3 were correlated
of BART multispliced transcripts (see above). Higher levels with low levels CXCL-11, a potent interferon-inducible T-
of BHRF1-3 were measured in latency type III Burkitt’s cellattractingchemokine.MiRNA-mediatedsuppressionof
lymphomas and in diffuse large B-cell lymphomas [66, 79, CXCL-11 may serve as an immunomodulating mechanism
98–100]. Another study demonstrated that induction of allowing the virus to survive in the host [79]. On the
EBV replication in latency I-infected cells was associated otherhand,enhancingCXCL-11expressioninEBV-positive
withincreased expression of miR-BHRF1-1, -2,and -3, but tumours by AMOs against miR-BHRF1-3 may increase
expression levels of miR-BART-1 and -2 did not change. susceptibility ofthetumourcellstotheimmune system.In
Ontheotherhand,inductionofEBVreplicationinlatency agreementwiththis,tworecentstudiesreportedantitumour
III-infected cells did hardly change the expression levels activityforCXCL-11inanimalmodels[104,105].
of BHRF1-1, -2, and -3 [98]. These observations suggest
that EBV miRNAs may be implicated in the oncogenic
propertiesofthevirus,butalsoinregulatingitsreplication.
4.4.6. Kaposi’s Sarcoma Virus (KSHV or Human Herpes
Moreover, a precise knowledge of the latency state of EBV
Virus Type 8; HHV-8). Kaposi’s sarcoma-associated virus,
andtheexpressionpatternofviralmiRNAsmayimprovethe
so named because it was detected in Kaposi’s sarcoma,
successfultreatmentofEBVinfectionwithAMOs.
belongstotheγ-herpesvirusesandisalsoknownashuman
The function of most EBV vmiRNA remains poorly
herpesvirus-8orHHV-8.HHV-8isassociatedwithKaposi’s
understood, but some targets of EBV miRNAs have been
sarcoma, as well as with two rare forms of B-cell malig-
recently identified. Several mi-BARTs prevent expression
nancy: primary effusion lymphoma (PEL) and the plasma
of viral latent infection membrane protein 1 (LMP1)
cell variant of multicentric Castleman’s disease. Like other
protein (see Table1). LMP1 functions as a constitutively
herpesviruses,KSHVcanestablishalifelonglatentinfection
active tumour necrosis receptor [101], and can activate
severalsignallingpathwaysincludingNFκB,AP1,JAK/STAT, characterizedbyalimitedviralgeneexpression[106].
MEK/ERK, and PKC. LMP1 can also interact with p53 A total of 17 KSHV miRNAs encoded by 12 distinct
andaffectscyclins,cyclin-dependentproteinkinases(CDK), miRNA genes have been reported and their sequences
and the CDK inhibitors p16 and p27 (reviewed in [102]). are highly conserved between different KSHV genomes
Furthermore,LMP1isexpressedinalltheEBVrelatedmalig- in PEL cell lines and in clinical samples. However, some
nanciesandpromotescellulartransformation.Itsoncogenic polymorphismwasobserved,particularlyinmiR-K12-5and
property makes LMP1 an attractive target for EBV therapy. miR-K12-9[80,107,108].TheentireKSHVmiRNAcluster
Interestingly, overexpression of LMP1 results in growth- resides within an approximately 4 kilobase-pairs region
inhibitoryandsensitizationtoapoptosisinducedbystressor betweenopenreadingframesORFK12(kaposin)andORF
chemotherapeuticagents([76]andreferencestherein).Thus 71(Figure2(e)).
AMO-mediatedneutralizationofmi-BARTsmayleadtoele- To elucidate the functions of the KSHV miRNAs,
vatedLMP1proteinlevelsandrenderEBV-positivetumour transcriptome analysis was performed from cells stably
cells more susceptible to chemotherapy. The viral miRNA expressing the miR-K12 cluster. Among the differentially
miR-BART2caninhibitexpressionofviralDNApolymerase expressed genes were genes encoding proteins implicated
BALF5 and may thus interfere with viral replication and in proliferation, immune modulation, angiogenesis, and
preventlyticinfection[51,77].SilencingmiR-BART2could apoptosis. The gene encoding thrombospondin-1 was tar-
thus allow the virus the complete its life cycle and produce geted by all ten KSHV miRNAs, but especially by miR-
new infectious virus particles, which then could offer the K12-1, miR-K12-3-3p, miR-K12-6-3p, and miR-K12-11.
immunesystemtheopportunitytodetectandeliminateEBV. Thrombospondin-1 possesses antiproliferative and anti-
UsingcomputationalpredictionprogramssuchasmiRanda angiogenic properties. Other transcripts that were reduced
and RNAhybrid (reviewed in [103]) allowed Choy and corresponded to the genes for osteopontin, S100 calcium
coworkers to envisage the cellular protein p53 up-regulator binding protein, plasticity related gene 1 product, and
of apoptosis (PUMA) as a target for miR-BART5 [78]. The integralmembraneprotein2A[80,81].ThemRNAforthe
authors demonstrated that PUMA levels were decreased in Bcl-2 interacting protein BCLAF1 was identified as a target
cellsexpressingmiR-BART5comparedtocellslackingmiR- formiR-K12-5.AdditionalinhibitionofBCLAF1expression
BART5. In accordance, when miR-BART5 was specifically wasobtainedinthepresenceofmiR-K9,-10a,and-10b.The