Analysis of the roles of HIV-derived microRNAs

1. miRNAs

2. Viral miRNAs

3. Conclusion

4. Expert opinion

Review

Analysis of the roles ofHIV-derived microRNAsAarthi Narayanan, Kylene Kehn-Hall, Charles Bailey & Fatah Kashanchi††George Mason University, National Center for Biodefense and Infectious Diseases, Discovery Hall,

Manassas, VA, USA

Importance of the field: HIV-1 is a retrovirus that has infected millions in

recent decades. The level of life cycle complexity and host control exerted

by this small virus with only nine proteins is astonishing. An interesting direc-

tion that has emerged in recent years is the role of small non-coding RNAs in

viral gene expression.

Areas covered in this review: We focus on HIV-1 produced microRNAs

(miRNAs), namely, TAR, Nef and miR-H1, and their roles in HIV-1 biogenesis.

The article provides insights into TAR miRNA-mediated downregulation of

viral and host gene expression by recruitment of chromatin remodeling

components (HDAC1).

What the reader will gain: We address the influence of TAR miRNA on host

cell cycle progression and apoptosis, and the role of Nef miRNA in the regula-

tion of viral and host gene expression. The review also highlights an intrigu-

ing connection between miR-H1 and HIV-1-associated neurological

pathogenesis, and the influence of the miRNAmachinery in the establishment

of latency. In the Expert Opinion section, we analyze the issue of host-

based therapeutics against HIV-1 and how transcription inhibitors are

influenced by viral miRNA production.

Take home message: HIV-derived miRNAs are of significance not only

to understand host-virus interactions, but also for the design of

effective therapeutics.

Keywords: cdk inhibitors, HIV-1, latency, microRNA, miR-H1, Nef, TAR

Expert Opin. Biol. Ther. (2011) 11(1):17-29

1. miRNAs

MicroRNAs (miRNAs) are small non-coding RNA molecules that are encoded bylife forms ranging from plants to higher order mammals [1]. Lin-4 [2] and let-7 [3]

miRNAs were the first identified miRNAs and they were demonstrated to nega-tively regulate gene expression in Caenorhabditis elegans. Since then, miRNAshave been discovered in plants and many metazoans, where they play crucial rolesin regulation of gene expression [4]. miRNAs exert their regulatory influence bydestruction of target mRNA molecules (transcriptional repression) or inhibitionof translation of target mRNAs (translational repression). Extensive research hasbeen conducted and multiple manuscripts have been published on cellular miRNAsand their roles in the regulation of gene expression pertinent to cellular processessuch as development, differentiation, stress response and apoptosis [5-9]. The focusof this review, however, is on the newly discovered HIV-1-derived miRNAs. Viralorigin of miRNAs is an accepted phenomenon and many DNA viruses producemiRNAs. Retroviral miRNAs, specifically HIV-1-derived miRNAs, are an excitingnovel discovery that have enormous implications in understanding viral regulationof susceptible host cells, cellular tropism, establishment of a latent state and,

10.1517/14712598.2011.540564 © 2011 Informa UK, Ltd. ISSN 1471-2598 17All rights reserved: reproduction in whole or in part not permitted

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ultimately, in their application towards the design of noveland effective inhibitors of viral multiplication.

1.1 The miRNA pathwayThe biogenesis of miRNAs has been very well characterizedand described in multiple articles [10-15]. Figure 1 illustratesthe miRNA pathway. miRNAs are usually transcribed byRNA polymerase II. A primary RNA transcript (pri-miRNA) [16] that is ~ 80 nucleotides long is processed inthe nucleus by the enzyme Drosha and its cofactorDGCR8 [17,18]. Drosha cleaves the miRNA to generatean ~ 60 nucleotide long pre-miRNA. The pre-miRNA isexported out of the nucleus by the exportin-5--RanGTP com-plex [19,20]. In the cytoplasm, the pre-miRNA is bound byDicer. Dicer cleaves the hairpin and the stem portion is car-ried on in the pathway as a miRNA duplex. One strand ofthis duplex (the ‘passenger strand’) is degraded while the other(the ‘guide strand’) is incorporated into RISC (RNA-inducedsilencing complex) and the HIV-1 TAR RNA binding protein(TRBP) is thought to assist in the loading process [21-23]. Thestrand that is less stably base-paired at the 5¢ end of the duplexis usually the guide strand [24]. The catalytic components ofRISC are the Argonaute proteins (Ago 1 -- 4). Ago 2 is shownto have endonuclease activity and can cleave target mRNAsthat show complementarity to the guide strand. The RISCcomplex and the associated miRNA usually bind to the 3¢UTR region of the target mRNAs. Nucleotides 2 -- 7 of themiRNA, called ‘the seed sequence’, play an important rolein the positioning of the RISC complex and the asso-ciated miRNA on the target mRNA [25,26]. In case of perfectcomplementarity between the target and the miRNA,the mRNA is degraded. However, when the complementa-rity is incomplete, the mRNA is translationally repressed.The mRNA--RISC complex is transported to P bodies thatare cytoplasmic structures containing RNA remodeling

components [27]. While the above mentioned processes occurat the post transcriptional level, RNA-mediated silencing canoperate at the chromatin level as well to regulate gene expres-sion. miRNAs can associate with the RNA-induced initiationof transcriptional silencing (RITS) complex and be guided tocomplementary regions in the chromosomal DNA [28,29]. Fol-lowing association with such genomic regions, the RITS com-plex recruits factors such as histone modifying enzymes,which alter the chromatin structure and induce transcriptionalsilencing [30].

2. Viral miRNAs

Viral encoded miRNAs could negatively regulate the virus.However, viral miRNAs could also confer significant advan-tages to the virus. For example, given that the requirementsof miRNA-mediated regulation of gene expression are flexiblein terms of target recognition, even a single mutation in themiRNA could sharply alter the target range thus increasingadaptability of the virus during viral evolution. In fact, it isestimated that the cellular pool of miRNAs could regulatethe expression of a third of the entire cellular genome becauseof the flexibility [31]. Timely suppression of viral multiplica-tion in the appropriate cell type is a crucial deciding factorin the establishment of latency. Indeed, viruses have alsoevolved means to suppress the cellular RNA interference(RNAi) machinery so that the repression can be relievedwhen no longer needed by the virus such as a switch fromlatency to lytic phase.

2.1 DNA viruses and miRNAsThe discovery of viral encoded miRNAs was initiated byPfeffer et al. when they reported in 2004 that they were ableto clone five Epstein--Barr virus (EBV) miRNAs from humanB cells that were latently infected with EBV [32]. Sincethen, > 140 virally encoded miRNAs have been discoveredin herpesviruses. A total of 25 EBV encoded miRNAs havebeen identified [33-35]. In the case of Kaposi’s sarcoma-associated herpesvirus (KSHV), a total of 12 pre-miRNAsthat resulted in 17 mature miRNAs have been reported [36].In the case of human cytomegalovirus (HCMV), 11 miRNAprecursors were identified that lead to the expression of14 mature RNA species [37]. In the case of a herpesviruses,HSV1 and 2, MDV1 and 2 have been demonstrated toencode for miRNAs [38]. Functionally, in a herpesviruses,similar miRNAs have been found in the latency-associatedtranscript regions of all a herpesviruses suggesting that animportant function of the only RNA species produced duringlatency is to express miRNAs. These miRNAs may contributeto the establishment and maintenance of latency [38]. BothHCMV and EBV miRNAs have been shown to target hostand viral genes and regulate their expression. In the case ofKSHV, although several host genes have been reported to beregulated by the viral miRNAs, there does not appear to beany report on viral genes being regulated. A general theme

Article highlights.

. MicroRNAs (miRNAs) are of cellular and viral origin.Retroviruses including HIV-1 are known to producemiRNAs in the infected host cells.

. The TAR miRNA of HIV is an abundant viral miRNA thatplays important roles in regulating viral and hostgene expression.

. The Nef miRNA and miR-H1 of HIV have beendemonstrated to be involved in disease progression.

. Viruses also encode suppressor of RNA silencingfunction to counter the effect of the host RNAsilencing machinery.

. Availability of a complete miRNA machinery could havesignificant implications for the establishment of virallatency in susceptible cells. Additionally, the cellularmiRNA machinery may be an important component ininfluencing viral inhibition by antivirals.

This box summarizes key points contained in the article.

Analysis of the roles of HIV-derived microRNAs

18 Expert Opin. Biol. Ther. (2011) 11(1)

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for viral miRNAs in herpesviruses appears to be latency/lytic switch, immune evasion, cell survival and proliferation.

2.2 Retroviruses and miRNAsWhile there have been numerous reports on miRNAsderived from DNA viruses, RNA virus-derived miRNAsappear to be somewhat controversial. It was first reportedin 2005 by Pfeffer et al. that cDNA cloning of smallRNAs from HIV-1 infected HeLa cells did not yield anyviral encoded miRNAs [39]. A second report was publishedin 2007 by Lin and Cullen [40] that small RNAs(18 -- 24 nt) cloned from HTLV-1 and HIV-1 infected cells

(MT-2 and ACH-2) cells did not yield any viral encodedmiRNAs. They report 1098 clones from ACH2 cells andstate that the majority of clones corresponded to cellularmiRNAs and a minor population of clones consisted ofbreakdown products of cellular mRNAs, tRNAs andrRNAs. The authors indicate that it may be possible forviral miRNAs to be present at very low levels (< 0.5% ofthe total cellular miRNAs). Another report [41] alsoexplained that the reason why no viral miRNAs were iden-tified in these experiments could be very low levels of viralmiRNA expression. Such low levels of miRNAs may rendertheir isolation by methods such as cloning difficult, more so

RNA pol II

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Figure 1. miRNA biogenesis. A primary miRNA (Pri-miRNA) with a 5’ cap and a 3’poly A tail is processed by Drosha to yield Pre-

miRNAs. They are transported to the cytoplasm by exportin-5 and the cytoplasmic pool of pre-miRNAs is processed by Dicer to

mature miRNAs. The mature miRNAs participate in transcriptional and translational silencing to regulate gene expression.RISC: RNA-induced silencing complex; RITS: RNA-induced initiation of transcriptional silencing; TRBP: TAR RNA binding protein.

Narayanan, Kehn-Hall, Bailey & Kashanchi

Expert Opin. Biol. Ther. (2011) 11(1) 19

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in the presence of abundant species such as ribosomal andtransfer RNAs. Also, expression of viral miRNAs may varybased on cell type and inability to detect viral miRNAs inone cell type does not mean they are absent in all. Hence,the successful capture of viral miRNAs by small RNA clon-ing methods is governed by the specific stage of infection,robustness of the infection and the infected cell type [42]

which could directly and indirectly affect total viral miRNAabundance. More sensitive methods such as RNase Protec-tion Assays (RPA) and next-generation sequencing [43]

could be advantageous in identifying low abundance smallnon-coding RNA species. Yeung et al. report that they iden-tified multiple small non-coding RNAs in HIV-1 infectedcells [43]. They sequenced a total of 47,773 clones of which60% were miRNAs. Within this population, they identified125 non-coding RNAs that were HIV-1 specific. They alsoreport that the TAR non-coding RNAs were the mostabundant followed by the RRE and NEF non-codingRNAs. These sensitive methods provide convincing evi-dence that HIV-1 encoded miRNAs do in fact exist andcan be effectively identified in infected cells. Additionally,recent evidence points to small viral RNAs (svRNAs)encoded by other RNA viruses such as influenza virus.The svRNAs were suggested to trigger the viral switchfrom transcription to replication through their interactionwith the viral polymerase machinery [44].

2.2.1 The TAR miRNAThe HIV-1 TAR element is a hairpin structure measuringabout 50 nucleotides in length that is found at the 5¢ end ofthe viral mRNA (Figure 2) [45,46]. The TAR element isencoded by the 5¢ and the 3¢ ends of HIV-1 RNA [47].Structural features and RNA--protein interactions providetantalizing clues to the potential of the TAR element to yieldmiRNAs. First, the hairpin structure of this element is inaccordance with the structure requirements of Dicer sub-strates. Second, computer modeling has predicted the TARelement as one of five potential Dicer substrates inHIV-1 [48]. Third, TAR being bound by two proteins thatare integral to the miRNA machinery, namely, TRBP andDicer argues convincingly for the involvement of TAR inmiRNA-mediated regulation of gene expression. Interest-ingly, a 124-mer TAR RNA domain present at the 5¢ end ofHIV-2 is considered to possibly encode pre-miRNAs andmay target a wide variety of cellular targets [49].

2.2.1.1 TRBPTRBP is the human homologue of the Drosophila Loquaciousprotein. Loquacious and TRBP bind Dicer and are required forthe efficient loading of the miRNA into the RISC complex.The importance of TRBP in RNAi was realized when intracel-lular depletion of TRBP resulted in a loss in cellular RNAsilencing function [21]. It is of interest that TRBP was discov-ered by its association with the TAR element [50] and wasdemonstrated to be involved in the transactivation and

inhibition of IFN-induced protein kinase R. It was demon-strated that HIV-1 may use TAR to target TRBP and sequesterit. This would result in reduced availability of TRBP to Dicerand hence affect miRNA processing.

2.2.1.2 Dicer and TAR miRNA biogenesisThe TAR element is bound by endogenous Dicer protein, asdemonstrated by biotin pull down assays [51]. Biotin labeledTAR was utilized to pull down ~ 0.3 -- 2.8% of the total cel-lular Dicer protein from infected and uninfected cell lines.Competition experiments with unlabeled TAR revealed thatthe TAR--Dicer association was specific to the presence ofthe TAR element and not to any generic RNA. A mutantversion of TAR called TAR-D (a shortened structure incapa-ble of Dicer binding) and a generic poly-U RNA moleculewere also used to demonstrate the specificity of theTAR--Dicer interaction.

It has also been demonstrated by ethidium bromide stain-ing and radioactive labeling that the TAR--Dicer associationresults in cleavage of the TAR RNA [51]. Both methodsrevealed the presence of an approximate 21 nucleotide frag-ment consistent with the cleavage of the TAR substrate. Theappearance of this 21 nucleotide fragment was contingenton the availability of enzymatically active Dicer as usingheat inactivated Dicer in in vitro studies did not yield thesame smaller processed RNA fragment. Knock down of cellu-lar Dicer expression using siRNAs showed that Dicer isindeed required for the processing of the TAR element.TAR mutants with alterations in structural elements (base-pair inversions, absence of a pyrimidine bulge and mutationsin the terminal loop element) while maintaining the overallintegrity of the stem loop structure were also processed byDicer comparably emphasizing the structural requirementfor this enzymatic processing reaction.

In addition to demonstrating that the TAR element couldbe bound and cleaved by Dicer in vitro, TAR-derived miRNA could also be detected in chronically infectedCD4+ T cells by RPA [51]. The RPA results revealed the pres-ence of a miRNA derived from the 5¢ end of the TAR ele-ment. TAR-derived miRNA was detectable in other celllines including latent cell lines (OM10.1 and HLM-1 cells).Recently, TAR miRNA has also been observed in HAARTtreated patient samples (F. Romerio, IHV Institute,unpublished data).

The 5¢ and the 3¢ arms of the TAR-derived miRNA havebeen cloned [50]. Comparison of the cloned sequences to thepredicted sequences in the Sanger miRNA database revealedthese clones to be different from previous predictions. Fourclones of the 5¢ stem (miR-TAR-5p) and about fourteenclones of the 3¢ stem (miR-TAR-3p) of TAR wereobtained [52] suggesting that the 3¢ stem of TAR may bemore abundant in infected cells. miR-TAR-3p exhibitedsuperior inhibition of gene expression comparatively and itis probably due to the preferential release and accumulationof the 3¢ element from the HIV-1 TAR RNA in vivo [53].



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2.2.1.3 Regulation of viral and host gene expression by

TAR miRNADownregulation of viral gene expression by TAR-derived miRNA has been demonstrated [51]. When utilizinga HIV-1 LTR controlled luciferase reporter, the TAR RNAwas able to suppress luciferase gene expression in aconcentration-dependent manner [51]. Additionally, it wasshown that the TAR-derived miRNA can downregulate geneexpression by recruiting chromatin remodeling componentsto the viral LTR. Using chromatin immunoprecipitationexperiments, the recruitment of HDAC-1 to the viral LTRof both transfected reporter plasmids and an integratedHIV-1 LTR was observed [49]. This recruitment ofHDAC-1 to the HIV-1 LTR by the TAR element suggestedthat the TAR element is capable of transcriptional silencingvia the RITS mechanism.

Additionally, it was found that the TAR miRNA affectedhost cell cycle [52]. 293T cells were transfected with wild-type and mutant TAR RNA molecules and effects on cellcycle progression were determined by flow cytometry. Theseexperiments showed that under conditions of stress, TARminus cells were arrested in G1 phase and subsequently pro-ceeded to apoptosis. In contrast, TAR miRNA-containingcells progressed through S and G2 phases, displayed a blockat G1/S and reduced apoptosis suggesting that the TARmiRNA protects the cells from stress-induced cell death [52].The same phenotype was also observed in chronically infectedcell lines (HLM-1 and ACH-2) suggesting that the anti-apoptotic effect of the TAR miRNA was independent of thesource of the miRNA. Using an antagomir with specificsequence complementarity to the mature TAR miRNA, thisanti-apoptotic effect was blocked thus underscoring the spec-ificity of TAR miRNA-mediated regulation of apoptosis ofthe host cell.

The fact that the TAR miRNA is expressed in bothlatent and actively infected cells suggests that the miRNAmay regulate a wide range of target genes including those

related to immune surveillance and evasion, cell survivalor increased viral production [52]. Microarray analysis ofRNA isolated from TAR transfected cells using an Affyme-trix human focus array identified 18 human genes thatwere downregulated and 14 genes that were upregulated.The downregulated genes were connected to several targetsrelated to replication, receptor signaling, DNA repair, apo-ptosis and mitochondrial function. Further validation offour of these genes showed possible links to apoptosisand cell survival including ERCC1 (excision repair crosscomplementing-group 1), PIASg (protein inhibitor of acti-vated STATg), GIT2 (GPCR interacting protein) andIER3 (intermediate early response 3). Interestingly, ininfected monocytes (which lack Dicer), the levels of theseproteins were upregulated. This suggests that the viralinfection may be upregulating the expression of these pro-teins while the viral miRNA was countering this effect.This serves as additional support for the notion that theviral miRNA downregulated ERCC1, GIT2 andIER3 expression (PIASg expression remained unaltered ininfected cells). As further evidence, it was demonstratedthat depletion of ERCC1 in 293T cells using siRNAsmade the cells more resistant to apoptosis. Interestingly,other analysis also revealed that the resistance to apoptosisby downregulation of ERCC1 involved activation of p53.Sequence analysis of the ERCC1 mRNA using the searchalgorithm miRanda revealed that there are six potentialtarget sites and that the TAR 5¢ miRNA was likely to bethe effector RNA strand. Cloning of these target sitesinto luciferase reporter constructs and analysis of reportergene expression in the presence of TAR miRNA revealeda suppression of luciferase expression thus confirmingthat TAR miRNA directed downregulation of theERCC1 gene. Additional studies revealed that the repres-sion of ERCC1 and IER3 expression involved alterationof protein expression without any effect on the respectivemRNAs (translational repression of the target). This

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Figure 2. HIV-1-derived miRNAs. Positions of the TAR, HIV-miR-H1, nef and RRE-derived miRNAs in the context of the viral

genome are indicated [45,52,53].



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downregulation of the host machinery by viral miRNAs is avital component to prolonging the life span of an infectedcell so as to allow ample time for viral multiplication orincreased life span in latently infected cells [52].

2.2.2 Nef miRNANef is a HIV-1 accessory gene located at the 3¢ end of the viralgenome, partially overlapping the 3¢ LTR (Figure 2) [48,54,55].It is important for viral replication in vivo and is conservedin HIV-1, HIV-2 and SIV. Nef is expressed during infectionand is said to account for up to 80% of HIV-specific RNAtranscripts during early stages of viral replication. Introduc-tion of defective variants of nef dsRNA [56] or cis expressionof nef [57] inhibits HIV-1 replication suggesting that nefmay function as a cis-regulatory element of HIV-1 replication.In fact, nef-derived miRNAs have been shown inHIV-1 infected cells [56]. Total RNA from MT-4 T cells per-sistently infected with HIV-1 IIIB strain was analyzed bynorthern blot with probes directed to the nef region. Theanalysis revealed small RNA molecules about 25 nucleotidesin length. These molecules were resistant to RNase A andT1 suggesting that they are likely to be double stranded.These miRNA species have been cloned and sequenced [56].Interestingly, nef miRNAs have been detected inHIV-1 infected long time non-progressors that display lowviremia. Using EGFP and luciferase reporter constructsdownstream of the nef target, nef miRNAs have been shownto downregulate nef expression in Jurkat T cells. Impressively,the nef miRNA (miR-N367) was effectively able to downre-gulate nef expression in the subcapsular regions of the spleenin mice, as determined by immunofluorescence andRT-PCR assays [58]. One of the downstream targets of nef isPPAR-g and nef suppresses PPAR-g expression. In miceexpressing nef miRNA, significant suppression of PPAR-gexpression was seen in the intestinal adipose tissue. Loss ofnef expression also appeared to afford protection to the miceby preventing fatigue and drastic weight loss. This opens thedoor to the possibility of utilizing HIV-1-derived miRNAsas therapeutics to control viral multiplication in vivo.

2.2.3 HIV miR-H1A third HIV-1-derived miRNA that has been reported ismiR-H1. This is an 81 nucleotide stem-loop structure thatis located downstream of the two NF-kB sites in the LTR(Figure 2) [48]. MiR-H1 has been demonstrated to degradethe apoptosis antagonizing transcription factor (AATF) geneproduct [41]. Downregulation of AATF is accompanied bylowered cell viability and lowered Bcl-2, c-myc, Par-4 andDicer levels. In this aspect, miR-H1 seems to be antagonisticto the anti-apoptotic effect afforded by TAR miRNA. It wasalso noted further that HIV miR-H1 downregulated expres-sion of the cellular miRNA miR149, which is considered totarget the Vpr gene encoded by HIV-1 [41]. Interestingly,the effects of miR-H1 have been examined in the context ofblood mononuclear cells. Monocytes not being able to sustain

a latent infection and miR-H1 negatively influencing Dicerlevels (hence, influencing HIV-1 pathogenesis) are discussedlater in the review. Strikingly, a study on miR-H1 variabilityin AIDS patients published earlier this year revealed that thereis a strong relationship between the miRNA sequence and thedevelopment of HIV-1-associated dementia (HAD) [59]. Allpatients with HAD shared a comparable miR-H1 sequencethat was similar to the published sequence. It is hypothesizedthat miRNAs that specifically target the AATF pathway couldbe involved in HAD development. The study also revealedthat another disease state, AIDS-related lymphoma, may beassociated with a less stable version of miR-H1 [59]. Theauthors hypothesize that the less stable versions may not beable to negatively regulate AATF; this may provide alternatemeans for activated macrophages to avoid apoptosis and pro-duce factors that could stimulate lymphoma growth. There-fore, the connection between distinct miRNA species andHIV-1-related pathology in patients with AIDS opens thepossibility for therapeutics.

2.3 Suppressor of RNA silencingThe presence of RNAi-based antiviral mechanisms in modernvertebrates, in additional to the protein-based immunity, con-tributes to a strong selection force that drives evolution ofviral genomes. The HIV-1 genome clearly has multipleregions that produce interfering RNAs that could elicit thecellular RNAi-based defense mechanism. However, virusesare also equipped with suppressor of RNA silencing (SRS).Jeang’s lab has published evidence for the existence of onesuch SRS [60]. While studying the ability of short hairpinRNA (shRNA) constructs to suppress reporter gene expres-sion when controlled by a Tat-independent promoter, it wasfound that whenever Tat was present in the experiment, theshRNAs were ineffective in suppressing the reporter gene.This lead to the conclusion that Tat was a suppressor ofRNAi-induced silencing. Because Tat is a protein that bindsto the TAR element, one could hypothesize that Tat-medi-ated suppression of the RNAi phenotype may be the resultof Tat coating the RNA and making it unavailable to theRNAi machinery. However, studies using non-HIV-1-derivedsiRNAs revealed that Tat is in fact a generic suppressor ofRNAi. Using Tat point mutants, it was shown that the twofunctions of Tat were distinct. Use of Tat deletion mutantsrevealed that amino acids 38 -- 72 in Tat contained its SRSfunction where this region is transcriptionally incompetent.It was also demonstrated that the SRS function of Tat wasnecessary for the virus to multiply in human cells. Impor-tantly, while Tat was able to suppress silencing mediated byshRNAs, it was ineffective against siRNAs. This suggeststhat the target of Tat is upstream of the siRNA--RISC com-plex, possibly at the level of Dicer. Tat was seen to affect thefunctionality of Dicer without influencing the total DicerRNA or protein levels. The connection of Tat with Dicer,Tat with the robust multiplication of the virus, lack of Dicerin monocytes and inability of monocytes to effectively sustain



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a latent state provide important clues about miRNA-mediated regulation of HIV-1 latency in Dicer containingtarget CD4+ T cells. However, there is some controversy sur-rounding the ability of Tat to function as a global RNAisuppressor. Lin and Cullen report that HIV-1 Tat andHTLV-Tax fail to inhibit RNAi in human cells. Their studiesdemonstrated that stable expression of Tat did not inhibitglobal miRNA production in infected human cells [40].

SRS function has been observed in other viruses as well [61].The viral 7a accessory protein of severe acute respiratory syn-drome coronavirus was found to suppress both transgene- andvirus-induced gene silencing. The SRS function was attrib-uted to the middle region (amino acids 32 -- 89) of 7a [62]

Interestingly, in case of adenovirus, viral-associated RNAsVA RNAI and VA RNAII function as suppressors of RNAiby interfering with Dicer function [63]. The ebolavirusVP35 protein has also been demonstrated to possess a SRSfunction that is equivalent to the HIV-1 Tat protein [64]. Incase of influenza virus, the NS1 protein of the strainA/WSN/33 (H1N1) was potent in suppressing RNAi-mediated silencing, although this function varied amonginfluenza strains and was suggested to contribute to alterationsin viral replication and pathogenicity [65]. It has also beendemonstrated that the NS3 protein encoded by rice hojablanca virus (plant virus) can complement the SRS functionof HIV-1 Tat [66].

2.4 Availability of the miRNA machinery and

implications for latencySuppression of viral replication and/or alteration of cellulargene expression (e.g., delay of apoptosis) may be importantmechanisms by which HIV-1 establishes latency. It is easy toconceive a mechanism by which HIV-1 miRNAs may bemore abundant in latent cells. As transcription is restricted inthe latent state, there is a low level of viral proteins producedincluding the viral transactivator Tat. This might result inlatent cells producing abortive short transcripts that arebetween 50 and 100 nucleotides in length and containing theTAR stem-loop structure. Such short transcripts have beenshown to be more abundant in non-viremic patients. As theseTAR containing transcripts are the only HIV-1 RNA speciesproduced in appreciable quantities in latent cells, it is possiblethat the miRNAs produced by the TAR element contributeto the maintenance of the latent state.

If the TAR miRNA is crucial to the maintenance of latency,it would follow that only cells that are competent to generatemiRNAs will be able to maintain the virus in the latent state.Dicer is present at strikingly low levels in monocytic cell linesand primary cells when compared to CD4+ T cells [67]. Analysisof CD4+ cell lines (Hut78, Molt4, H9, Jurkat, CEM) andHIV-1 infected cell lines (J1.1, ACH2, BE5) revealed thatDicer expression is similar in these multiple cells and remainsunaffected during HIV infection. Studies of cells of the mye-loid lineage including monocytes (THP-1), pro-monocytes(U937) and HIV-1 infected pro-monocytes (U1) revealed

that there was no detectable level of Dicer in these cells. Suchanalyses have been carried out in PBMCs obtained fromhealthy donors as well. Monocytes and lymphocytes from thesedonor cells and primary cells of the myeloid lineage were alsofound to be sub-optimal for Dicer protein. The levels of detect-able Dicer in these monocytic cells were the same irrespectiveof the method of preparation of cell lysates. Interestingly,RT-PCR analysis revealed that 40 -- 60% less Dicer mRNA ispresent in U937 and U1 cells than CEM and ACH2 cells.However, Dicer protein becomes detectable in monocyteswhen they are differentiated to macrophages. When U937 cellswere treated with increasing amounts of phorbol-myristateacetate (PMA) for 48 h and then probed for Dicer expression,Dicer protein was detected, although two to four times lowerthan what is seen in T cells. The absence of a complete, well-defined miRNA machinery in monocytes coincides with theinability of monocytes to sustain a latent state while theCD4+ T cells are targets of latency. This striking difference inDicer levels observed between monocytes and macrophagesraises questions about other miRNA components in mono-cytes. Drosha, Ago 1 and 2 were detectable in monocytic cells,but at lower levels than what were present in macrophages.When these cells were treated with PMA or M-CSF, Drosha,Ago 1 and 2 could be observed readily. Extensive analysiswith multiple cell types revealed a clear distinction betweencells of the lymphoid and myeloid lineage with respect to themiRNA machinery. Specifically, cells of the myeloid lineagewere deficient for proteins relevant to miRNA biogenesis untilinduced to terminally differentiate. The absence of Dicerexpression in monocytes brought up the question whether therewas a transcriptional or a translational block. RT-PCR analysisof monocyte extracts revealed that Dicer mRNA was present atall times and did not show any particular increase followingPMA treatment. This indicates a post-transcriptional blockwhich could be the result of a specific miRNA--mRNA inter-action. Interestingly, the cellular miRNA miR106a wasinvolved in the suppression of Dicer expression in monocytes.While monocyte-derived macrophages (MDMs) produceddetectable Dicer protein, HIV-1 infection of MDMssuppressed Dicer expression.

If monocytes are deficient for Dicer protein, do the cellsproduce any host cellular miRNAs? Microarray analysis ofRNA isolated from monocytes and MDMs using an AgilentV2 Human miRNA chip capable of detecting 722 uniquehuman miRNAs revealed that miRNAs are in fact producedin monocytes and both PMA treatment and HIV-1 infectionaltered the miRNAs [67]. Consistent with HIV-1-mediatedsuppression of Dicer in MDMs, the array studies revealedthat cells transfected with an HIV-1 clone produced fewermature miRNAs when compared with control untransfectedcells. Additionally, using mutant HIV-1 constructs, it wasdetermined that the viral protein Vpr contributes to loweredDicer levels in MDMs. Tat, as mentioned earlier, was the firstdiscovered viral suppressor of Dicer activity. Vpr and nef havebeen demonstrated to be additional HIV-1 proteins that can



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suppress the miRNA machinery by suppressing Dicer proteinproduction [67].The presence of multiple host cell miRNAs in a cellular

environment that is defective for Dicer expression raises ques-tions about the origin of these miRNAs. In addition tosiRNAs and miRNAs, another group of small RNA moleculesthat are detected in multiple cell types are the piRNAs. Theseare generated by members of the PIWI family in a Dicer-inde-pendent manner. PIWI does not function in an enzymaticcapacity. Instead, it protects double-stranded RNAs fromribonuclease degradation. Contrary to initial reports thatPIWI expression is restricted to reproductive tissues, PIWIexpression has been reported in somatic cells as well. Corrob-orating this, PIWIL4 expression has been observed in manycells of the lymphocyte and myeloid lineage and HIV-1 infec-tion had no effect on endogenous PIWIL4 levels [67]. MaturemiRNAs ranging in size between 26 and 33 base-pairs wereproduced in U937 cells, which were processed by PIWIL4.Collectively, analysis of the RNAi machinery in lymphoidand myeloid cells revealed that HIV-1 suppresses the miRNAmachinery in both cell types, but by alternate means.

3. Conclusion

An array of literature in the recent 5 -- 7 years has demon-strated the existence of HIV-1-derived miRNAs. ThesemiRNAs appear to be abundantly expressed and derivedfrom coding and non-coding regions of the viral genome.The miRNAs regulate both viral and host gene expression toalter the host milieu to support viral multiplication andestablishment of latency. HIV-1, in addition to coding formiRNAs, also encodes a suppressor of the RNAi machinery.Finally, the availability of a functional, miRNA machinery isan important factor in the establishment of latency.

4. Expert opinion

4.1 The influence of endogenous miRNA machinery

on effectiveness of anti-HIV inhibitorsHIV-1 continues to be the subject of much scrutiny when itcomes to therapeutics. Anti-HIV compounds directedagainst viral proteins eventually contribute to the evolutionof resistance. While HAART treatment does save many livesand suppresses evolution of resistance, there is continuingconcern on this matter [68,69]. A portion of the viral genomethat appears to be highly conserved even in the presence ofantivirals is the TAR element. The TAR element is thetarget for the viral transactivator protein Tat and both Tatand TAR are essential for HIV-1 replication [70,71]. Com-parison of human and simian immunodeficiency virusesreveals that there is significant structural conservation (byco-variations of base-pairs) in spite of sequence divergencein the viral repeat (R) region that codes for the TAR RNAmotif [72]. It was demonstrated that mutant virusesharboring base substitution mutations in the lower portion

of the TAR RNA stem were ‘dead viruses’ and that thebase-pairing at the lower portion of the stem was criticalfor viral multiplication [73]. The critical role played by theTAR element in viral multiplication may be the reason forthe observed resistance to mutation and this may make theTAR element more suited for the development of longterm RNAi-based therapeutics. There has been mixedreports of success in utilizing the TAR region as a target.For example, in the study by Das et al., only one out of eighttarget regions (including the 5¢ and the 3¢ UTRs) was capa-ble of inhibiting HIV-1 and the successful siRNA wasdirected against the Nef region [74]. While Leonard et al. sug-gested that HIV-1 could potentially evade the TAR directedsiRNA inhibition by developing compensatory mechanismsbased on the SP1 sites [75], Berkhout had suggested in anevaluation of the report that the HIV-1 strain used in theirexperiments modulates SP1 sites even when there is noRNAi-based inhibitory pressure [76].

Cellular miRNAs have been shown to function in an anti-viral capacity. There is a large body of literature that focuseson the interactions between the host miRNA machinery andHIV-1 [77,78]. The cellular miRNAs display distinct antiviralroles against HIV-1 and there are multiple references in theliterature relevant to this subject [79-82]. However, in this arti-cle, we restrict the focus to virally derived miRNAs and theirinfluence on HIV-1.

The cellular RNAi machinery has also been utilized in ananti-HIV-1 manner by introducing antiviral RNA molecules.For example, Son et al. have demonstrated that HIV-1 can beeffectively suppressed by co-targeting multiple conservedsequence elements in the HIV-1 genome. When co-expressedas an artificial miRNA molecule from a miR-155-based vec-tor, sequences directed against Tat and Vif genes mediatedeffective and sustained inhibition of HIV-1 [83]. In anothersuch approach to target multiple HIV-1 regions and to attackescape-prone viruses, Liu et al. tested a multiplex miRNAexpression system by inserting multiple anti-HIV-1 siRNAsequences into the miRNA polycistron mir-17 -- 92 and dem-onstrated that there is efficient inhibition of HIV-1 replica-tion [84]. A similar study is described by Zhang and Rossiwhere they express multiple miRNA effector molecules as asingle Pol II driven polycistronic transcript [85].

An alternate way to overcome the problem of drug resis-tance is to develop antivirals by targeting host componentsthat are essential for viral replication. Important host candi-date proteins that have been the subject of extensive researchas anti-HIV-1 targets are the chemokine receptors CCR5and CXCR4 (co-receptors for HIV-1 entry). Among theCCR5 inhibitors developed so far, maraviroc is the firstdrug that has been approved by the FDA. ManyCXCR4 inhibitors have been studied, but have not reachedthe stage of FDA approval and research efforts in this arenacontinue [86]. Chable-Bessia et al. have demonstrated thatdownregulation of miRNA effector RCK/p54 protein in theinfected cells also affects HIV-1 multiplication [87].



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Another example of such targets is cyclin-dependent kin-ases (cdks). HIV-1 is influenced by both cdk2/cyclin E com-plex and cdk9/cyclin T1 [88-91]. Thus, cdk/cyclin inhibitorsare ideal candidates for host-based antivirals againstHIV-1 [92,93]. Roscovitine and flavopiridol are two cdk inhib-itors that are most studied in relation to HIV-1 [94-99].Cyc202 (R-roscovitine) targets the cdk2--cyclin E complexand causes apoptosis in HIV infected cells. CR8 is a derivativeof Cyc202 with an alteration in the purine ring. CR8#13 is athird generation derivative that is a potent inhibitor ofHIV-1 transcription without affecting host gene expressionor cell viability [100].

The cellular miRNA machinery was necessary for effectiveviral inhibition by Cyc202 and CR8#13 as the drugs were

more effective in T cells than in monocytes. Specifically, the5¢ TAR miRNA was required for CR8#13-mediated inhibi-tion. CR8#13 was shown to decrease RNA polymerase IIphosphorylation, which in turn inhibits transcription elonga-tion. This leads to increased TAR production which results inthe recruitment of the miRNA machinery and chromatinremodeling complexes to the viral LTR. The outcome ofrecruitment of remodeling complexes to the LTR is transcrip-tional repression mediated by the cdk inhibitors. This modelhas been illustrated in Figure 3. This dependence of antiviralson the endogenous miRNA machinery for effective inhibitionof HIV-1 demonstrates that inhibitors that are effective insuppressing HIV-1 in lymphoid cells may not work inmyeloid cells. Understanding viral pathology in multiple

HDAC Chp1

HP1

Me Me Me

Me

Me

MeMe

MeMe

Me

RITS

RITS

Non codingRNA (either bound

to DNA or nascent RNA)

Cdk Inhibitors

P

Pol II

RITS complexrecruitment

Chromatin RemodelingFactor recruitment

Repressive Histone methylation

Possible LTRDNA methylation(heterochromatin)

Abortive transcription(noncoding RNAs)

NoncodingRNA (TAR)

+1

?

DNMT

(TAR)

?

Figure 3. Cdk inhibitors and transcriptional repression. Abortive transcription due to cdk inhibitors results in increased

production of TAR miRNAs. The TAR miRNAs are incorporated into the RITS complex, which leads to recruitment of repressive

chromatin. This ultimately culminates in repressive histone methylation, which could potentially lead to DNAmethylation and

formation of heterochromatin.DNMT: DNA methyltransferase; HDAC: Histone deacetylase; RITS: RNA-induced initiation of transcriptional silencing.



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target cells is hence an important prerequisite to designingeffective anti-HIV-1 therapeutics.An important avenue for future research is to understand if

the TAR miRNA affects the translational machinery in anymanner. As mentioned above, while the role played by TARin regulating transcription by controlling heterochromatinformation is being addressed, the potential role of TAR inmodulating the host translational machinery to influenceboth viral and host translation requires attention. Also, itwould be interesting to determine what happens to the hostmiRNA machinery (including Dicer) during later stages ofan infectious process. It is not known if Dicer undergoes a

suppression of function as the viral infection progresses. Tack-ling these questions will be crucial for developing a well-rounded and coherent picture of miRNA-mediated regulationof the retroviral life cycle.

Declaration of interest

This paper was supported by NIH grants AI 078859,AI 074410 and Department of Energy grant numberDE-SC0001599 awarded to F. Kashanchi. The other authorsdeclare no conflict of interest and have received no paymentin preparation of this manuscript.

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100. Carpio L, Klase Z, Coley W, et al.

MicroRNA machinery is an integral

component of drug-induced transcription

inhibition in HIV-1 infection. J RNAi

Gene Silencing 2010;6(1):386-400

AffiliationAarthi Narayanan1 PhD,

Kylene Kehn-Hall2 PhD, Charles Bailey3 PhD &

Fatah Kashanchi†4 PhD†Author for correspondence1Research Assistant Professor,

George Mason University,

National Center for Biodefense and

Infectious Diseases,

Discovery Hall, Room 306,

10900 University Blvd. MS 1H8,

Manassas, VA 20110, USA2Assistant Professor,






Manassas, VA 20110, USA3Center Director,






Manassas, VA 20110, USA4Director of Research,

Professor of Microbiology,






Manassas, VA 20110, USA

Tel: +1 703 993 9160; Fax: +1 703 993 7022;

E-mail: [email protected]



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Documents

Analysis of the roles of HIV-derived microRNAs