A Novel Assay for Viral MicroRNA Function Identifies a Single Nucleotide Polymorphism That Affects Drosha Processing

MicroRNAs (miRNAs) are a class of $~$ 22-nucleotide noncoding RNAsthat inhibit the expression of specific target genes at theposttranscriptional level. Recently, 11 miRNAs encoded by thepathogenic human herpesvirus Kaposi's sarcoma-associated herpesvirus(KSHV) were cloned from latently infected cells. While the expressionof these miRNAs has been confirmed by Northern analysis, theirability to inhibit target gene expression has not been demonstrated.We have devised a novel assay for miRNA function that uses lentiviralindicator vectors carrying two perfectly complementary targetsites for each given miRNA in the 3' untranslated region ofthe Renilla luciferase gene. This assay allowed us to demonstratethe activity of each viral miRNA upon cotransduction of cellswith the Renilla luciferase indicator vector together with afirefly luciferase control vector. In KSHV-infected BC-1 andBCBL-1 cells, but not uninfected control cells, Renilla luciferaseexpression was selectively reduced up to 10-fold. Interestingly,one of the viral miRNAs (miR-K5) exhibited much higher activityin BC-1 cells than in BCBL-1 cells. Sequence analysis of bothviral genomes revealed a single nucleotide polymorphism in themiR-K5 precursor stem-loop, which inhibits the expression ofmature miR-K5 in BCBL-1 cells. We show that the primary miR-K5sequence present in BCBL-1 results in diminished processingby Drosha both in vivo and in vitro. This is the first reportof a naturally occurring sequence polymorphism in an miRNA precursorthat results in reduced processing and therefore lower levelsof mature miRNA expression and function.

INTRODUCTION

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Introduction

Animals, plants, and some viruses encode microRNAs (miRNAs),a class of small ( $~$ 22-nucleotide [nt]) regulatory RNAs (1). Aspart of RNA-induced silencing complexes (RISCs), miRNAs guidethe sequence-specific posttranscriptional repression of targetmRNAs containing sequences with various degrees of complementarityto the miRNA (1, 11). The RISC was initially discovered as themediator of RNA interference (15), and it is now clear that,at least in vertebrates, miRNAs and small interfering RNAs utilizeoverlapping cellular pathways (10, 19, 37). miRNAs are initiallytranscribed as part of long primary miRNA (pri-miRNA) precursorsin which they form part of one arm of an RNA stem-loop (4, 8,23). In animals, the processing of the pri-miRNA is initiatedby the RNase III enzyme Drosha, which acts in concert with itspartner, DGCR8, to release a $~$ 65-nt pre-miRNA hairpin characterizedby a 2-nt 3' overhang and an imperfect $~$ 20-bp stem (9, 12, 16,20, 22, 34). The 3' overhang and RNA stem allow the recognitionand nuclear export of the pre-miRNA by exportin 5 (25, 32, 35).Once in the cytoplasm, cleavage of the pre-miRNA by anotherRNase enzyme, Dicer, yields an imperfectly base-paired RNA duplex(2, 14, 18). Generally, one strand of this duplex is then selectivelyincorporated into the RISC to form the mature miRNA, while theother strand, referred to as the passenger strand, is typicallydegraded (17). Target recognition by the RISC results in targetdegradation if the miRNA is perfectly complementary to the target(15). While this is mainly the case in the related RNA interferencepathway, this seems to be unusual for animal miRNAs (1). However,it has been shown that miRNAs can direct the cleavage of mRNAscontaining perfectly matched target sequences (19, 37). Morecommonly, the miRNA and its cellular target sequences are imperfectlymatched, and the binding of the RISC to such targets leads mainlyto a block of mRNA translation (1). It has been suggested that $~$ 30% of mammalian genes are subject to regulation by miRNAs (24).

Given the emerging importance of miRNA-mediated gene regulation,it is perhaps not surprising that several viruses also encodemiRNAs. The gammaherpesvirus Epstein-Barr virus was the firstvirus shown to express miRNAs, and subsequently, the gammaherpesvirusesKaposi's sarcoma-associated herpesvirus (KSHV) and murine herpesvirus68 as well as the betaherpesvirus human cytomegalovirus (CMV)were also shown to express miRNAs (5, 13, 26, 27, 30). Moreover,the polyomavirus simian virus 40 encodes a miRNA that targetsand downregulates the expression of simian virus 40 T antigens,the coding region of which overlaps the pri-miRNA gene in thereverse orientation (31). While several groups have previouslyidentified 11 KSHV miRNAs that are expressed in latently KSHV-infectedBC-1 and BCBL-1 cells, the biological activity of these KSHVmiRNAs, or indeed any herpesviral miRNA, has not been demonstrated(5, 26, 30). Here, we utilize a novel indicator system for miRNAfunction to show that the majority of the KSHV miRNAs expressedin BC-1 and BCBL-1 cells can induce robust downregulation ofan mRNA bearing artificial target sequences. We also provideevidence for differential expression of one KSHV miRNA, miR-K5,in the latently infected human cell lines BC-1 and BCBL-1. Wedemonstrate that this difference arises from a sequence polymorphismin KSHV that results in the inefficient processing of the pri-miR-K5precursor in BCBL-1 cells.

MATERIALS AND METHODS

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Materials and Methods

Molecular clones. Full-length firefly luciferase (FLuc) and Renilla luciferase(RLuc) cDNAs were amplified by PCR and used to replace the SnaB1-XhoIfragment of the previously described human immunodeficiencyvirus type 1-based vector pNL-SIN-CMV-BLR (21). Oligonucleotidesbearing two target sequences were annealed and inserted betweenthe ClaI and XbaI sites of pNL-SIN-CMV-RL. The N-terminal ClaIsite was destroyed by ligation of the insert, and a second ClaIsite 4 nt upstream of the XbaI site was used to insert two moretarget sites in the case of the vectors carrying four targetsequences. KSHV miRNA expression constructs for miR-K1 (bp 121762to 122003), miR-K3 (bp 121455 to 121703), miR-K8 (bp 119855to 120100), and miR-K9 (bp 119212 to 119447) were cloned byPCR amplification of the genomic KSHV DNA present in BC-1 cells(29) and inserted into the BglII and HindIII sites of a modifiedpSUPER construct (3). Termination was achieved by a run of fiveT's inserted 5' to the HindIII site. pTre-miR-K5(BC-1) and pTre-miR-K5(BCBL-1)were cloned by the insertion of PCR-generated DNA fragmentscorresponding to bp 121418 to 120947 of the KSHV genome intothe BamHI and SalI sites of pTre2hyg (BD Clontech). All PCR-amplifiedfragments were verified by sequencing.

Virus production and transduction and target site assays. 293T, BJAB, BC-1, and BCBL-1 cells were maintained as previouslydescribed (5). Transfections were carried out using the calciumphosphate method. Virus was produced from 293T cells by cotransfectionof the reporter vectors with pcRev, pcTat, and pHIT/G, as previouslydescribed (21). Virus was harvested 2 days after transfection,and control virus and indicator virus were mixed at a 2:1 ratioand used to transduce BJAB, BC-1, and BCBL-1 cells in the presenceof polybrene. Twenty-four hours after transduction, the cellswere recovered by centrifugation, washed with phosphate-bufferedsaline, and subjected to a dual luciferase assay (Promega).For the transient cotransfection of indicator constructs withKSHV expression constructs, cells were transfected in 24-wellplates with 2.5 ng control vector, 2.5 ng indicator vector,and 0.4 µg of the KSHV miRNA expression construct. Dualluciferase assays were carried out 3 days after transfection.

Northern blotting and in vitro Drosha cleavage. Mature miRNAs were detected by Northern blotting as previouslydescribed (5). The primary transcript from the pTre-miR-K5 constructswas detected using a $~$ 250-bp PCR product derived from the KSHVgenome present in BC-1 cells, which was labeled using a random-primedDNA labeling kit (Roche). For in vitro Drosha processing, 293Tcells were cotransfected using Drosha-Flag (22) and DGCR8-Flag(12) expression plasmids at a 9:1 ratio. After 48 h, Flag-taggedDrosha and DGCR8 proteins were copurified as described previously(33). Drosha processing experiments were performed as describedpreviously except that drained beads were used directly in thereaction mixture (33). The substrate was prepared by in vitrotranscription from T7-added, $~$ 270-bp PCR products, encompassingthe entire pre-miRNA stem-loop as well as flanking regions onboth sites.

RESULTS AND DISCUSSION

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Results and Discussion

Previously established assays for miRNA activity rely on thetransfection of indicator constructs bearing multiple copiesof complementary target sites in the 3' untranslated region(3'UTR) of a reporter gene (10, 36, 37). This approach is difficultto apply to hard-to-transfect cell lines or primary cells. WhileKSHV-infected B cells can be transfected by electroporation,this method is not suitable for large sample numbers and/orsmall sample volumes. We therefore established a new indicatorassay for miRNA activity that relies on the delivery of indicatorconstructs by transduction rather than by transfection. Thisassay is based on the previously described self-inactivatinghuman immunodeficiency virus type 1-based lentiviral vectorpNL-SIN-CMV (21), which was reengineered to express either FLucor RLuc under the control of the CMV immediate-early promoter.The resulting vectors were designated pNL-SIN-CMV-FL and pNL-SIN-CMV-RL.Unique ClaI and XbaI sites within pNL-SIN-CMV-RL allow the insertionof target site sequences into the 3'UTR of the Renilla luciferasegene (Fig. 1A). In the presence of the miRNA in question, theratio of RLuc activity to FLuc activity is expected to be reducedcompared to an RLuc indicator vector containing no target siteor an unrelated target site. Similarly, this same ratio shouldalso be reduced compared to the value obtained from controlcell lines that do not express the relevant miRNA.

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FIG. 1. (A) Schematic of the control vector pNL-SIN-CMV-FL, which encodes FLuc, and the indicator vector pNL-SIN-CMV-RL, which encodes RLuc. In each RLuc-based indicator construct, two perfectly complementary target sequences for one KSHV miRNA were inserted into the 3'UTR of the Renilla luciferase gene. (B) BJAB, BC-1, and BCBL-1 cells were transduced with a mixture of the control and indicator viruses, and dual luciferase assays were performed 24 h later. RLuc activities were normalized to the observed FLuc activity. Normalized RLuc activities in KSHV-positive BC-1 and BCBL-1 cells are shown relative to those obtained in KSHV-negative BJAB cells. Values from cells transduced with the parental RLuc-expressing vector were set at 100%. LTR, long terminal repeat.

To assess the biological activity of the various KSHV miRNAs,we constructed an indicator vector for each KSHV miRNA by theinsertion of two perfectly complementary target sites. The parentalRLuc vector, lacking any inserted target sites, served as anegative control (Fig. 1A). Production of vesicular stomatitisvirus G pseudotyped lentiviral vector particles was carriedout as described previously (21), and virus preparations fromeach of the 13 RLuc expression vectors were mixed with an aliquotof the FLuc control virus. These mixed virus stocks were thenused to infect the KSHV-negative B-cell line BJAB as well asthe latently KSHV-infected B-cell lines BC-1 and BCBL-1. At24 h after transduction, dual luciferase assays were performed.Based on control transductions with a green fluorescent protein(GFP)-expressing vector, we estimate transduction efficienciesto be >90% in the case of BJAB and $~$ 10% in the case of BC-1and BCBL-1 cells (data not shown). The ratios of RLuc activitiesto FLuc activities obtained for BC-1 and BCLB-1 cells were normalizedto the ratio obtained using the control RLuc vector that didnot contain any target sequences and to the ratios obtainedin BJAB cells (Fig. 1B). Phenotypically, the KSHV miRNAs fellinto three groups. miR-K1, miR-K3, and miR-K4-3p reduced theexpression of RLuc from the corresponding target site vectorby up to 10-fold. While less active, miR-K6-3p, miR-K5 in BC-1,miR-K11, and miR-K7 nevertheless induced a readily detectableinhibition (three- to fivefold) of RLuc expression, while miR-K2induced only about a twofold inhibition. The remaining miRNAs(miR-K4-5p, miR-K5 in BCBL-1, miR-K8, miR-K9-5p, and miR-K10)induced only a modest reduction ( $≤$ 40%) of RLuc activity.

The differences between the activities measured for the variousKSHV miRNAs could reflect either differences in their expressionlevels or inaccessibility of the target sites in a subset ofthe lentiviral indicator constructs. It seems unlikely, however,that some target site sequences would be readily accessiblewhile others at the same location are not. If the differencein activity indeed reflects differences in viral miRNA expressionlevels, it should be possible to achieve robust reporter geneinhibition upon overexpression of the relevant miRNA in allcases. To test this, we generated expression constructs formiR-K1 and miR-K3, two miRNAs that had high levels of activityin BC-1 and BCBL-1 cells, as well as for miR-K8 and miR-K9,which were only poorly active in these cell types. In each case,we placed a $~$ 250-bp genomic fragment, encompassing the miRNAstem-loop at a central position, under the control of the H1polymerase III promoter in a modified pSUPER construct (3).The miRNA signals detected by Northern blotting for 293T cellstransfected with these constructs were comparable to those ofBC-1 cells (Fig. 2A). However, since less than 60% of the 293Tcells were transfected (estimated using GFP control transfection),the expression levels of the miRNAs in 293T cells are presumablyhigher, on a per-cell basis, than those in BC-1 cells.

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FIG. 2. Overexpression of selected KSHV miRNAs results in enhanced reporter gene repression. (A) Northern analysis of miR-K1, miR-K3, miR-K8, and miR-K9 expression in total RNA from untransfected 293T cells (left lane), BC-1 cells, and 293T cells transfected with the relevant miRNA expression construct (right lane). (B to E) Reporter assay using 293T cells cotransfected with the indicated RLuc target site vector, the control FLuc vector, and expression constructs for miRK-1 (B), miR-K3 (C), miR-K8 (D), or miR-K9 (E). (F) Effect of different amounts of a transfected miR-K1 expression construct (pSUPER/250K1) on indicator activity. In this case, six-well plates were transfected with 12.5 ng of each luciferase vector and the indicated amounts of pSUPER/250K1. The total amount of transfected plasmid was adjusted by adding pSUPER to 2 µg total DNA.

To test miRNA activity, we cotransfected the miRNA expressionconstructs with the FLuc control vector as well as RLuc vectorscontaining zero, two, or four perfectly complementary targetsites for each miRNA in the 3'UTR of the RLuc gene. Dual luciferaseassays were carried out 3 days after transfection. As predicted,we saw robust target downregulation in all cases. While miR-K1was still the most active miRNA (Fig. 2B), we now achieved levelsof target gene repression for miR-K8 and miR-K9 that were comparableto that of miR-K3 (Fig. 2C and D), which was much more activein BC-1 and BCBL-1 cells (Fig. 1B). This finding strongly suggeststhat miR-K8 and miR-K9 are not expressed at sufficiently highlevels in BC-1 and BCBL-1 cells to induce efficient inhibitionof reporter gene expression. It is also evident from this experimentthat four perfectly matched target sites in the 3'UTR of theRLuc gene do not result in significantly greater inhibitionof target gene expression than do two target sites. This isconsistent with RLuc downregulation by mRNA cleavage, whichis believed to require only one perfectly matched target site,while downregulation by translational repression may requireseveral copies of imperfectly matched target sequences (10).To confirm that the observed RLuc activity indeed correlateswith the level of miRNA expression, we performed a dose-responseexperiment by transfecting different amounts of an miR-K1 expressionplasmid, pSUPER/250 K1 (Fig. 2F). Within a concentration rangeof 5 to 50 ng pSUPER/250K1 per six-well dish, the RLuc activitywas found to be approximately linearly related to the plasmidinput.

miR-K5 is differentially expressed in BC-1 and BCBL-1 cells. miR-K5 reduced reporter gene expression by $~$ 80% in BC-1 cells,while repression was much more moderate ( $~$ 35%) in BCBL-1 cells(Fig. 1B). If our target site assay accurately reflects theactivity and/or expression level of viral miRNAs, one wouldpredict that miR-K5 is less abundant in BCBL-1 cells than inBC-1 cells or that its activity is selectively inhibited bysome other mechanism. We performed Northern analysis to comparemiR-K5 expression levels in BC-1 and BCBL-1 cells and also probedmiR-K6-3p expression as a control (Fig. 3A). Clearly, miR-K5expression was much lower in BCBL-1 cells than in BC-1 cells,while levels of miR-K6-3p were virtually identical. The relativeabundance of pre-miR-K5, which was also detected, mirrored thatof the mature miR-K5. Therefore, the results obtained in thetarget site assay indeed accurately reflect the expression levelof miR-K5 in both cell types.

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FIG. 3. Differential expression of miR-K5 in BC-1 and BCBL-1 cells. (A) Northern analysis of miR-K5 (upper panel) and miR-K6-3p expression in total RNA preparations from 293T cells, BC-1 cells, and BCBL-1 cells. (B) Predicted (Mfold) pre-miR-K5 stem-loop structures encoded by BC-1-derived (upper panel) or BCBL-1-derived (lower panel) KSHV. The mature miR-K5 sequence is shown in boldface type, while the single nucleotide difference is shown in boldface type and italics. (C) Northern analysis of total RNA preparations from 293T cells transfected with the indicated plasmids and cultured in the presence or absence of doxycycline (Dox). The miR-K5 pri-miRNA and pre-miRNA precursors as well as mature miR-K5 are indicated. The quotation marks flanking "pri-miR-K5" signify that this RNA represents a truncated form of the authentic viral pri-miR-K5 transcript.

Sequence analysis of the genomic context of the miR-K5 precursorindicated a single nucleotide polymorphism in the stem-loopstructure of the miR-K5 precursor in BCBL-1 cells. This changeis located in the predicted stem sequence of the pre-miR-K5precursor, opposite the mature miR-K5, and is predicted to introducean A:C mismatch in place of a G:C base pair. This is expectedto result in an enlarged RNA bulge compared to the homologouspri-miR-K5 stem-loop structure found in the KSHV isolate presentin BC-1 cells (Fig. 3B). Conceivably, the presence of this largerbulge might inhibit the processing of the miR-K5 pri-miRNA precursorby Drosha or the efficiency of nuclear export of the pre-miRNAor might alter pre-miRNA stability.

To test whether Drosha cleavage is indeed reduced, we cloneda $~$ 470-bp fragment of the KSHV genome encompassing the miR-K5precursor from both BC-1 cells and BCBL-1 cells and expressedthis fragment under the control of a tetracycline-responsivepromoter element. These constructs were transfected into 293Tcells and examined by Northern analysis for expression of theprimary transcript, pre-miR-K5, and mature miR-K5. As expected,we detected high levels of pri-miRNA only when the pTet-Offtransactivator was coexpressed and in the absence of doxycycline,thus providing a control for specificity (Fig. 3C). Importantly,the BC-1- or BCBL-1-derived expression constructs yielded comparablelevels of the predicted primary miR-K5 transcript (Fig. 3C,upper panel). In contrast, the pre-miR-K5 precursor and maturemiR-K5 were readily detected in cells transfected with the BC-1-derivedKSHV sequence but not in cells transfected with the BCBL-1-derivedconstruct (Fig. 3C, lower panel).

The lack of pre-miR-K5 expression in BCBL-1 cells could representeither a block in pri-miRNA processing by Drosha or reducedpre-miRNA stability (e.g., due to a defect in recognition byits nuclear export receptor, exportin 5). To address whetherDrosha processing directly contributed to the observed phenotype,we subjected in vitro-transcribed pri-miR-K5 substrates, obtainedfrom either the BC-1- or BCBL-1-derived KSHV genome, to processingusing recombinant Drosha/DGCR8. This experiment (Fig. 4) essentiallyrecapitulated the difference seen in RNA preparations from BC-1and BCBL-1 cells or upon overexpression of the pTre-miR-K5 expressionconstructs. While the BC-1-derived substrate yielded readilydetectable levels of pre-miR-K5, this was clearly not the casefor the analogous BCBL-1-derived substrate. Therefore, subtledifferences in the RNA structure adopted by the miR-K5 RNA stem-loopcan modulate Drosha processing efficiency and thereby affectthe level of miRNA expression. This is the first report of anaturally occurring polymorphism in an miRNA precursor stem-loopthat modulates the level of expression of the mature miRNA.

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FIG. 4. The BCBL-1-derived miR-K5 precursor is deficient in Drosha processing. BC-1- or BCBL-1-derived pri-miR-K5 transcripts were incubated in vitro with bead-bound Flag-Drosha/Flag-DGCR-8 (+) or beads incubated with extracts prepared from GFP-transfected cells (–). The processed pre-miRNA is marked by an arrow.

We conducted a BLAST search using the pre-miR-K5 sequence fromthe KSHV genome contained in BCBL-1 as input. Interestingly,the same sequence found in BCBL-1 was also present in the onlytwo other previously published KSHV sequences encompassing thisregion, both derived from Kaposi's sarcoma biopsies (GenBankaccession numbers AF148805 and U93872). We also extended ouranalysis to another latently Epstein-Barr/KSHV-coinfected cellline, JSC-1 (6). The biological activity of miR-K5 in JSC-1cells was comparable to that in BCBL-1 cells (data not shown),and, consistent with this finding, sequencing of pre-miR-K5from JSC-1 cells yielded a sequence identical to that foundin BCBL-1 cells. Therefore, BC-1 cells contain the only miR-K5sequence presently known to be fully active.

We also performed BLAST searches on the pre-miRNA sequencesof all other KSHV miRNAs. For most miRNAs, only four sequenceshave been published. These include the BC-1 sequence (GenBankaccession numbers NC_003409 and U75698), the miRNA cluster fromBCBL-1 (accession number AY973824), and the two sequences fromKaposi's sarcoma biopsies mentioned above. Our analysis identifiedfurther polymorphisms in miR-K3, miR-K6, and miR-K10 (Fig. 5).In the case of miR-K3, only KSHV from BC-1 cells has a G nearthe base of the stem, while the remaining three sequences havean A at this position. In this case, however, the disruptionof base pairing does not result in a pronounced effect on miR-K3activity in BCBL-1 (Fig. 1B). In the case of miR-K6-3p, thesesame three sequences showed a 1-nt insertion in the terminalloop of the pre-miRNA. Based on previously published data, thischange is not expected to affect miRNA production (34), andindeed, the levels of miR-K6-3p activity in BC-1 and BCBL-1cells are indistinguishable (Fig. 1). For miR-K10, 15 otherKSHV sequences are available in addition to the sequences mentionedabove. The position of reported sequence polymorphisms is shownin Fig. 5. While a U at the base of the pre-miRNA stem may disruptbase pairing, this bulge might be too far away from the Droshacleavage site to affect processing. The other polymorphismsin the stem actually all stabilize the stem and are thereforenot predicted to negatively affect Drosha processing. As discussedabove for miR-K6-3p, the sequence in the terminal loop is largelyirrelevant to miRNA biogenesis. Interestingly, however, in severalcases, the sequence of the mature miRNA is altered. One of thesevariable residues actually lies in the miRNA seed region thatis expected to be most relevant for target recognition (1).While A and G might both base pair with U, it is currently unclearwhether a wobble G:U base pair can function in a seed region.Notably, the previously reported sequences could also encodedefective miRNAs and/or have been selected for other reasons(e.g., changes in the overlapping K12 reading frame) (5, 24).

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FIG. 5. Predicted pre-miRNA structures of BC-1-derived miR-K3 (top), miR-K6-3p (middle), and miR-K10 (bottom). Polymorphisms are indicated by arrows, and numbers refer to the accession numbers of the sequences carrying the identified nucleotides. In the case of miR-K10, a total of 18 pre-miR-K10 sequences were available. Of these sequences, nine differed from the sequence in BC-1 by at least 1 nt. GenBank accession numbers are as follows: 1, AY973824 (BCBL-1); 2, AF148805 (GK18); 3, U93872; 4, KSU66522; 5, AY157025; 6, AY042963; 7, AY042957; 8, AY042958; 9, AY042963; 10, AY042959; 11, AY042960.

In this paper, we describe a novel indicator assay in whichlentivirus-based indicator vectors are delivered by transductionrather than transfection. We used this assay to show that themajority of the KSHV miRNAs are biologically active in latentlyKSHV-infected BC-1 and BCBL-1 cells. We predict that this assaywill also be useful to evaluate the biological activity of artificiallyintroduced miRNAs (e.g., in miRNA-expressing cell lines) orthe differential expression of miRNAs in different types ofprimary cells, including nondividing cells such as neurons.Guided by the observed differences in the activity of miR-K5in the indicator system, we identified a naturally occurringsequence polymorphism in the passenger strand of the miR-K5pre-miRNA precursor that leads to reduced miR-K5 expressionin BCBL-1 cells due to diminished Drosha cleavage. While ourgroup previously showed that the artificial enlargement of aninternal bulge in a pre-miRNA stem-loop can abolish miRNA expression(34), the example of KSHV miR-K5 shows that subtle changes withina pre-miRNA precursor can greatly affect the level of maturemiRNA expression. Moreover, these polymorphisms can clearlyarise naturally. Since the mRNA targets and biological functionsof miR-K5 remain unknown, the consequences of reduced miR-K5expression for KSHV replication and pathogenesis are unclear.While it is possible that miR-K5 function is dispensable, itis also possible that the loss of miR-K5 expression might accountfor differences in the properties of BCBL-1 cells compared tothose of BC-1, such as the higher rate of spontaneous KSHV lyticreplication in BCBL-1 cells (5, 7, 28).

As in the case of the viral miRNA miR-K5, it is easy to imaginethat subtle sequence changes in cellular miRNA precursors, producedeither by mutation or by RNA editing, might also either inhibitor enhance mature miRNA expression and hence affect the progressionof diseases such as cancer.

ACKNOWLEDGMENTS

This research was supported by National Institutes of Healthgrant GM071408 awarded to B.R.C. and by a Feodor Lynen researchfellowship from the Alexander von Humboldt Foundation to E.G.

We thank V. Narry Kim and R. Shiekhattar for plasmids used inthe research.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Genetics and Microbiology, Box 3025, Duke University Medical Center, Durham, NC 27710. Phone: (919) 684-3369. Fax: (919) 681-8979. E-mail: culle002{at}mc.duke.edu.

REFERENCES

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