Gene 570 (2015) 108–114
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Research paper
Human cytomegalovirus microRNA miR-US25-1-5p inhibits viral replication by targeting multiple cellular genes during infection Shujuan Jiang a,b, Ying Qi a, Rong He b,⁎, Yujing Huang a, Zhongyang Liu a, Yanping Ma a, Xin Guo a, Yaozhong Shao a, Zhengrong Sun a, Qiang Ruan a,⁎⁎ a b
Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, China Clinical Genetics, The Affiliated Shengjing Hospital, China Medical University, China
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 29 May 2015 Accepted 3 June 2015 Available online 6 June 2015 Keywords: Human cytomegalovirus hcmv-miR-US25-1-5p Down-regulation Target genes DNA replication
a b s t r a c t MicroRNAs (miRNAs) play important roles in regulating various cellular processes in plants, animals, and viruses. This mechanism is also utilized by human cytomegalovirus (HCMV) in the process of infection and pathogenesis. The HCMV-encoded miRNA, hcmv-miR-US25-1-5p, was highly expressed during lytic and latent infections, and was found to inhibit viral replication. Identification of functional target genes of this microRNA is important in that it will enable a better understanding of the function of hcmv-miR-US25-1-5p during HCMV infection. In the present study, 35 putative cellular transcript targets of hcmv-miR-US25-1-5p were identified. Down-regulation of the targets YWHAE, UBB, NPM1, and HSP90AA1 by hcmv-miR-US25-1-5p was validated by luciferase reporter assay and Western blot analysis. In addition, we showed that hcmv-miR-US25-1-5p could inhibit viral replication by interacting with these targets, the existence of which may impact virus replication directly or indirectly. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus that can cause significant morbidity and mortality in newborns and immunocompromised individuals (Kesson and Kakakios, 2007). The pathogenic mechanism of HCMV is complex, and microRNAs (miRNAs) not only function as post-transcriptional regulators, but also are utilized by HCMV in the process of infection. HCMV encodes at least 16 pre-miRNAs corresponding to a total of 26 mature miRNA species (Babu et al., 2014). Previous studies demonstrated that hcmv-miRUS25-1-5p is highly expressed during lytic and latent infections, and it was found to inhibit HCMV DNA replication through the reduction of IE72 and pp65 expressions (Stern-Ginossar et al., 2009; Shen et al., Abbreviations: miRNAs, microRNAs; HCMV, human cytomegalovirus; siRNA, small interference RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MEM, minimal essential medium; MOI, multiplicity of infection; hpi, hours post infection; RT, reverse transcription; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3phosphate dehydrogenase; ECL, electrochemiluminescence; CDS, coding sequence; mfe, minimum free energy; MICB, major histocompatibility complex class I-related chain B; IFNs, type I interferons; IL-32, interleukin-32; eIF4A1, eukaryotic translation initiation factor 4A1; STX3, Syntaxin3; RISC-IP, RNA induced silencing complex immunoprecipitation; CSF, cerebral spinal fluid; LMP1, latency-associated membrane protein 1; CHPK, conserved protein kinase. ⁎ Correspondence to: R. He, Clinical Genetics, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, China. ⁎⁎ Correspondence to: Q. Ruan, Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, China. E-mail addresses: [email protected] (R. He), [email protected] (Q. Ruan).
http://dx.doi.org/10.1016/j.gene.2015.06.009 0378-1119/© 2015 Elsevier B.V. All rights reserved.
2014; Dunn et al., 2005). These findings suggest that it may play an important role in the establishment and maintenance of viral latency and persistence. Identification of the target genes and regulatory functions of hcmv-miR-US25-1-5p may provide further insight into the pathogenesis of HCMV. In the present study, hcmv-miR-US25-1-5p was confirmed to inhibit HCMV DNA replication during infection. A series of cellular genes (including YWHAE, UBB, NPM1, and HSP90AA1) were identified as direct targets of hcmv-miR-US25-1-5p. Down-regulation of these targets by hcmv-miR-US25-1-5p was validated by luciferase reporter assay and Western blot analysis. Inhibition of these targets by hcmvmiR-US25-1-5p or corresponding specific small interference RNAs (siRNAs) can reduce viral replication. 2. Materials and methods 2.1. Cell culture, transfection, and virus preparation Human embryonic kidney cells (HEK 293) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 units/ml each of penicillin and streptomycin. MRC-5 cells were maintained in a minimal essential medium (MEM) supplemented with 15% FBS and 100 units/ml each of penicillin and streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . Transfection was performed using the Lipofectamine 2000 (Invitrogen) kit according to the manufacturer's protocol.
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A low-passage HCMV isolate, Han, was originally isolated from a urine sample of a 5-month-old infant hospitalized in Shengjing Hospital of China Medical University (He et al., 2012). The urine sample was collected in the course of routine laboratory diagnosis. The collection was undertaken with the understanding and written consent of the subject's parents, and was approved by the ethical committee of Shengjing Hospital. The virus was propagated in MRC-5 cells maintained in MEM supplemented with 2% FBS and 100 units/ml each of penicillin and streptomycin. Then, the supernatant was harvested and stored at −80 °C before use.
2.2. Quantitative real-time PCR For the quantitative analysis of mature hcmv-miR-US25-1-5p in infected cells, MRC-5 cells growing on 60-mm plates were inoculated with the HCMV Han strain at a multiplicity of infection (MOI) of 3. Total RNAs were extracted from uninfected and infected cells at 24, 48, 72, 96, 120, and 144 h post infection (hpi), then were treated with a TURBO DNA-free™ Kit (Ambion). Reverse transcription (RT) was carried out with hcmv-miR-US25-1-5p specific primers (Applied Biosystems) and a TaqMan miRNA reverse transcription kit (Applied Biosystems). Real-time PCR was performed using the Universal PCR Master Mix Kit and a specific probe for hcmv-miR-US25-1-5p (Applied Biosystems) on the Applied Biosystems 7300 Fast Real-time PCR System. U6 snRNA was amplified as a normalization control. The relative expression level was calculated using the comparative threshold (2−ΔΔCT) method (Schmittgen et al., 2008).
2.3. Viral DNA quantification MRC-5 cells were transfected with 100 nM miRNA as negative control, hcmv-miR-US25-1-5p mimics, or hcmv-miR-US25-1-5p specific inhibitor (RiboBio). 24 h later, the transfected cells were inoculated with HCMV Han strain at an MOI of 3. Total DNA was extracted from the infected cells at 48 hpi. HCMV DNA genome copy number was then determined by real-time PCR using a commercial kit (Da An Gene). All measurements were done in triplicate and the results from three independent repetitions were presented as mean ± SE.
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2.5. Plasmid construction All primer sequences used in plasmid construction are listed in Table S1. For the luciferase assay, partial sequences of identified targets containing putative binding sites for hcmv-miR-US25-1-5p were amplified from mRNA-derived cDNA using primers listed in Table S1. Products were inserted into the pMIR vector (Ambion) to generate pMIR-YWHAE, pMIR-UBB, pMIR-NPM1, and pMIR-HSP90AA1. Mutations in the hcmv-miR-US25-1-5p binding site were made using a site-directed gene mutagenesis kit (Beyotime), and the resulting mutants were named pMIR-YWHAE-Mut, pMIR-UBB-Mut, pMIR-NPM-Mut, and pMIR-HSP90AA1-Mut. The full-length ORFs of YWHAE, UBB, and NPM1 were amplified from mRNA-derived cDNA, and an in-frame Myc epitope tag sequence was introduced into the PCR products. The PCR products were then inserted into the pBI-CMV2 vector (Clontech) to construct pBI-YWHAE-Myc, pBI-UBB-Myc, and pBINPM1-Myc, which constitutively expressed the proteins of interest as well as green fluorescent protein (GFP). For knockdown of these targets, siRNA sequences against YWHAE (Tang et al., 2013), UBB (Oh et al., 2013), NPM1 (Wang et al., 2006), or HSP90AA1 (Chatterjee et al., 2007) were synthesized, annealed, and inserted into a BamHI/HindIII-digested pSilencer4.1 plasmid (Ambion) to generate pS-siYWHAE, pS-siUBB, pS-siNPM1, and pS-siHSP90AA1, respectively. All constructs were confirmed by DNA sequencing. 2.6. Luciferase assay A total of 100 nM hcmv-miR-US25-1-5p mimics (RiboBio) was co-transfected into HEK293 cells with 0.2 μg of empty vector pMIR, pMIR containing the putative target sequences or their mutants, and 0.2 μg control renilla plasmid pRL-TK (Promega). As a negative control, a miRNA mimic (RiboBio) capable of producing a random small RNA was transfected. Cells were collected 48 h post transfection and luciferase activity levels were measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. All measurements were done in triplicate wells and signals were normalized for transfection efficiency against the renilla control. Data from three independent repetitions were presented as the mean ± SE for use in statistical analysis. 2.7. Western blotting
2.4. Hybrid PCR and sequencing Target genes of hcmv-miR-US25-1-5p in HCMV Han-infected MRC-5 cells were screened by hybrid PCR as previously described, using a 3′-Full RACE Core Set (TakaRa) (Huang et al., 2011). The hybrid PCR primer for hcmv-miR-US25-1-5p was designed as follows: 5′-GGRCCGRGCCRCTGRGCGGT-3′ (R = A / G). A previous study demonstrated that hcmv-miR-US25-1-5p could down-regulate its targets through mRNA 5′ UTRs (Grey et al., 2010). In this study, another hybrid PCR-derived method was used to screen targets with a binding site located in the 5′ UTR or coding sequence. In this method, another specific primer, 5′-TCAAGTTAGATAAACCGCRCAG-3′ (R = A/G), was designed according to the sequence of hcmv-miR-US25-1-5p, using the 5′-Full RACE Core Set (TakaRa). PCR products were purified using the Promega Wizard SV Gel and PCR Clean-up System (Promega), and were cloned into pMD-19T vectors (TaKaRa). The recombinants were then transformed into Escherichia coli to produce a pool which should contain specific sequences of putative target mRNAs that hcmv-miR-US25-1-5p can bind to. One hundred and forty five clones were randomly selected and the inserts were sequenced on an ABI 3730 automated sequencer. The specific mRNA sequences located between the hcmv-miR-US25-1-5p hybrid primer and the poly-A tail were intercepted and used to identify the putative target genes online (http://www.ncbi.nlm.nih.gov/blast).
Total protein was extracted from HEK293 and MRC-5 cells with a mammalian protein extraction reagent M-PER (ThermoScientific) according to the manufacturer's instructions, and then protein levels were quantified using a BCA protein assay kit (Beyotime). The extracted proteins were separated in a 10% acrylamide gel and transferred onto a nitrocellulose membrane. Western blot analyses were performed using HSP90AA1 (Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam), and Myc- or GFP-specific antibodies (Clontech). Immunoblots were visualized using an electrochemiluminescence (ECL) detection system. 2.8. Statistics Data are shown as mean ± SE. Statistical significance was determined by Student's t-test, with a P-value of b0.05 considered to be statistically significant. 3. Results 3.1. hcmv-miR-US25-1-5p is highly expressed in HCMV-infected MRC-5 cells Previous studies demonstrated that hcmv-miR-US25-1-5p was abundantly expressed during HCMV infection. In the present study,
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the expression kinetics of hcmv-miR-US25-1-5p was determined in MRC-5 cells at different timepoints post-HCMV infection. As shown in Fig. 1, hcmv-miR-US25-1-5p was highly expressed at 24 hpi, followed by a gradual, further increase in expression with prolonged HCMV infection. Moreover, it remained highly expressed even at 144 hpi. 3.2. Over-expression of miR-US25-1-5p reduces HCMV DNA replication HCMV genome copies were determined, to validate the effect of hcmv-miR-US25-1-5p on HCMV DNA replication. MRC-5 cells transfected with miR-NC, hcmv-miR-US25-1-5p, or hcmv-miR-US251-5p inhibitor were infected with HCMV Han strain. The amount of viral DNA post-HCMV infection was measured by real-time PCR at 48 hpi. As shown in Fig. 2, over-expression of hcmv-miR-US25-1-5p resulted in a significant reduction in HCMV DNA synthesis compared with cells transfected with miR-NC. Conversely, silencing of hcmvmiR-US25-1-5p by its inhibitor increased HCMV DNA synthesis in infected cells. 3.3. Putative targets of hcmv-miR-US25-1-5p identified by hybrid PCR Thirty-five putative target mRNAs for hcmv-miR-US25-1-5p were obtained using hybrid PCR and a hybrid PCR-derived method (described in Section 2.4). Detailed information on the identified putative targets is shown in Tables 1 and 2. The corresponding original mRNAs were successfully identified in GenBank (http://www.ncbi.nlm.nih.gov/ GenBank). All identified putative target mRNAs of hcmv-miR-US25-15p were from the human genome. Most of the putative targets had been previously reported (Grey et al., 2010). Of these, YWHAE, UBB, NPM1, and HSP90AA1 were chosen for further validation. The predicted binding sites for hcmv-miR-US25-1-5p were located within the coding sequences (CDS) of YWHAE and UBB, and the 5′ UTRs of NPM1 and HSP90AA1. These binding sites were also predicted by RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/ rnahybrid/submission.html) with minimum free energy (mfe) values of −33.3 kcal/mol, −29.4 kcal/mol, −26.3 kcal/mol, and −27.4 kcal/mol, respectively (Fig. 3A). 3.4. YWHAE, UBB, NPM1, and HSP90AA1 are direct targets of hcmv-miR-US25-1-5p To validate whether or not YWHAE, UBB, NPM1, and HSP90AA1 are direct targets of hcmv-miR-US25-1-5p, we initially identified the binding ability of hcmv-miR-US25-1-5p to these targets. As shown in Fig. 3B, transfection of hcmv-miR-US25-1-5p mimics in HEK293 cells significantly decreased the luciferase activities of YWHAE (39.1%), UBB (46.2%), NPM1 (49.8%), and HSP90AA1 (34.5%) compared with the
Fig. 1. Expression kinetics of hcmv-miR-US25-1-5p in HCMV-infected MRC-5 cells. MRC-5 cells were infected with HCMV at an MOI of 3. Expression levels of hcmv-miR-US25-1-5p in HCMV-infected cells were quantified by RT-qPCR at 0, 24, 48, 72, 96, 120, and 144 hpi. Data from three independent repetitions were used for statistical analysis.
miRNA negative control (NC) transfected cells. This decrease was not seen in mutant constructs of each of these four putative targets. We next investigated the effect of hcmv-miR-US25-1-5p on ectopically expressed YWHAE, UBB, and NPM1 or endogenous HSP90AA1 in HEK293 cells. Western blot demonstrated that the protein levels of these targets were significantly decreased after transfection with hcmv-miR-US25-1-5p mimics compared with the miRNA negative control (NC)-transfected cells (Fig. 3C). Therefore, these results indicated that hcmv-miR-US25-1-5p repressed these targets via the predicted binding sites. 3.5. hcmv-miR-US25-1-5p-mediated repression of the targets YWHAE, UBB, NPM1, and HSP90AA1 inhibits HCMV DNA replication Next, we sought to investigate whether hcmv-miR-US25-1-5pmediated down-regulation of its targets could further influence HCMV DNA replication. HCMV genome copy number, as well as expression of the targets, was quantified in MRC-5 cells infected by HCMV after cotransfection with vectors expressing the target genes along with the following non-coding RNAs: miRNA negative control, hcmv-miRUS25-1-5p mimics, hcmv-miR-US25-1-5p inhibitor, or specific siRNA against the corresponding targets. Our data showed that decreased expression of these targets caused by hcmv-miR-US25-1-5p or specific siRNA (Fig. 4B) markedly reduced HCMV DNA levels (Fig. 4A). Conversely, silencing of hcmv-miR-US25-1-5p by its specific inhibitor counteracted the inhibition effect of hcmv-miR-US25-1-5p on the expression of these targets (Fig. 4B), and the corresponding HCMV copy number was increased (Fig. 4A). These results indicated that hcmv-miR-US25-1-5p-mediated down-regulation of its targets could inhibit virus replication during HCMV infection. 4. Discussion Since the discovery of HCMV-encoded miRNAs (Dunn et al., 2005; Stark et al., 2012; Grey et al., 2005; Grey and Nelson, 2008; Pfeffer et al., 2005), more and more targets of these miRNAs have been identified. An increasing number of studies have attempted to elucidate the function of these miRNAs during HCMV infection. It has been reported that hcmv-miR-UL112-1 restricted viral acute replication through targeting of the genes IE72, (UL123, IE1), UL112/113, UL120/121, and UL144, which are encoded by HCMV itself and are involved in viral replication (Stern-Ginossar et al., 2009; Grey et al., 2007). Meanwhile, hcmv-miR-UL112-1 can also reduce the expression of major histocompatibility complex class I-related chain B (MICB), type I interferons (IFNs), and HCMV-activated interleukin-32 (IL-32), which may enhance viral immune evasion (Huang et al., 2013, 2014; Stern-Ginossar et al., 2007). Moreover, hcmv-miR-US25-2 can reduce HCMV replication by repressing IE72 and pp65 expression or by targeting eukaryotic translation initiation factor 4A1 (eIF4A1) (Stern-Ginossar et al., 2009; Qi et al., 2013), while hcmv-miR-US33 can reduce HCMV US29 mRNA levels and inhibit viral DNA synthesis by down-regulating expression of the host Syntaxin3 (STX3) (Shen et al., 2014; Guo et al., 2015). In addition, hcmv-miR-US4-1 was reported to inhibit CD8 + T cell responses by targeting the immunoevasion protein aminopeptidase ERAP1 (Kim et al., 2011). Furthermore, hcmv-miR-US5-1 has been reported to target secretary pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine secretion synergistically with hcmv-miR-UL112-1 and hcmv-miR-US5-2 (Hook et al., 2014). Collectively, these results suggest that HCMV might establish the mechanism of immune escape or regulate viral DNA replication during infection by targeting its own genes as well as host cellular genes via self-encoded miRNAs. Mature hcmv-miR-US25-1-5p is derived from the 5′ arm of the pre-hcmv-miR-US25-1 stem-loop structure encoded by the intergenic region between the HCMV US24 and US26 genes (Grey and Nelson, 2008; Dolken et al., 2009; Buck et al., 2007; Fannin et al., 2008).
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Fig. 2. Over-expression of hcmv-miR-US25-1-5p reduces HCMV DNA replication in MRC-5 cells at 48 hpi. (A) MRC-5 cells transfected with miRNA negative control (NC), hcmv-miR-US25-1-5p mimics (US25-1-5p), or hcmv-miR-US25-1-5p inhibitor (Inhi) were infected with HCMV at an MOI of 3, 24 h after transfection. Expression levels of hcmv-miR-US25-1-5p were measured by RT-qPCR, and normalized to those of cells transfected with a miRNA negative control at 48 hpi.(B) Viral genome copy number in cells treated as above was also determined using real-time PCR. Data from three independent repetitions were used for statistical analysis. * indicates P b 0.05 compared with data from miRNA negative control transfected cells.
It has been reported that hcmv-miR-US25-1-5p has the ability to downregulate multiple cellular genes associated with cell cycle control, such as cyclin E2, BRCC3, EID1, MAPRE2, and CD147, by interacting with the 5′ UTR of the targets (Grey et al., 2010). In addition, hcmv-miR-US251-5p was demonstrated to inhibit HCMV DNA replication through the reduction of IE72 and pp65 expressions (Stern-Ginossar et al., 2009). It has also been reported to target ATP6V0C, an essential host factor for HCMV replication (Pavelin et al., 2013). A recent study revealed that hcmv-miR-US25-1-5p aggravates the ox-LDL-induced apoptosis of endothelial cells via targeting and down-regulating BRCC3 (Fan et al., 2014). Collectively, these results suggest that hcmv-miR-US251-5p is involved in diverse processes; not only cell cycle control, but also virus replication inhibition as well as apoptosis regulation. Identifying putative targets of hcmv-miR-US25-1-5p is crucial for a comprehensive understanding of its function during HCMV infection.
In the present study, 35 putative targets of hcmv-miR-US25-1-5p were successfully identified using hybrid PCR and a hybrid PCR-derived method. All of these targets were from the human genome, and most of them were identified by Finn Grey (Grey et al., 2010) using RNAinduced silencing complex immunoprecipitation (RISC-IP) techniques. However, they were not subsequently validated. In our study, four host cellular genes (YWHAE, UBB, NPM1, and HSP90AA1) were demonstrated to be direct targets of hcmv-miR-US25-1-5p by luciferase assay and Western blot. As shown in Fig. 1, cotransfection of these targets with hcmv-miR-US25-1-5p mimics showed lower luciferase activity compared with cotransfection with the miRNA negative control. However, mutation of the hcmv-miR-US5-1 binding site in these targets' sequence abolished this effect. Moreover, the protein levels of these targets were significantly decreased in the presence of hcmv-miR-US25-1-5p. This indicates that
Table 2 Putative hcmv-miR-US25-1-5p targets identified by a hybrid PCR-derived method. Table 1 Putative hcmv-miR-US25-1-5p targets identified by hybrid PCR. Putative target mRNA
Accession number
Position of binding site
Ribosomal protein L8 (RPL8) Adaptor-related protein complex 3, delta 1 subunit (AP3D1) Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon (YWHAE) Ubiquitin B (UBB) S100 calcium binding protein A11 (S100A11) Myosin, light chain 5, regulatory (MYL5) Ring finger protein 31 (RNF31) Epidermal growth factor receptor (EGFR) Ribosomal protein S4, X-linked (RPS4X) PRELI domain containing 1 (PRELID1) Trafficking protein, kinesin binding 2 (TRAK2) Leucine carboxyl methyltransferase 1 (LCMT1) Ribosomal protein S6 kinase, 90 kDa, polypeptide 2 (RPS6KA2) Importin 9 (IPO9) Ring finger protein 187 (RNF187) AHNAK nucleoprotein (AHNAK) Dystroglycan 1 (DAG1) Ubiquitin-conjugating enzyme E2M (UBE2M) Mitochondrial ribosomal protein L46 (MRPL46) MAX dimerization protein 3 (MXD3) Activating transcription factor 7 (ATF7)
NM_033301.1 NM_001077523.1
CDS CDS
NM_006761.4
CDS
BC046123.2 NM_005620.1 BC040050.1 BC012077.1 NM_005228.3 BC000472.2 BC000007.2 AB038951.1 BC014217.2 NM_001006932.1
CDS CDS CDS CDS CDS CDS CDS 3′ UTR 3′ UTR 3′ UTR
NM_018085.4 BC012758.2 NM_001620.1 NM_004393.4 NM_003969.3 NM_022163.3 NM_001142935.1 NM_001130060.1
3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR
Putative target mRNA
Accession number
Position of binding site
Nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1) Heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1) Myristoylated alanine-rich protein kinase C substrate (MARCKS) RNA polymerase II, TATA box binding protein (TBP)-associated factor, 80 kDa (TAF6) Ribosomal protein L18 (RPL18) FERM and PDZ domain containing 4 (FRMPD4) Apoptosis enhancing nuclease (AEN) Dephospho-CoA kinase domain containing (DCAKD) NADH dehydrogenase Fe–S protein 7, 20 kDa (NDUFS7) Lysosomal-associated membrane protein 2 (LAMP2) RGD motif, leucine rich repeats, tropomodulin domain and proline-rich containing (RLTPR) Canopy FGF signaling regulator 4 (CNPY4) C-type lectin domain family 2, member D (CLEC2D) Lysophosphatidylcholine acyltransferase 1 (LPCAT1)
NM_002520.6
5′ UTR
NM_005348.3
5′ UTR
NM_002356.5
5′ UTR
NM_001190415.1
5′ UTR
NM_000979.2 NM_014728.3
CDS CDS
NM_022767.3 NM_024819.4
CDS CDS
NM_024407.4
CDS
NM_002294.2
CDS
NM_001013838.1
CDS
NM_152755.1 NM_013269.4
CDS 3′ UTR
NM_024830.3
3′ UTR
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Fig. 3. Hcmv-miR-US25-1-5p targets YWHAE, UBB, NPM1, and HSP90AA1 through predicted binding sites. (A) Schematic diagrams of predicted target sites of hcmv-miR-US25-1-5p in YWHAE, UBB, NPM1, and HSP90AA1. (B) Dual luciferase assay with cotransfection of the empty PMIR reporter vector, the reporter vectors containing the putative target sequences, or their mutants, along with miR-NC or hcmv-miR-US25-1-5p, in HEK293 cells. Data from three independent repetitions were used for statistical analysis. * indicates P b 0.05 compared with data from miRNA negative control transfected cells. (C) Western blot analysis of ectopically expressed YWHAE, UBB, and NPM1 or endogenous HSP90AA1 in HEK293 cells.
hcmv-miR-US25-1-5p has the ability to specifically repress these targets via the predicted binding sites. These four cellular genes identified in our study could encode proteins involved in diverse cellular processes such as metabolism, cell proliferation, apoptosis, cell cycle regulation, signal transduction, and protein chaperoning. The YWHAE protein is present in the cerebral spinal fluid (CSF) of those with cytomegalovirus or HIV infection, and the expression levels also correlated with the viral load of HIV-1 in the brain and CSF (Wakabayashi et al., 2001; Gelman and Nguyen, 2010). The UBB gene encodes ubiquitin, one of the most conserved proteins known. It is involved in the maintenance of chromatin structure as well as regulation of gene expression and the stress response (Conaway et al., 2002). Importantly, the intracellular ubiquitin level could affect the stability of the HIV-1 Rev protein, which plays a key role in virus replication (Vitte et al., 2006). NPM1 is a multifunctional chaperone which plays important roles in ribosome biogenesis and chromatin remodeling (Lindstrom, 2011). Interaction between NPM1 and HBV core protein was reported to increase HBV capsid assembly, while inhibition of NPM1 reduced intracellular capsid formation, resulting in a decrease in HBV production in HepG2.2.15 cells (Jeong et al., 2014). In addition, NPM1 was demonstrated to play important roles related to the establishment and maintenance of EBV latency in
B cells (Liu et al., 2012). HSP90, which aids in the folding of multiple client proteins, displays pleiotropic functions through its interaction with various cellular client proteins. It has been implicated in the replication of several different viruses, including HIV-1 (Anderson et al., 2014), HCV (Kim et al., 2012; Taguwa et al., 2009), HBV (Shim et al., 2011), EBV (Murata et al., 2013), KSHV (Qin et al., 2010), HVMV (Basha et al., 2005), HSV-1, and HSV-2 (Li et al., 2004, 2012). Furthermore, pharmacological inhibitors of HSP90 display broad antiviral effects. Geldanamycin, a potent and specific inhibitor of HSP90, was reported to inhibit gene expression and replication of human cytomegalovirus (Basha et al., 2005). A recent study demonstrated that HSP90 associates with the conserved protein kinase (CHPK) encoded by each of the 8 human herpesviruses, and treatment with the HSP90 inhibitor 17-DMAG decreases expression of these virally encoded kinases in cells transfected with expression vectors for each kinase. In addition, the expression of the EBV-encoded and HCMV-encoded kinases is markedly decreased in EBV-infected and HCMV-infected cells, respectively, when treated with 17-DMAG. Furthermore, 17-DMAG reduces production in Epstein–Barr virus-infected cells (Sun et al., 2013). Based on the studies above, we can speculate that all four of the cellular genes identified in our study may play important roles during virus infection, and may impact virus replication directly or indirectly.
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Fig. 4. Over-expression of hcmv-miR-US25-1-5p inhibits HCMV DNA replication by targeting YWHAE, UBB, NPM1, and HSP90AA1.(A) MRC-5 cells cotransfected with YWHAE, UBB, or NPM1 expression vectors with miRNA negative control (NC), hcmv-miR-US25-1-5p mimics (US25-1-5p), hcmv-miR-US25-1-5p inhibitor (Inhi), or specific siRNA against corresponding targets, were infected with HCMV at an MOI of 3, 24 h after transfection. Viral genome copy number was measured by real-time PCR at 48 hpi. Data from three independent repetitions were used for statistical analysis. * indicates P b 0.05 compared with data from miRNA negative control transfected cells. (B) Ectopically expressed YWHAE, UBB, and NPM1, or endogenous HSP90AA1 in cells treated as above were measured by Western blot.
Given the roles of these targets, and the fact that hcmv-miRUS25-1-5p could inhibit HCMV DNA replication by targeting the viral genes IE72 and pp65, it is of interest whether hcmv-miRUS25-1-5p could also affect viral replication by targeting these host cellular genes. Our results revealed that viral DNA genome copies, as well as protein levels of these four host target genes, were reduced in cells transfected with hcmv-miR-US25-1-5p compared with levels in cells transfected with a miRNA control. This reduction was eliminated when an hcmv-miR-US25-1-5p inhibitor was also used. In addition, the use of specific siRNAs against these targets ruled out possible off-target effects of hcmv-miR-US25-1-5p on HCMV DNA replication. This result indicated that hcmv-miR-US25-1-5pmediated down-regulation of these host cellular genes could inhibit virus replication during HCMV infection. However, more important biological functions of hcmv-miR-US25-1-5p during HCMV infection remain to be defined; hcmv-miR-US25-1-5p could play a wide range of roles by targeting a large number of host cellular genes.
In conclusion, our study showed that hcmv-miR-US25-1-5p could reduce virus replication by targeting multiple cellular transcripts that might impact virus replication directly or indirectly. These findings provide new insight into hcmv-miR-US25-1-5p. Further studies are required to investigate the biological function of hcmv-miRUS25-1-5p during HCMV infection. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.06.009. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was supported by the National Natural Science Foundation of China (81171580 and 81371788) and the Specialized
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Gene journal homepage: www.elsevier.com/locate/gene
Research paper
Human cytomegalovirus microRNA miR-US25-1-5p inhibits viral replication by targeting multiple cellular genes during infection Shujuan Jiang a,b, Ying Qi a, Rong He b,⁎, Yujing Huang a, Zhongyang Liu a, Yanping Ma a, Xin Guo a, Yaozhong Shao a, Zhengrong Sun a, Qiang Ruan a,⁎⁎ a b
Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, China Clinical Genetics, The Affiliated Shengjing Hospital, China Medical University, China
a r t i c l e
i n f o
Article history: Received 25 March 2015 Received in revised form 29 May 2015 Accepted 3 June 2015 Available online 6 June 2015 Keywords: Human cytomegalovirus hcmv-miR-US25-1-5p Down-regulation Target genes DNA replication
a b s t r a c t MicroRNAs (miRNAs) play important roles in regulating various cellular processes in plants, animals, and viruses. This mechanism is also utilized by human cytomegalovirus (HCMV) in the process of infection and pathogenesis. The HCMV-encoded miRNA, hcmv-miR-US25-1-5p, was highly expressed during lytic and latent infections, and was found to inhibit viral replication. Identification of functional target genes of this microRNA is important in that it will enable a better understanding of the function of hcmv-miR-US25-1-5p during HCMV infection. In the present study, 35 putative cellular transcript targets of hcmv-miR-US25-1-5p were identified. Down-regulation of the targets YWHAE, UBB, NPM1, and HSP90AA1 by hcmv-miR-US25-1-5p was validated by luciferase reporter assay and Western blot analysis. In addition, we showed that hcmv-miR-US25-1-5p could inhibit viral replication by interacting with these targets, the existence of which may impact virus replication directly or indirectly. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Human cytomegalovirus (HCMV) is a ubiquitous β-herpesvirus that can cause significant morbidity and mortality in newborns and immunocompromised individuals (Kesson and Kakakios, 2007). The pathogenic mechanism of HCMV is complex, and microRNAs (miRNAs) not only function as post-transcriptional regulators, but also are utilized by HCMV in the process of infection. HCMV encodes at least 16 pre-miRNAs corresponding to a total of 26 mature miRNA species (Babu et al., 2014). Previous studies demonstrated that hcmv-miRUS25-1-5p is highly expressed during lytic and latent infections, and it was found to inhibit HCMV DNA replication through the reduction of IE72 and pp65 expressions (Stern-Ginossar et al., 2009; Shen et al., Abbreviations: miRNAs, microRNAs; HCMV, human cytomegalovirus; siRNA, small interference RNA; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; MEM, minimal essential medium; MOI, multiplicity of infection; hpi, hours post infection; RT, reverse transcription; GFP, green fluorescent protein; GAPDH, glyceraldehyde-3phosphate dehydrogenase; ECL, electrochemiluminescence; CDS, coding sequence; mfe, minimum free energy; MICB, major histocompatibility complex class I-related chain B; IFNs, type I interferons; IL-32, interleukin-32; eIF4A1, eukaryotic translation initiation factor 4A1; STX3, Syntaxin3; RISC-IP, RNA induced silencing complex immunoprecipitation; CSF, cerebral spinal fluid; LMP1, latency-associated membrane protein 1; CHPK, conserved protein kinase. ⁎ Correspondence to: R. He, Clinical Genetics, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, China. ⁎⁎ Correspondence to: Q. Ruan, Virus Laboratory, The Affiliated Shengjing Hospital, China Medical University, Shenyang, Liaoning 110004, China. E-mail addresses: [email protected] (R. He), [email protected] (Q. Ruan).
http://dx.doi.org/10.1016/j.gene.2015.06.009 0378-1119/© 2015 Elsevier B.V. All rights reserved.
2014; Dunn et al., 2005). These findings suggest that it may play an important role in the establishment and maintenance of viral latency and persistence. Identification of the target genes and regulatory functions of hcmv-miR-US25-1-5p may provide further insight into the pathogenesis of HCMV. In the present study, hcmv-miR-US25-1-5p was confirmed to inhibit HCMV DNA replication during infection. A series of cellular genes (including YWHAE, UBB, NPM1, and HSP90AA1) were identified as direct targets of hcmv-miR-US25-1-5p. Down-regulation of these targets by hcmv-miR-US25-1-5p was validated by luciferase reporter assay and Western blot analysis. Inhibition of these targets by hcmvmiR-US25-1-5p or corresponding specific small interference RNAs (siRNAs) can reduce viral replication. 2. Materials and methods 2.1. Cell culture, transfection, and virus preparation Human embryonic kidney cells (HEK 293) were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 units/ml each of penicillin and streptomycin. MRC-5 cells were maintained in a minimal essential medium (MEM) supplemented with 15% FBS and 100 units/ml each of penicillin and streptomycin. Cells were maintained at 37 °C in a humidified atmosphere containing 5% CO 2 . Transfection was performed using the Lipofectamine 2000 (Invitrogen) kit according to the manufacturer's protocol.
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A low-passage HCMV isolate, Han, was originally isolated from a urine sample of a 5-month-old infant hospitalized in Shengjing Hospital of China Medical University (He et al., 2012). The urine sample was collected in the course of routine laboratory diagnosis. The collection was undertaken with the understanding and written consent of the subject's parents, and was approved by the ethical committee of Shengjing Hospital. The virus was propagated in MRC-5 cells maintained in MEM supplemented with 2% FBS and 100 units/ml each of penicillin and streptomycin. Then, the supernatant was harvested and stored at −80 °C before use.
2.2. Quantitative real-time PCR For the quantitative analysis of mature hcmv-miR-US25-1-5p in infected cells, MRC-5 cells growing on 60-mm plates were inoculated with the HCMV Han strain at a multiplicity of infection (MOI) of 3. Total RNAs were extracted from uninfected and infected cells at 24, 48, 72, 96, 120, and 144 h post infection (hpi), then were treated with a TURBO DNA-free™ Kit (Ambion). Reverse transcription (RT) was carried out with hcmv-miR-US25-1-5p specific primers (Applied Biosystems) and a TaqMan miRNA reverse transcription kit (Applied Biosystems). Real-time PCR was performed using the Universal PCR Master Mix Kit and a specific probe for hcmv-miR-US25-1-5p (Applied Biosystems) on the Applied Biosystems 7300 Fast Real-time PCR System. U6 snRNA was amplified as a normalization control. The relative expression level was calculated using the comparative threshold (2−ΔΔCT) method (Schmittgen et al., 2008).
2.3. Viral DNA quantification MRC-5 cells were transfected with 100 nM miRNA as negative control, hcmv-miR-US25-1-5p mimics, or hcmv-miR-US25-1-5p specific inhibitor (RiboBio). 24 h later, the transfected cells were inoculated with HCMV Han strain at an MOI of 3. Total DNA was extracted from the infected cells at 48 hpi. HCMV DNA genome copy number was then determined by real-time PCR using a commercial kit (Da An Gene). All measurements were done in triplicate and the results from three independent repetitions were presented as mean ± SE.
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2.5. Plasmid construction All primer sequences used in plasmid construction are listed in Table S1. For the luciferase assay, partial sequences of identified targets containing putative binding sites for hcmv-miR-US25-1-5p were amplified from mRNA-derived cDNA using primers listed in Table S1. Products were inserted into the pMIR vector (Ambion) to generate pMIR-YWHAE, pMIR-UBB, pMIR-NPM1, and pMIR-HSP90AA1. Mutations in the hcmv-miR-US25-1-5p binding site were made using a site-directed gene mutagenesis kit (Beyotime), and the resulting mutants were named pMIR-YWHAE-Mut, pMIR-UBB-Mut, pMIR-NPM-Mut, and pMIR-HSP90AA1-Mut. The full-length ORFs of YWHAE, UBB, and NPM1 were amplified from mRNA-derived cDNA, and an in-frame Myc epitope tag sequence was introduced into the PCR products. The PCR products were then inserted into the pBI-CMV2 vector (Clontech) to construct pBI-YWHAE-Myc, pBI-UBB-Myc, and pBINPM1-Myc, which constitutively expressed the proteins of interest as well as green fluorescent protein (GFP). For knockdown of these targets, siRNA sequences against YWHAE (Tang et al., 2013), UBB (Oh et al., 2013), NPM1 (Wang et al., 2006), or HSP90AA1 (Chatterjee et al., 2007) were synthesized, annealed, and inserted into a BamHI/HindIII-digested pSilencer4.1 plasmid (Ambion) to generate pS-siYWHAE, pS-siUBB, pS-siNPM1, and pS-siHSP90AA1, respectively. All constructs were confirmed by DNA sequencing. 2.6. Luciferase assay A total of 100 nM hcmv-miR-US25-1-5p mimics (RiboBio) was co-transfected into HEK293 cells with 0.2 μg of empty vector pMIR, pMIR containing the putative target sequences or their mutants, and 0.2 μg control renilla plasmid pRL-TK (Promega). As a negative control, a miRNA mimic (RiboBio) capable of producing a random small RNA was transfected. Cells were collected 48 h post transfection and luciferase activity levels were measured using the Dual Luciferase Reporter Assay System (Promega) according to the manufacturer's instructions. All measurements were done in triplicate wells and signals were normalized for transfection efficiency against the renilla control. Data from three independent repetitions were presented as the mean ± SE for use in statistical analysis. 2.7. Western blotting
2.4. Hybrid PCR and sequencing Target genes of hcmv-miR-US25-1-5p in HCMV Han-infected MRC-5 cells were screened by hybrid PCR as previously described, using a 3′-Full RACE Core Set (TakaRa) (Huang et al., 2011). The hybrid PCR primer for hcmv-miR-US25-1-5p was designed as follows: 5′-GGRCCGRGCCRCTGRGCGGT-3′ (R = A / G). A previous study demonstrated that hcmv-miR-US25-1-5p could down-regulate its targets through mRNA 5′ UTRs (Grey et al., 2010). In this study, another hybrid PCR-derived method was used to screen targets with a binding site located in the 5′ UTR or coding sequence. In this method, another specific primer, 5′-TCAAGTTAGATAAACCGCRCAG-3′ (R = A/G), was designed according to the sequence of hcmv-miR-US25-1-5p, using the 5′-Full RACE Core Set (TakaRa). PCR products were purified using the Promega Wizard SV Gel and PCR Clean-up System (Promega), and were cloned into pMD-19T vectors (TaKaRa). The recombinants were then transformed into Escherichia coli to produce a pool which should contain specific sequences of putative target mRNAs that hcmv-miR-US25-1-5p can bind to. One hundred and forty five clones were randomly selected and the inserts were sequenced on an ABI 3730 automated sequencer. The specific mRNA sequences located between the hcmv-miR-US25-1-5p hybrid primer and the poly-A tail were intercepted and used to identify the putative target genes online (http://www.ncbi.nlm.nih.gov/blast).
Total protein was extracted from HEK293 and MRC-5 cells with a mammalian protein extraction reagent M-PER (ThermoScientific) according to the manufacturer's instructions, and then protein levels were quantified using a BCA protein assay kit (Beyotime). The extracted proteins were separated in a 10% acrylamide gel and transferred onto a nitrocellulose membrane. Western blot analyses were performed using HSP90AA1 (Abcam), glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Abcam), and Myc- or GFP-specific antibodies (Clontech). Immunoblots were visualized using an electrochemiluminescence (ECL) detection system. 2.8. Statistics Data are shown as mean ± SE. Statistical significance was determined by Student's t-test, with a P-value of b0.05 considered to be statistically significant. 3. Results 3.1. hcmv-miR-US25-1-5p is highly expressed in HCMV-infected MRC-5 cells Previous studies demonstrated that hcmv-miR-US25-1-5p was abundantly expressed during HCMV infection. In the present study,
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the expression kinetics of hcmv-miR-US25-1-5p was determined in MRC-5 cells at different timepoints post-HCMV infection. As shown in Fig. 1, hcmv-miR-US25-1-5p was highly expressed at 24 hpi, followed by a gradual, further increase in expression with prolonged HCMV infection. Moreover, it remained highly expressed even at 144 hpi. 3.2. Over-expression of miR-US25-1-5p reduces HCMV DNA replication HCMV genome copies were determined, to validate the effect of hcmv-miR-US25-1-5p on HCMV DNA replication. MRC-5 cells transfected with miR-NC, hcmv-miR-US25-1-5p, or hcmv-miR-US251-5p inhibitor were infected with HCMV Han strain. The amount of viral DNA post-HCMV infection was measured by real-time PCR at 48 hpi. As shown in Fig. 2, over-expression of hcmv-miR-US25-1-5p resulted in a significant reduction in HCMV DNA synthesis compared with cells transfected with miR-NC. Conversely, silencing of hcmvmiR-US25-1-5p by its inhibitor increased HCMV DNA synthesis in infected cells. 3.3. Putative targets of hcmv-miR-US25-1-5p identified by hybrid PCR Thirty-five putative target mRNAs for hcmv-miR-US25-1-5p were obtained using hybrid PCR and a hybrid PCR-derived method (described in Section 2.4). Detailed information on the identified putative targets is shown in Tables 1 and 2. The corresponding original mRNAs were successfully identified in GenBank (http://www.ncbi.nlm.nih.gov/ GenBank). All identified putative target mRNAs of hcmv-miR-US25-15p were from the human genome. Most of the putative targets had been previously reported (Grey et al., 2010). Of these, YWHAE, UBB, NPM1, and HSP90AA1 were chosen for further validation. The predicted binding sites for hcmv-miR-US25-1-5p were located within the coding sequences (CDS) of YWHAE and UBB, and the 5′ UTRs of NPM1 and HSP90AA1. These binding sites were also predicted by RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/ rnahybrid/submission.html) with minimum free energy (mfe) values of −33.3 kcal/mol, −29.4 kcal/mol, −26.3 kcal/mol, and −27.4 kcal/mol, respectively (Fig. 3A). 3.4. YWHAE, UBB, NPM1, and HSP90AA1 are direct targets of hcmv-miR-US25-1-5p To validate whether or not YWHAE, UBB, NPM1, and HSP90AA1 are direct targets of hcmv-miR-US25-1-5p, we initially identified the binding ability of hcmv-miR-US25-1-5p to these targets. As shown in Fig. 3B, transfection of hcmv-miR-US25-1-5p mimics in HEK293 cells significantly decreased the luciferase activities of YWHAE (39.1%), UBB (46.2%), NPM1 (49.8%), and HSP90AA1 (34.5%) compared with the
Fig. 1. Expression kinetics of hcmv-miR-US25-1-5p in HCMV-infected MRC-5 cells. MRC-5 cells were infected with HCMV at an MOI of 3. Expression levels of hcmv-miR-US25-1-5p in HCMV-infected cells were quantified by RT-qPCR at 0, 24, 48, 72, 96, 120, and 144 hpi. Data from three independent repetitions were used for statistical analysis.
miRNA negative control (NC) transfected cells. This decrease was not seen in mutant constructs of each of these four putative targets. We next investigated the effect of hcmv-miR-US25-1-5p on ectopically expressed YWHAE, UBB, and NPM1 or endogenous HSP90AA1 in HEK293 cells. Western blot demonstrated that the protein levels of these targets were significantly decreased after transfection with hcmv-miR-US25-1-5p mimics compared with the miRNA negative control (NC)-transfected cells (Fig. 3C). Therefore, these results indicated that hcmv-miR-US25-1-5p repressed these targets via the predicted binding sites. 3.5. hcmv-miR-US25-1-5p-mediated repression of the targets YWHAE, UBB, NPM1, and HSP90AA1 inhibits HCMV DNA replication Next, we sought to investigate whether hcmv-miR-US25-1-5pmediated down-regulation of its targets could further influence HCMV DNA replication. HCMV genome copy number, as well as expression of the targets, was quantified in MRC-5 cells infected by HCMV after cotransfection with vectors expressing the target genes along with the following non-coding RNAs: miRNA negative control, hcmv-miRUS25-1-5p mimics, hcmv-miR-US25-1-5p inhibitor, or specific siRNA against the corresponding targets. Our data showed that decreased expression of these targets caused by hcmv-miR-US25-1-5p or specific siRNA (Fig. 4B) markedly reduced HCMV DNA levels (Fig. 4A). Conversely, silencing of hcmv-miR-US25-1-5p by its specific inhibitor counteracted the inhibition effect of hcmv-miR-US25-1-5p on the expression of these targets (Fig. 4B), and the corresponding HCMV copy number was increased (Fig. 4A). These results indicated that hcmv-miR-US25-1-5p-mediated down-regulation of its targets could inhibit virus replication during HCMV infection. 4. Discussion Since the discovery of HCMV-encoded miRNAs (Dunn et al., 2005; Stark et al., 2012; Grey et al., 2005; Grey and Nelson, 2008; Pfeffer et al., 2005), more and more targets of these miRNAs have been identified. An increasing number of studies have attempted to elucidate the function of these miRNAs during HCMV infection. It has been reported that hcmv-miR-UL112-1 restricted viral acute replication through targeting of the genes IE72, (UL123, IE1), UL112/113, UL120/121, and UL144, which are encoded by HCMV itself and are involved in viral replication (Stern-Ginossar et al., 2009; Grey et al., 2007). Meanwhile, hcmv-miR-UL112-1 can also reduce the expression of major histocompatibility complex class I-related chain B (MICB), type I interferons (IFNs), and HCMV-activated interleukin-32 (IL-32), which may enhance viral immune evasion (Huang et al., 2013, 2014; Stern-Ginossar et al., 2007). Moreover, hcmv-miR-US25-2 can reduce HCMV replication by repressing IE72 and pp65 expression or by targeting eukaryotic translation initiation factor 4A1 (eIF4A1) (Stern-Ginossar et al., 2009; Qi et al., 2013), while hcmv-miR-US33 can reduce HCMV US29 mRNA levels and inhibit viral DNA synthesis by down-regulating expression of the host Syntaxin3 (STX3) (Shen et al., 2014; Guo et al., 2015). In addition, hcmv-miR-US4-1 was reported to inhibit CD8 + T cell responses by targeting the immunoevasion protein aminopeptidase ERAP1 (Kim et al., 2011). Furthermore, hcmv-miR-US5-1 has been reported to target secretary pathway genes to facilitate formation of the virion assembly compartment and reduce cytokine secretion synergistically with hcmv-miR-UL112-1 and hcmv-miR-US5-2 (Hook et al., 2014). Collectively, these results suggest that HCMV might establish the mechanism of immune escape or regulate viral DNA replication during infection by targeting its own genes as well as host cellular genes via self-encoded miRNAs. Mature hcmv-miR-US25-1-5p is derived from the 5′ arm of the pre-hcmv-miR-US25-1 stem-loop structure encoded by the intergenic region between the HCMV US24 and US26 genes (Grey and Nelson, 2008; Dolken et al., 2009; Buck et al., 2007; Fannin et al., 2008).
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Fig. 2. Over-expression of hcmv-miR-US25-1-5p reduces HCMV DNA replication in MRC-5 cells at 48 hpi. (A) MRC-5 cells transfected with miRNA negative control (NC), hcmv-miR-US25-1-5p mimics (US25-1-5p), or hcmv-miR-US25-1-5p inhibitor (Inhi) were infected with HCMV at an MOI of 3, 24 h after transfection. Expression levels of hcmv-miR-US25-1-5p were measured by RT-qPCR, and normalized to those of cells transfected with a miRNA negative control at 48 hpi.(B) Viral genome copy number in cells treated as above was also determined using real-time PCR. Data from three independent repetitions were used for statistical analysis. * indicates P b 0.05 compared with data from miRNA negative control transfected cells.
It has been reported that hcmv-miR-US25-1-5p has the ability to downregulate multiple cellular genes associated with cell cycle control, such as cyclin E2, BRCC3, EID1, MAPRE2, and CD147, by interacting with the 5′ UTR of the targets (Grey et al., 2010). In addition, hcmv-miR-US251-5p was demonstrated to inhibit HCMV DNA replication through the reduction of IE72 and pp65 expressions (Stern-Ginossar et al., 2009). It has also been reported to target ATP6V0C, an essential host factor for HCMV replication (Pavelin et al., 2013). A recent study revealed that hcmv-miR-US25-1-5p aggravates the ox-LDL-induced apoptosis of endothelial cells via targeting and down-regulating BRCC3 (Fan et al., 2014). Collectively, these results suggest that hcmv-miR-US251-5p is involved in diverse processes; not only cell cycle control, but also virus replication inhibition as well as apoptosis regulation. Identifying putative targets of hcmv-miR-US25-1-5p is crucial for a comprehensive understanding of its function during HCMV infection.
In the present study, 35 putative targets of hcmv-miR-US25-1-5p were successfully identified using hybrid PCR and a hybrid PCR-derived method. All of these targets were from the human genome, and most of them were identified by Finn Grey (Grey et al., 2010) using RNAinduced silencing complex immunoprecipitation (RISC-IP) techniques. However, they were not subsequently validated. In our study, four host cellular genes (YWHAE, UBB, NPM1, and HSP90AA1) were demonstrated to be direct targets of hcmv-miR-US25-1-5p by luciferase assay and Western blot. As shown in Fig. 1, cotransfection of these targets with hcmv-miR-US25-1-5p mimics showed lower luciferase activity compared with cotransfection with the miRNA negative control. However, mutation of the hcmv-miR-US5-1 binding site in these targets' sequence abolished this effect. Moreover, the protein levels of these targets were significantly decreased in the presence of hcmv-miR-US25-1-5p. This indicates that
Table 2 Putative hcmv-miR-US25-1-5p targets identified by a hybrid PCR-derived method. Table 1 Putative hcmv-miR-US25-1-5p targets identified by hybrid PCR. Putative target mRNA
Accession number
Position of binding site
Ribosomal protein L8 (RPL8) Adaptor-related protein complex 3, delta 1 subunit (AP3D1) Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, epsilon (YWHAE) Ubiquitin B (UBB) S100 calcium binding protein A11 (S100A11) Myosin, light chain 5, regulatory (MYL5) Ring finger protein 31 (RNF31) Epidermal growth factor receptor (EGFR) Ribosomal protein S4, X-linked (RPS4X) PRELI domain containing 1 (PRELID1) Trafficking protein, kinesin binding 2 (TRAK2) Leucine carboxyl methyltransferase 1 (LCMT1) Ribosomal protein S6 kinase, 90 kDa, polypeptide 2 (RPS6KA2) Importin 9 (IPO9) Ring finger protein 187 (RNF187) AHNAK nucleoprotein (AHNAK) Dystroglycan 1 (DAG1) Ubiquitin-conjugating enzyme E2M (UBE2M) Mitochondrial ribosomal protein L46 (MRPL46) MAX dimerization protein 3 (MXD3) Activating transcription factor 7 (ATF7)
NM_033301.1 NM_001077523.1
CDS CDS
NM_006761.4
CDS
BC046123.2 NM_005620.1 BC040050.1 BC012077.1 NM_005228.3 BC000472.2 BC000007.2 AB038951.1 BC014217.2 NM_001006932.1
CDS CDS CDS CDS CDS CDS CDS 3′ UTR 3′ UTR 3′ UTR
NM_018085.4 BC012758.2 NM_001620.1 NM_004393.4 NM_003969.3 NM_022163.3 NM_001142935.1 NM_001130060.1
3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR 3′ UTR
Putative target mRNA
Accession number
Position of binding site
Nucleophosmin (nucleolar phosphoprotein B23, numatrin) (NPM1) Heat shock protein 90 kDa alpha, class A member 1 (HSP90AA1) Myristoylated alanine-rich protein kinase C substrate (MARCKS) RNA polymerase II, TATA box binding protein (TBP)-associated factor, 80 kDa (TAF6) Ribosomal protein L18 (RPL18) FERM and PDZ domain containing 4 (FRMPD4) Apoptosis enhancing nuclease (AEN) Dephospho-CoA kinase domain containing (DCAKD) NADH dehydrogenase Fe–S protein 7, 20 kDa (NDUFS7) Lysosomal-associated membrane protein 2 (LAMP2) RGD motif, leucine rich repeats, tropomodulin domain and proline-rich containing (RLTPR) Canopy FGF signaling regulator 4 (CNPY4) C-type lectin domain family 2, member D (CLEC2D) Lysophosphatidylcholine acyltransferase 1 (LPCAT1)
NM_002520.6
5′ UTR
NM_005348.3
5′ UTR
NM_002356.5
5′ UTR
NM_001190415.1
5′ UTR
NM_000979.2 NM_014728.3
CDS CDS
NM_022767.3 NM_024819.4
CDS CDS
NM_024407.4
CDS
NM_002294.2
CDS
NM_001013838.1
CDS
NM_152755.1 NM_013269.4
CDS 3′ UTR
NM_024830.3
3′ UTR
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Fig. 3. Hcmv-miR-US25-1-5p targets YWHAE, UBB, NPM1, and HSP90AA1 through predicted binding sites. (A) Schematic diagrams of predicted target sites of hcmv-miR-US25-1-5p in YWHAE, UBB, NPM1, and HSP90AA1. (B) Dual luciferase assay with cotransfection of the empty PMIR reporter vector, the reporter vectors containing the putative target sequences, or their mutants, along with miR-NC or hcmv-miR-US25-1-5p, in HEK293 cells. Data from three independent repetitions were used for statistical analysis. * indicates P b 0.05 compared with data from miRNA negative control transfected cells. (C) Western blot analysis of ectopically expressed YWHAE, UBB, and NPM1 or endogenous HSP90AA1 in HEK293 cells.
hcmv-miR-US25-1-5p has the ability to specifically repress these targets via the predicted binding sites. These four cellular genes identified in our study could encode proteins involved in diverse cellular processes such as metabolism, cell proliferation, apoptosis, cell cycle regulation, signal transduction, and protein chaperoning. The YWHAE protein is present in the cerebral spinal fluid (CSF) of those with cytomegalovirus or HIV infection, and the expression levels also correlated with the viral load of HIV-1 in the brain and CSF (Wakabayashi et al., 2001; Gelman and Nguyen, 2010). The UBB gene encodes ubiquitin, one of the most conserved proteins known. It is involved in the maintenance of chromatin structure as well as regulation of gene expression and the stress response (Conaway et al., 2002). Importantly, the intracellular ubiquitin level could affect the stability of the HIV-1 Rev protein, which plays a key role in virus replication (Vitte et al., 2006). NPM1 is a multifunctional chaperone which plays important roles in ribosome biogenesis and chromatin remodeling (Lindstrom, 2011). Interaction between NPM1 and HBV core protein was reported to increase HBV capsid assembly, while inhibition of NPM1 reduced intracellular capsid formation, resulting in a decrease in HBV production in HepG2.2.15 cells (Jeong et al., 2014). In addition, NPM1 was demonstrated to play important roles related to the establishment and maintenance of EBV latency in
B cells (Liu et al., 2012). HSP90, which aids in the folding of multiple client proteins, displays pleiotropic functions through its interaction with various cellular client proteins. It has been implicated in the replication of several different viruses, including HIV-1 (Anderson et al., 2014), HCV (Kim et al., 2012; Taguwa et al., 2009), HBV (Shim et al., 2011), EBV (Murata et al., 2013), KSHV (Qin et al., 2010), HVMV (Basha et al., 2005), HSV-1, and HSV-2 (Li et al., 2004, 2012). Furthermore, pharmacological inhibitors of HSP90 display broad antiviral effects. Geldanamycin, a potent and specific inhibitor of HSP90, was reported to inhibit gene expression and replication of human cytomegalovirus (Basha et al., 2005). A recent study demonstrated that HSP90 associates with the conserved protein kinase (CHPK) encoded by each of the 8 human herpesviruses, and treatment with the HSP90 inhibitor 17-DMAG decreases expression of these virally encoded kinases in cells transfected with expression vectors for each kinase. In addition, the expression of the EBV-encoded and HCMV-encoded kinases is markedly decreased in EBV-infected and HCMV-infected cells, respectively, when treated with 17-DMAG. Furthermore, 17-DMAG reduces production in Epstein–Barr virus-infected cells (Sun et al., 2013). Based on the studies above, we can speculate that all four of the cellular genes identified in our study may play important roles during virus infection, and may impact virus replication directly or indirectly.
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Fig. 4. Over-expression of hcmv-miR-US25-1-5p inhibits HCMV DNA replication by targeting YWHAE, UBB, NPM1, and HSP90AA1.(A) MRC-5 cells cotransfected with YWHAE, UBB, or NPM1 expression vectors with miRNA negative control (NC), hcmv-miR-US25-1-5p mimics (US25-1-5p), hcmv-miR-US25-1-5p inhibitor (Inhi), or specific siRNA against corresponding targets, were infected with HCMV at an MOI of 3, 24 h after transfection. Viral genome copy number was measured by real-time PCR at 48 hpi. Data from three independent repetitions were used for statistical analysis. * indicates P b 0.05 compared with data from miRNA negative control transfected cells. (B) Ectopically expressed YWHAE, UBB, and NPM1, or endogenous HSP90AA1 in cells treated as above were measured by Western blot.
Given the roles of these targets, and the fact that hcmv-miRUS25-1-5p could inhibit HCMV DNA replication by targeting the viral genes IE72 and pp65, it is of interest whether hcmv-miRUS25-1-5p could also affect viral replication by targeting these host cellular genes. Our results revealed that viral DNA genome copies, as well as protein levels of these four host target genes, were reduced in cells transfected with hcmv-miR-US25-1-5p compared with levels in cells transfected with a miRNA control. This reduction was eliminated when an hcmv-miR-US25-1-5p inhibitor was also used. In addition, the use of specific siRNAs against these targets ruled out possible off-target effects of hcmv-miR-US25-1-5p on HCMV DNA replication. This result indicated that hcmv-miR-US25-1-5pmediated down-regulation of these host cellular genes could inhibit virus replication during HCMV infection. However, more important biological functions of hcmv-miR-US25-1-5p during HCMV infection remain to be defined; hcmv-miR-US25-1-5p could play a wide range of roles by targeting a large number of host cellular genes.
In conclusion, our study showed that hcmv-miR-US25-1-5p could reduce virus replication by targeting multiple cellular transcripts that might impact virus replication directly or indirectly. These findings provide new insight into hcmv-miR-US25-1-5p. Further studies are required to investigate the biological function of hcmv-miRUS25-1-5p during HCMV infection. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.06.009. Competing interests The authors declare that they have no competing interests. Acknowledgments This work was supported by the National Natural Science Foundation of China (81171580 and 81371788) and the Specialized
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