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• W2164403493 abstract "Human (Homo sapiens) micro-RNAs (hsa-miRNAs) regulate virus and host-gene translation, but the biological impact in patients with human cytomegalovirus (hCMV) infection is not well defined in a clinically relevant model. First, we compared hsa-miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847 hsa-miRNAs. This approach demonstrated a set of 142 differentially expressed hsa-miRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast-1) infected with the hCMV Towne strain, identifying a subset of proviral and antiviral hsa-miRNAs. miRNA-target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV-UL52 and -UL100 [UL = unique long]) and host-genes (e.g., interleukin-1 receptor, IRF1). Luciferase-expressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa-miRNAs in 242 transplant recipients with hCMV-viremia. We measured hsa-miRNAs before and after antiviral therapy and correlated hsa-miRNA expression levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa-miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa-miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication. Human (Homo sapiens) micro-RNAs (hsa-miRNAs) regulate virus and host-gene translation, but the biological impact in patients with human cytomegalovirus (hCMV) infection is not well defined in a clinically relevant model. First, we compared hsa-miRNA expression profiles in peripheral blood mononuclear cells from 35 transplant recipients with and without CMV viremia by using a microarray chip covering 847 hsa-miRNAs. This approach demonstrated a set of 142 differentially expressed hsa-miRNAs. Next, we examined the effect of each of these miRNAs on viral growth by using human fibroblasts (human foreskin fibroblast-1) infected with the hCMV Towne strain, identifying a subset of proviral and antiviral hsa-miRNAs. miRNA-target prediction software indicated potential binding sites within the hCMV genome (e.g., hCMV-UL52 and -UL100 [UL = unique long]) and host-genes (e.g., interleukin-1 receptor, IRF1). Luciferase-expressing plasmid constructs and immunoblotting confirmed several predicted miRNA targets. Finally, we determined the expression of selected proviral and antiviral hsa-miRNAs in 242 transplant recipients with hCMV-viremia. We measured hsa-miRNAs before and after antiviral therapy and correlated hsa-miRNA expression levels to hCMV-replication dynamics. One of six antiviral hsa-miRNAs showed a significant increase during treatment, concurrent with viral decline. In contrast, six of eight proviral hsa-miRNAs showed a decrease during viral decline. Our results indicate that a complex and multitargeted hsa-miRNA response occurs during CMV replication in immunosuppressed patients. This study provides mechanistic insight and potential novel biomarkers for CMV replication. Patients undergoing transplantation are at high risk for cytomegalovirus (CMV) reactivation (1Humar A Snydman D. Cytomegalovirus in solid organ transplant recipients.Am J Transplant. 2009; 9: S78-S86Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,2Kotton CN Kumar D Caliendo AM International consensus guidelines on the management of cytomegalovirus in solid organ transplantation.Transplantation. 2010; 89 (et al): 779-795Crossref PubMed Scopus (498) Google Scholar). High-risk transplant recipients often receive prolonged antiviral prophylaxis, which is only partially effective in controlling CMV reactivation (3Humar A Lebranchu Y Vincenti F The efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients.Am J Transplant. 2010; 10 (et al): 1228-1237Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar,4Humar A Limaye AP Blumberg EA Extended valganciclovir prophylaxis in D+/R- kidney transplant recipients is associated with long-term reduction in cytomegalovirus disease: Two-year results of the IMPACT study.Transplantation. 2010; 90 (et al): 1427-1431Crossref PubMed Scopus (162) Google Scholar,5Humar A Mazzulli T Moussa G Clinical utility of cytomegalovirus (CMV) serology testing in high-risk CMV D+/R- transplant recipients.Am J Transplant. 2005; 5 (et al): 1065-1070Crossref PubMed Scopus (123) Google Scholar). A subset of patients will develop CMV replication with high viral loads and severe tissue-invasive disease (1Humar A Snydman D. Cytomegalovirus in solid organ transplant recipients.Am J Transplant. 2009; 9: S78-S86Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar,6Egli A Binggeli S Bodaghi S Cytomegalovirus and polyomavirus BK posttransplant.Nephrol Dial Transplant. 2007; 22 (et al): viii72-viii82PubMed Google Scholar). Aside from antiviral drugs, control of CMV replication involves a complex interplay between innate and adaptive immunity. CMV-specific T cells have been associated with control of CMV replication after transplantation (7Egli A Binet I Binggeli S Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients.J Transl Med. 2008; 6 (et al): 29Crossref PubMed Scopus (96) Google Scholar,8Egli A Humar A Kumar D. State-of-the-art monitoring of cytomegalovirus-specific cell-mediated immunity after organ transplant: a primer for the clinician.Clin Infect Dis. 2012; 55: 1678-1689Crossref PubMed Scopus (112) Google Scholar). The priming of a naïve immune response and the development of immunological memory with efficient CMV-specific T cells are significantly reduced after transplantation (6Egli A Binggeli S Bodaghi S Cytomegalovirus and polyomavirus BK posttransplant.Nephrol Dial Transplant. 2007; 22 (et al): viii72-viii82PubMed Google Scholar,7Egli A Binet I Binggeli S Cytomegalovirus-specific T-cell responses and viral replication in kidney transplant recipients.J Transl Med. 2008; 6 (et al): 29Crossref PubMed Scopus (96) Google Scholar,8Egli A Humar A Kumar D. State-of-the-art monitoring of cytomegalovirus-specific cell-mediated immunity after organ transplant: a primer for the clinician.Clin Infect Dis. 2012; 55: 1678-1689Crossref PubMed Scopus (112) Google Scholar,9Egli A Kumar D Broscheit C Comparison of the effect of standard and novel immunosuppressive drugs on CMV-specific T-cell cytokine profiling.Transplantation. 2013; 95 (et al): 448-455Crossref PubMed Scopus (34) Google Scholar,10Sester M Sester U Gartner B Levels of virus-specific CD4 T cells correlate with cytomegalovirus control and predict virus-induced disease after renal transplantation.Transplantation. 2001; 71 (et al): 1287-1294Crossref PubMed Scopus (211) Google Scholar,11Kumar D Chernenko S Moussa G Cell-mediated immunity to predict cytomegalovirus disease in high-risk solid organ transplant recipients.Am J Transplant. 2009; 9 (et al): 1214-1222Crossref PubMed Scopus (204) Google Scholar). Current clinical and virological biomarkers fail to adequately predict the risk for CMV replication or CMV disease. A more recently characterized regulatory layer that influences host–pathogen interaction includes micro-RNAs (miRNAs). Both viral and host-encoded miRNAs play an important role in the pathogenesis of viruses (12Lisboa LF Egli A O’Shea D hCMV-miR-UL22A-5p: A biomarker in transplantation with broad impact on host gene expression and potential immunological implications.Am J Transplant. 2015; 15 (et al): 1893-1902Abstract Full Text Full Text PDF Scopus (27) Google Scholar). The miRNAs are short RNA sequences, which can bind to complementary messenger RNA (mRNA). Traditionally, the miRNA-binding site is a 7- to 9-nucleotide-long sequence called the “seed” region, which specifically interacts with the 3’-untranslated region (3’UTR) of the mRNA (13Rehmsmeier M Steffen P Hochsmann M Giegerich R. Fast and effective prediction of microRNA/target duplexes.RNA. 2004; 10: 1507-1517Crossref PubMed Scopus (1850) Google Scholar). The binding interaction can result in either target repression or cleavage of the mRNA (14Ambros V. microRNAs: Tiny regulators with great potential.Cell. 2001; 107: 823-826Abstract Full Text Full Text PDF PubMed Scopus (1445) Google Scholar,15Cullen BR. RNA interference: Antiviral defense and genetic tool.Nat Immunol. 2002; 3: 597-599Crossref PubMed Scopus (112) Google Scholar,16Cullen BR. MicroRNAs as mediators of viral evasion of the immune system.Nat Immunol. 2013; 14: 205-210Crossref PubMed Scopus (188) Google Scholar). The impact of the human (Homo sapiens) miRNA (hsa-miRNA) response to CMV infection and its potential biological proviral or antiviral effects in patients with CMV replication has not been well defined in a clinically relevant model. The human genome encodes more than 1000 hsa-miRNAs that may regulate one-third of all human gene transcripts (17Chang TC Mendell JT. MicroRNAs in vertebrate physiology and human disease.Annu Rev Genomics Hum Genet. 2007; 8: 215-239Crossref PubMed Scopus (379) Google Scholar). hsa-miRNAs may serve various roles in non–virus-infected cells by suppressing interferon-stimulated gene expression, regulating apoptosis and proliferation, and maintaining homeostasis. In virus infection, miRNA downregulation may increase interferon-stimulated gene expression to control virus replication (18Seo GJ Kincaid RP Phanaksri T Reciprocal inhibition between intracellular antiviral signaling and the RNAi machinery in mammalian cells.Cell Host Microbe. 2013; 14 (et al): 435-445Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). During virus infection, hsa-miRNA results in antiviral or proviral effects in cell culture experiments (19Wen BP Dai HJ Yang YH MicroRNA-23b inhibits enterovirus 71 replication through downregulation of EV71 VPl protein.Intervirology. 2013; 56 (et al): 195-200Crossref PubMed Scopus (56) Google Scholar,20Zheng Z Ke X Wang M Human microRNA hsa-miR-296-5p suppresses enterovirus 71 replication by targeting the viral genome.J Virol. 2013; 87 (et al): 5645-5656Crossref PubMed Scopus (140) Google Scholar,21Song L Liu H Gao S Cellular microRNAs inhibit replication of the H1N1 influenza A virus in infected cells.J Virol. 2010; 84 (et al): 8849-8860Crossref PubMed Scopus (245) Google Scholar,22Terrier O Textoris J Carron C Host microRNA molecular signatures associated with human H1N1 and H3N2 influenza A viruses reveal an unanticipated antiviral activity for miR-146a.J Gen Virol. 2013; 94 (et al): 985-995Crossref PubMed Scopus (66) Google Scholar,23Fang J Hao Q Liu L Epigenetic changes mediated by microRNA miR29 activate cyclooxygenase 2 and lambda-1 interferon production during viral infection.J Virol. 2012; 86 (et al): 1010-1020Crossref PubMed Scopus (70) Google Scholar,24Kakumani PK Ponia SS Rajgokul KS Role of RNA interference (RNAi) in dengue virus replication and identification of NS4B as an RNAi suppressor.J Virol. 2013; 87 (et al): 8870-8883Crossref PubMed Scopus (155) Google Scholar,25Li Y Xie J Xu X MicroRNA-548 down-regulates host antiviral response via direct targeting of IFN-lambda1.Prot Cell. 2013; 4 (et al): 130-141Crossref PubMed Scopus (85) Google Scholar,26Hou J Wang P Lin L MicroRNA-146a feedback inhibits RIG-I-dependent type I IFN production in macrophages by targeting TRAF6, IRAK1, and IRAK2.J Immunol. 2009; 183 (et al): 2150-2158Crossref PubMed Scopus (622) Google Scholar,27Zhang X Daucher M Armistead D MicroRNA expression profiling in HCV-infected human hepatoma cells identifies potential anti-viral targets induced by interferon-alpha.PLoS One. 2013; 8 (et al): e55733Crossref PubMed Scopus (60) Google Scholar,28Li Y Fan X He X MicroRNA-466l inhibits antiviral innate immune response by targeting interferon-alpha.Cell Mol Immunol. 2012; 9 (et al): 497-502Crossref PubMed Scopus (47) Google Scholar,29Bakre A Mitchell P Coleman JK Respiratory syncytial virus modifies microRNAs regulating host genes that affect virus replication.J Gen Virol. 2012; 93 (et al): 2346-2356Crossref PubMed Scopus (75) Google Scholar,30Buggele WA Johnson KE Horvath CM. Influenza A virus infection of human respiratory cells induces primary microRNA expression.J Biol Chem. 2012; 287: 31027-31040Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar,31Zawislak CL Beaulieu AM Loeb GB Stage-specific regulation of natural killer cell homeostasis and response against viral infection by microRNA-155.Proc Natl Acad Sci U S A. 2013; 110 (et al): 6967-6972Crossref PubMed Scopus (90) Google Scholar). The objective of our study was to define the host miRNA response to CMV replication by using clinical datasets and bioinformatics. We hypothesized that (i) the host response to CMV replication likely includes a complex set of hsa-miRNAs that may have both antiviral and proviral effects; (ii) antiviral hsa-miRNAs may target viral genes, thereby inhibiting viral replication; and (iii) proviral miRNAs may target antiviral host-gene expression, thereby promoting viral replication. The first cohort consisted of solid organ transplant recipients (N = 35) with CMV viremia (n = 24) and controls without CMV viremia (n = 11). Table S1A shows patient characteristics. Peripheral blood mononuclear cells (PBMCs) were isolated, and hsa-miRNA expression was analyzed by using an miRNA microarray (see later). The sample taken at detection of viremia before the initiation of antiviral therapy was used. CMV viremia was quantified with the use of polymerase chain reaction (PCR) from plasma (32Lisboa LF Asberg A Kumar D The clinical utility of whole blood versus plasma cytomegalovirus viral load assays for monitoring therapeutic response.Transplantation. 2011; 91 (et al): 231-236Crossref PubMed Scopus (79) Google Scholar). The second cohort consisted of samples from solid organ transplant recipients (N = 242) enrolled in the VICTOR study (A Study of Valcyte (Valganciclovir PO) Compared to Ganciclovir IV in Patients With Cytomegalovirus [CMV] Disease Who Are Solid Organ Transplant Recipients) (33Asberg A Humar A Jardine AG Long-term outcomes of CMV disease treatment with valganciclovir versus IV ganciclovir in solid organ transplant recipients.Am J Transplant. 2009; 9 (et al): 1205-1213Crossref PubMed Scopus (143) Google Scholar). CMV blood viral loads were available at days 0, 3, 7, 14, and 21, after antiviral therapy, and during a 1-year follow-up period (34Humar A Kumar D Boivin G Caliendo AM. Cytomegalovirus (CMV) virus load kinetics to predict recurrent disease in solid-organ transplant patients with CMV disease.J Infect Dis. 2002; 186: 829-833Crossref PubMed Scopus (161) Google Scholar). Table S1B shows patient characteristics. All studies were approved by respective local institutional review board (University of Alberta and University of Oslo; S-04011 and 2010/3464), and all patients gave written informed consent. A detailed miRNA profile was determined by using microarray technology (Genechip miRNA 1.0, Affymetrix, Santa Clara, CA). The microarray is based on the miRbase v11 and contains 847 miRNAs. Detection sensitivity was 1 amol with a dynamic range of greater than 3 log10. Expression levels of miRNAs were determined by using GeneSpring software (Agilent Technologies, Santa Clara, CA), as shown here later. Single miRNAs were mixed with Lipofectamine RNAiMax (Invitrogen, Carlsbad, CA) and OptiMEM (Gibco, Grand Island, NY), and 30,000 cells were added to the transfection solution. Effects of miRNA were always compared with the controls. An apoptosis inducing small interfering RNA (AllStars Hs Cell Death, Qiagen, Hilden, Germany) was used as a positive transfection control. Negative controls were Lipofectamine without miRNA and a nontarget miRNA (HMC0002, Sigma-Aldrich, St. Louis, MO) at the same concentrations and conditions as specific miRNAs (25 nmol/L, all from Sigma-Aldrich). The effects of miRNA transfection on CMV replication capacity and protein expression were determined in human foreskin fibroblasts (HFF-1; SCRC1041, ATCC, Manassas, VA). All cells were grown in Dulbecco’s modified Eagle’s medium (with 10% heat-inactivated fetal calf serum, containing 2% NaHCO3, 1% N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid, and 1% l-glutamine). The laboratory-adapted CMV Towne strain was used for all assays (ATCC VR-977). At 72 h after transfection, we infected HFF-1 cells with CMV as described in the individual experiments (multiplicity of infection, 0.03). Cells and supernatants were harvested at days 4, 6, and 9 postinfection. Next, we used a standard 14-day plaque assay to determine CMV replication and individual growth curves for each miRNA in triplicate. We used a putative 3’UTR database covering all AD169 strain CMV genes (database provided by Dr. Finn Grey, University of Edinburgh, Edinburgh, UK). To decrease the likelihood of false-positive results, both RNAhybrid (13Rehmsmeier M Steffen P Hochsmann M Giegerich R. Fast and effective prediction of microRNA/target duplexes.RNA. 2004; 10: 1507-1517Crossref PubMed Scopus (1850) Google Scholar) and PITA (35Kertesz M Iovino N Unnerstall U The role of site accessibility in microRNA target recognition.Nat Genet. 2007; 39 (et al): 1278-1284Crossref PubMed Scopus (1900) Google Scholar) computer algorithms were independently used to predict target sequences. Only targets predicted at the same position by both algorithms were interpreted as potential gene targets. The “RNAhybrid” algorithm determines the minimum free energy between miRNA seed region and mRNA target site. A minimum free energy value of –25 kcal/mol or less was used. In contrast, the “PITA” algorithm calculates the difference between the free energy gained from the formation of the miRNA target duplex and the energetic cost of unpairing the target to make it accessible to the miRNA (ΔΔG score). The algorithm incorporates the role of target-site accessibility. A ΔΔG score of –10 or less, allowing for one G:U wobble or loop, was used. We screened the human genome by using a two-step approach. First, a screening using Targetscan (36Lewis BP Burge CB Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets.Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9882) Google Scholar) and myMIR (37Corrada D Viti F Merelli I myMIR: A genome-wide microRNA targets identification and annotation tool.Brief Bioinform. 2011; 12 (et al): 588-600Crossref PubMed Scopus (21) Google Scholar) was performed. The “Targetscan” algorithm predicts biological targets of miRNAs by searching for the presence of sites that match the miRNA seed region. It includes sequence conservation across species in the seed-matching process. We sorted the predictions considering the total context and overall score. The top predictions were further screened with a literature research in PubMed using the search terms “name of the predicted target gene” and “virus” and/or “immune response.” The “myMIR” algorithm combines several prediction softwares including Targetscan, PITA, and RNAhybrid. Again, the top 50 predicted gene targets using the same literature search algorithm as previously mentioned were included. Next, for genes predicted by either “Targetscan” or “myMIR,” the 3’UTR was determined using the University of California Santa Cruz genome browser (http://genome.ucsc.edu). Then, 3’UTR sequences were included in a more detailed prediction using a combination of RNAhybrid and PITA algorithms as just described here. pMIR target vector–based luciferase-expressing plasmids were used to confirm the miRNA–mRNA interaction. Luciferase was under an internal ribosome entry site promoter, and the predicted 3’UTR target sequence was attached to the luciferase mRNA coding sequence (all Blue Heron Biotechnology, Bothell, WA, Figure S1A). Binding of miRNA to the predicted mRNA resulted in reduced luciferase expression. At 24 h after miRNA transfection, we transfected the luciferase plasmid constructs at a final concentration of 100 ng/mL using EndoFectin (GeneCopoeia, Rockville, MD). After 24 h, the luciferase expression was measured using a development kit (britelite plus, PerkinElmer, Hopkinton, MA) with an ELISA reader (EnSpire Multimode Plate Reader, PerkinElmer). Baseline signal (mock-transfected cells) was subtracted from specific signals. The results were normalized to negative control miRNA (set as 100% luciferase expression). Every plasmid construct was also challenged by nonbinding miRNAs, thereby allowing unspecific controls. A high specificity of miRNA to predicted targets sites could be observed (data not shown). Proteins from miRNA-transfected fibroblasts were harvested by using RIPA lysis buffer (Santa Cruz Biotechnology, Santa Cruz, CA). Samples were loaded at 10 µg of protein/lane in standard loading buffer to a discontinuous (4.0%/7.5%) Laemmli/SDS-PAGE gel. Standard running conditions and wet transfer were used. Primary antibodies against virus targets: CMV-UL52 [where UL = unique long] (1:200; Dr. Stipan Jonjic, University of Tijeka, Croatia) and CMV IE-1 (1:1000; Millipore, Billerica, MA). Primary antihuman antibodies against host targets were interleukin 1 receptor subunit 1 (IL1R1), eukaryotic translation initiation factor 4E-binding protein 1 (4EBP1), interleukin-1 receptor-associated kinase (IRAK1), tuberous sclerosis 1 (TSC1), Facotr II receptor (FIIR), and Tumor necrosis factor receptor associated factor 6 (TRAF6) (all 1:1000). All protein expressions were normalized against glyceraldehyde-3-phosphate dehydrogenase or β-tubulin. CMV-UL52 was in addition normalized to IE-1 expression. Western blot experiments were repeated at least three times and summarized in bar graphs. The hsa-miRNA was extracted from 484 blood samples (242 patients each at two time points; day 0 and day 21) using the miRNeasy Mini Kit (Qiagen). Each sample was quantified and diluted to 2 ng/μL. From these 484 RNA samples, cDNA was synthesized with miRNA-specific primers using the TaqMan microRNA protocol (Applied Biosystems, Foster City, CA). Briefly, 10 μL of reverse transcription master mix and 5 µL of RNA (10 ng) were pipetted by using the PerkinElmer Janus Automated Workstation 96 with modular dispense technology. Reverse transcription was performed with a modular dispense technology thermal cycler. Quantitative PCR setup was performed as described in the TaqMan miRNA protocol (Applied Biosystems). Every quantitative PCR plate included a negative control and two positive controls (with the highest copy number standard). Each of the 14 miRNAs had its unique standard curve, ranging from 10 to 106 copies included in triplicate. These standards were highly purified RNA oligonucleotides (synthesized by Integrated DNA Technologies, IDT, Coralville, IA) of each miRNA. Statistical analyses were performed using SPSS (version 18.0, SPSS, Chicago, IL) and GraphPad Prism (version 4.0, GraphPad, La Jolla, CA). Categorical variables were evaluated using a chi-square test. Non-normally distributed data were evaluated with a Mann-Whitney U test. A two-tailed p value of <0.05 was considered significant. For repeated measures within the same subject, a Wilcoxon sign rank test was used. GeneSpring GX version 12 (Agilent Technologies Canada, Saint-Laurent, Quebec, Canada) was used for cluster and principal component analysis (PCA) of the miRNA data measured in microarray and quantitative PCR. Percentile shift was used as normalization algorithm, and baseline transformation was performed to the median value for all samples. Hierarchical clustering of conditions and miRNA was done using euclidean as similarity measure and centroid as linkage rule. Figure 1A summarizes the overall study layout. First, to identify a subset of biologically relevant miRNAs, we compared the differences in hsa-miRNA expression in PBMCs isolated from transplant recipients (n = 35) with or without CMV viremia (all samples in the viremic group were taken at the onset of viremia identification). Figure 1B shows a heat map that includes all hsa-miRNAs segregated by virus replication state. Microarray analysis showed that 142 of 847 hsa-miRNAs had significantly different expression patterns between the viremic and control groups (Table S2). Of 142, 131 were downregulated by a median of 3.33-fold (range, 2.01- to 8.97-fold). Examples of downregulated hsa-miRNAs were hsa-miRNA-301a (8.97-fold), -30e (7.75-fold), -326 (7.11-fold), -199a-5p (7.04-fold), and -31 (7.04-fold). Of 142, 11 were upregulated by a median of 3.48-fold (range, 2.09- to 19.13-fold). Examples of upregulated hsa-miRNAs were hsa-miRNA-1244 (19.13-fold), -1281 (6.60-fold), -1825 (5.35-fold), -940 (4.05-fold), and -1228 (3.62-fold). Figure 1C shows a PCA of patients with or without CMV replication based on the miRNA expression profile (Figure 1C). The PCA identified two clusters of miRNA expression between patients with and without CMV replication. Next, we determined which of the 142 differentially expressed hsa-miRNAs were associated with proviral or antiviral effects during CMV replication. We used HFF-1 cells as an infection model for the CMV Towne strain. HFF-1 cells were transfected with specific hsa-miRNAs and cell lysates were harvested. Plaque assays were then used to generate individual growth curves for each single transfected hsa-miRNA. Figure S1B provides an example for a proviral and antiviral effect on viral growth for two selected miRNAs. Figure 2A shows the full range of transfected hsa-miRNAs and their effect on CMV replication relative to a negative control miRNA (Figure 2). The most antiviral hsa-miRNAs were miRNA-324-5p, -185*, -29b, -1287, and -24-1, which ranged from 90% to 95% reduction in CMV replication compared with the negative control miRNA. The most proviral hsa-miRNAs were miRNA-1301, -328, -454, -181C, and -146b-5p. These increased CMV replication between 570% and 886%. In the initial discovery cohort, all of these miRNAs were found to be downregulated in the viremic patients compared with nonviremic controls. Table S3 shows the data for each of the 142 miRNAs and their respective effects on CMV replication at day 6 (Table S3). Antiviral effects were not due to cytotoxicity as indicated by cell morphology and salt metabolism 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromid (MTT) assay after transfection (data not shown). A putative 3’UTR database including all CMV genes of the AD169 strain was used to predict potential target sequences for six “antiviral” (i.e., inhibiting CMV replication) hsa-miRNAs. The 3’UTR database was provided by Dr. Finn Grey (University of Edinburgh). For prediction of targets, the PITA and RNAhybrid algorithms were used as indicated earlier (see Methods). The PITA algorithm predicted 494 potential targets within 127 CMV genes. The RNAhybrid algorithm predicted 556 potential targets within 130 CMV genes. By combining both algorithms the targets were reduced to 125 potential targets within 70 CMV genes (Figure S2A). Figure 3B shows an example of a typical interaction between the “antiviral” hsa-miRNA-185* and CMV-UL70 (Figure S2B). We mapped the binding regions within the CMV genome based on a published gene knockout map of CMV (Figure S2C and Figure S3, Table S4A) (38Dunn W Chou C Li H Functional profiling of a human cytomegalovirus genome.Proc Natl Acad Sci U S A. 2003; 100 (et al): 14223-14228Crossref PubMed Scopus (526) Google Scholar). As indicated, a high number of predicted targets lay within CMV genes essential for viral replication. For 11 “proviral” (i.e., increasing CMV replication) hsa-miRNAs, we hypothesized that targets may be present in regulatory host-genes, and an initial screening was performed with two prediction algorithms. Targetscan revealed 119 potential targets, and myMIR revealed 1823 potential targets (see Table S4B for all predicted human targets). We next screened the literature (MEDLINE search September 2012) for the predicted gene targets that were also associated with virus replication and/or immune response. The 3’UTR mRNA sequences of target genes associated with virus replication or immune response to viruses were further analyzed by using the PITA and RNA hybrid software (Figure S2D). A combination of both target prediction algorithms gave a list of 26 genes. Figure 3E shows an example of atypical interaction between hsa-miRNA-454 and TRAF6 (Figure S2E). Next, we performed a Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis including all miRNAs and predicted gene targets (Table S5A–C). Some miRNAs targeted genes that are directly involved in sensing of viral infection such as nuclear factor–κB signaling (p < 0.0001) or Toll-like receptor signaling pathways (p < 0.0001). To confirm the predicted target sequences of various hsa-miRNAs, we first used luciferase-expressing plasmid constructs. The 3’UTR of the luciferase gene contained the predicted target sequence flanked by 100 nucleotides, allowing the formation of secondary RNA structures (Figure S1A). Binding of a specific miRNA to the 3’UTR mRNA target sequence resulted in significantly reduced luciferase expression. All effects of hsa-miRNA were expressed relative to the negative control miRNA, baseline expression was subtracted, and all experiments were independently repeated at least five times in triplicate. Nonpredicted targets sequences and nonspecific miRNAs were used as additional controls (data not shown). Figure 4A shows selected luciferase expression after transfection with “antiviral” miRN" @default.
• W2164403493 created "2016-06-24" @default.
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• W2164403493 date "2016-02-01" @default.
• W2164403493 modified "2023-10-18" @default.
• W2164403493 title "Complexity of Host Micro-RNA Response to Cytomegalovirus Reactivation After Organ Transplantation" @default.
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• W2164403493 doi "https://doi.org/10.1111/ajt.13464" @default.
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