A Human Cytomegalovirus-Encoded microRNA Regulates Expression of Multiple Viral Genes Involved in Replication
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{"title"=>"A human cytomegalovirus-encoded microRNA regulates expression of multiple viral genes involved in replication", "type"=>"journal", "authors"=>[{"first_name"=>"Finn", "last_name"=>"Grey", "scopus_author_id"=>"8557365000"}, {"first_name"=>"Heather", "last_name"=>"Meyers", "scopus_author_id"=>"7004445319"}, {"first_name"=>"Elizabeth A.", "last_name"=>"White", "scopus_author_id"=>"8574443400"}, {"first_name"=>"Deborah H.", "last_name"=>"Spector", "scopus_author_id"=>"7102752494"}, {"first_name"=>"Jay", "last_name"=>"Nelson", "scopus_author_id"=>"35461231000"}], "year"=>2007, "source"=>"PLoS Pathogens", "identifiers"=>{"scopus"=>"2-s2.0-37349007653", "sgr"=>"37349007653", "issn"=>"15537366", "doi"=>"10.1371/journal.ppat.0030163", "pmid"=>"17983268", "isbn"=>"1553-7374 (Electronic)\\r1553-7366 (Linking)", "pui"=>"350291597"}, "id"=>"aabce3da-d362-395d-9094-cd83f1a78268", "abstract"=>"Although multiple studies have documented the expression of over 70 novel virus-encoded microRNAs (miRNAs), the targets and functions of most of these regulatory RNA species are unknown. In this study a comparative bioinformatics approach was employed to identify potential human cytomegalovirus (HCMV) mRNA targets of the virus-encoded miRNA miR-UL112-1. Bioinformatics analysis of the known HCMV mRNA 3' untranslated regions (UTRs) revealed 14 potential viral transcripts that were predicted to contain functional target sites for miR-UL112-1. The potential target sites were screened using luciferase reporters that contain the HCMV 3'UTRs in co-transfection assays with miR-UL112-1. Three of the 14 HCMV miRNA targets were validated, including the major immediate early gene encoding IE72 (UL123, IE1), UL112/113, and UL120/121. Further analysis of IE72 regulation by miR-UL112-1 with clones encoding the complete major immediate early region revealed that the IE72 3'UTR target site is necessary and sufficient to direct miR-UL112-1-specific inhibition of expression in transfected cells. In addition, miR-UL112-1 regulation is mediated through translational inhibition rather than RNA degradation. Premature expression of miR-UL112-1 during HCMV infection resulted in a significant decrease in genomic viral DNA levels, suggesting a functional role for miR-UL112-1 in regulating the expression of genes involved in viral replication. This study demonstrates the ability of a viral miRNA to regulate multiple viral genes.", "link"=>"http://www.mendeley.com/research/human-cytomegalovirusencoded-microrna-regulates-expression-multiple-viral-genes-involved-replication", "reader_count"=>82, "reader_count_by_academic_status"=>{"Unspecified"=>2, "Professor > Associate Professor"=>10, "Researcher"=>25, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>2, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>9, "Lecturer"=>2, "Professor"=>3}, "reader_count_by_user_role"=>{"Unspecified"=>2, "Professor > Associate Professor"=>10, "Researcher"=>25, "Student > Doctoral Student"=>3, "Student > Ph. D. Student"=>22, "Student > Postgraduate"=>2, "Student > Master"=>3, "Other"=>1, "Student > Bachelor"=>9, "Lecturer"=>2, "Professor"=>3}, "reader_count_by_subject_area"=>{"Unspecified"=>2, "Biochemistry, Genetics and Molecular Biology"=>7, "Agricultural and Biological Sciences"=>50, "Medicine and Dentistry"=>14, "Neuroscience"=>1, "Veterinary Science and Veterinary Medicine"=>2, "Computer Science"=>4, "Immunology and Microbiology"=>2}, "reader_count_by_subdiscipline"=>{"Medicine and Dentistry"=>{"Medicine and Dentistry"=>14}, "Neuroscience"=>{"Neuroscience"=>1}, "Immunology and Microbiology"=>{"Immunology and Microbiology"=>2}, "Agricultural and Biological Sciences"=>{"Agricultural and Biological Sciences"=>50}, "Computer Science"=>{"Computer Science"=>4}, "Biochemistry, Genetics and Molecular Biology"=>{"Biochemistry, Genetics and Molecular Biology"=>7}, "Unspecified"=>{"Unspecified"=>2}, "Veterinary Science and Veterinary Medicine"=>{"Veterinary Science and Veterinary Medicine"=>2}}, "reader_count_by_country"=>{"United States"=>6, "Brazil"=>1, "United Kingdom"=>1, "Italy"=>1, "Israel"=>1}, "group_count"=>4}

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  • {"files"=>["https://ndownloader.figshare.com/files/942942"], "description"=>"<div><p>(A) Luciferase screening of HCMV 3′UTR candidate sequences. Predicted target sequences were tested for their ability to inhibit expression of a luciferase reporter construct in the presence of miR-UL112-1. Either the entire 3′UTR (in the case of IE72, UL112/113 and UL120) or the predicted target sequence within 500 bases of flanking sequence were cloned downstream of the luciferase reporter gene and co-transfected with either a miR-UL112-1 expression plasmid or the same expression plasmid containing a random hairpin sequence as a negative control (pSIREN Neg). Results are shown as percentage expression of negative control sample following correction for transfection levels according to control renilla luciferase expression. Each transfection was carried out in duplicate.</p><p>(B) Predicted miR-UL112-1 binding to functional target sites determined using mfold algorithm [<a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030163#ppat-0030163-b060\" target=\"_blank\">60</a>]. Seed region indicated by box surrounding nucleotide 2–6 of miRNA/target.</p></div>", "links"=>[], "tags"=>["hcmv"], "article_id"=>613344, "categories"=>["Virology", "Molecular Biology"], "users"=>["Finn Grey", "Heather Meyers", "Elizabeth A White", "Deborah H Spector", "Jay Nelson"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.0030163.g002", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Luciferase_Screening_of_HCMV_3_8242_UTR_Candidate_Sequences_/613344", "title"=>"Luciferase Screening of HCMV 3′UTR Candidate Sequences", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2007-11-02 00:55:44"}
  • {"files"=>["https://ndownloader.figshare.com/files/943090"], "description"=>"<div><p>(A) 3′UTR sequence of IE72 indicating predicted target sequence shown in red and the underlined region deleted in pSVH-1 deletion.</p><p>(B) Luciferase construct containing either the IE72 3′UTR or the IE72 3′UTR deleted for miR-UL112-1 target sequence or luciferase construct containing the IE86 3′UTR were co-transfected with pSIREN miR-UL112-1 or the control pSIREN expressing miR-UL22A-1. Luciferase levels only inhibited in construct containing full IE72 3′UTR in the context of miR-UL112-1 expression.</p><p>(C) IE72 and IE86 protein levels determined following co-transfection with genomic plasmid, pSVH-1, containing IE72 and IE86 exons and miR-UL112-1 expression plasmid or miR-UL22A-1 expression plasmid. miR-UL112-1 significantly reduces IE72 expression levels but not IE86 levels. The pSVH-1 plasmid containing deletion mutation depicted in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030163#ppat-0030163-g004\" target=\"_blank\">Figure 4</a>A was unaffected by miR-UL112-1 expression.</p><p>(D) Densitometry analysis of western blot depicted in <a href=\"http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0030163#ppat-0030163-g004\" target=\"_blank\">Figure 4</a>C.</p><p>(E) IE72 and IE86 RNA levels were determined by RT-PCR following co-transfection of pSVH-1 or pSVH-1 deletion with pSIREN miR-UL112-1 using primers that span intronic regions. IE72 and IE86 RNA levels were unaffected despite inhibition of IE72 protein expression in pSVH-1 construct, indicating the mechanism is post-transcriptional. Reactions that did not include RT enzyme showed no detectable signal.</p><p>(F) Insertion of predicted target sequence into 3′UTR of IE86 directs miR-UL112-1-dependent inhibition, indicating the predicted sequence is functionally sufficient.</p></div>", "links"=>[], "tags"=>["inhibits", "ie72", "mrna"], "article_id"=>613492, "categories"=>["Virology", "Molecular Biology"], "users"=>["Finn Grey", "Heather Meyers", "Elizabeth A White", "Deborah H Spector", "Jay Nelson"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.0030163.g004", "stats"=>{"downloads"=>0, "page_views"=>5, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_miR_UL112_1_Specifically_Inhibits_IE72_Protein_Expression_but_Not_mRNA_Transcription_/613492", "title"=>"miR-UL112-1 Specifically Inhibits IE72 Protein Expression but Not mRNA Transcription", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2007-11-02 00:58:12"}
  • {"files"=>["https://ndownloader.figshare.com/files/943024"], "description"=>"<p>The MIE region showing IE72 and IE86 transcripts resulting from alternative splicing are shown as well as splicing to downstream exons UL120/UL121. Position of miR-UL112-1 target sites in 3′UTR region of IE72 and UL120/UL121 indicated by red boxes.</p>", "links"=>[], "tags"=>["mir-ul112-1", "targets", "sites", "mie"], "article_id"=>613424, "categories"=>["Virology", "Molecular Biology"], "users"=>["Finn Grey", "Heather Meyers", "Elizabeth A White", "Deborah H Spector", "Jay Nelson"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.0030163.g003", "stats"=>{"downloads"=>0, "page_views"=>28, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Genomic_Position_of_miR_UL112_1_Targets_Sites_within_the_MIE_Region_/613424", "title"=>"Genomic Position of miR-UL112-1 Targets Sites within the MIE Region", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2007-11-02 00:57:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/464721", "https://ndownloader.figshare.com/files/464803", "https://ndownloader.figshare.com/files/464908", "https://ndownloader.figshare.com/files/465012", "https://ndownloader.figshare.com/files/465146", "https://ndownloader.figshare.com/files/465197"], "description"=>"<div><p>Although multiple studies have documented the expression of over 70 novel virus-encoded microRNAs (miRNAs), the targets and functions of most of these regulatory RNA species are unknown. In this study a comparative bioinformatics approach was employed to identify potential human cytomegalovirus (HCMV) mRNA targets of the virus-encoded miRNA miR-UL112-1. Bioinformatics analysis of the known HCMV mRNA 3′ untranslated regions (UTRs) revealed 14 potential viral transcripts that were predicted to contain functional target sites for miR-UL112-1. The potential target sites were screened using luciferase reporters that contain the HCMV 3′UTRs in co-transfection assays with miR-UL112-1. Three of the 14 HCMV miRNA targets were validated, including the major immediate early gene encoding IE72 (UL123, IE1), UL112/113, and UL120/121. Further analysis of IE72 regulation by miR-UL112-1 with clones encoding the complete major immediate early region revealed that the IE72 3′UTR target site is necessary and sufficient to direct miR-UL112-1-specific inhibition of expression in transfected cells. In addition, miR-UL112-1 regulation is mediated through translational inhibition rather than RNA degradation. Premature expression of miR-UL112-1 during HCMV infection resulted in a significant decrease in genomic viral DNA levels, suggesting a functional role for miR-UL112-1 in regulating the expression of genes involved in viral replication. This study demonstrates the ability of a viral miRNA to regulate multiple viral genes.</p></div>", "links"=>[], "tags"=>["cytomegalovirus-encoded", "microrna", "regulates", "viral", "genes", "replication"], "article_id"=>151598, "categories"=>["Molecular Biology", "Cancer"], "users"=>["Finn Grey", "Heather Meyers", "Elizabeth A White", "Deborah H Spector", "Jay Nelson"], "doi"=>["https://dx.doi.org/10.1371/journal.ppat.0030163.sg001", "https://dx.doi.org/10.1371/journal.ppat.0030163.sg002", "https://dx.doi.org/10.1371/journal.ppat.0030163.sg003", "https://dx.doi.org/10.1371/journal.ppat.0030163.sg004", "https://dx.doi.org/10.1371/journal.ppat.0030163.st001", "https://dx.doi.org/10.1371/journal.ppat.0030163.st002"], "stats"=>{"downloads"=>19, "page_views"=>23, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_Human_Cytomegalovirus_Encoded_microRNA_Regulates_Expression_of_Multiple_Viral_Genes_Involved_in_Replication/151598", "title"=>"A Human Cytomegalovirus-Encoded microRNA Regulates Expression of Multiple Viral Genes Involved in Replication", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2007-11-02 00:26:38"}
  • {"files"=>["https://ndownloader.figshare.com/files/942872"], "description"=>"<p>3′UTR sequences were determined as the 3′ end of each annotated ORF to the first AATAAA polyadenylation site. Following analysis using RNAhybrid, 14 candidate 3′UTRs contained target sites for miR-UL112-1 were identified within corresponding HCMV and CCMV 3′UTR sequences.</p>", "links"=>[], "tags"=>["mir-ul112-1", "targets", "hcmv"], "article_id"=>613272, "categories"=>["Virology", "Molecular Biology"], "users"=>["Finn Grey", "Heather Meyers", "Elizabeth A White", "Deborah H Spector", "Jay Nelson"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.0030163.g001", "stats"=>{"downloads"=>0, "page_views"=>1, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Bioinformatic_Strategy_for_the_Identification_of_miR_UL112_1_Targets_in_HCMV_Genome_/613272", "title"=>"Bioinformatic Strategy for the Identification of miR-UL112-1 Targets in HCMV Genome", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2007-11-02 00:54:32"}
  • {"files"=>["https://ndownloader.figshare.com/files/943197"], "description"=>"<div><p>U373 cells were transfected with either synthetic miR-UL112-1 or a random sequence negative pre-miR RNA followed by infection with AD169gfp at a multiplicity of 0.5 plaque-forming units per cell.</p><p>(A) Total protein was harvested and analyzed by western blot using an antibody that cross reacts with MIE genes. miR-UL112-1 transfection resulted in significant reduction in immediate early gene expression.</p><p>(B) Levels of gfp were determined by microscopy at 24 and 48 h post infection, demonstrating a reduction in gfp fluorescence in cells transfected with miR-UL112-1 at later time points.</p><p>(C) DNA was isolated from infected cells at the indicated time points and viral DNA levels were determined by real-time PCR and corrected against beta-actin copies. Transfection of miR-UL112-1 (syn miR-UL112-1) results in consistently lower levels of HCMV DNA at later time points compared to cells transfected with randomized control miRNA (syn negative).</p><p>(D) DNA replication of HSV-1 is unaffected by miR-UL112-1 expression. U373 Cells were transfected with either miR-UL112-1 or the negative control RNA and infected with HSV at a multiplicity of 0.1. DNA levels were determined by PCR and corrected against beta-actin copies.</p></div>", "links"=>[], "tags"=>["inhibits", "viral", "dna"], "article_id"=>613599, "categories"=>["Virology", "Molecular Biology"], "users"=>["Finn Grey", "Heather Meyers", "Elizabeth A White", "Deborah H Spector", "Jay Nelson"], "doi"=>"https://dx.doi.org/10.1371/journal.ppat.0030163.g005", "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_miR_UL112_1_Inhibits_Immediate_Early_Gene_Expression_and_Viral_DNA_Replication_/613599", "title"=>"miR-UL112-1 Inhibits Immediate Early Gene Expression and Viral DNA Replication", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2007-11-02 00:59:59"}

PMC Usage Stats | Further Information

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  • {"unique-ip"=>"13", "full-text"=>"11", "pdf"=>"5", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2014", "month"=>"12"}
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  • {"unique-ip"=>"29", "full-text"=>"20", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"8", "cited-by"=>"1", "year"=>"2015", "month"=>"10"}
  • {"unique-ip"=>"11", "full-text"=>"12", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"4", "year"=>"2015", "month"=>"11"}
  • {"unique-ip"=>"11", "full-text"=>"5", "pdf"=>"6", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"3", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2015", "month"=>"12"}
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  • {"unique-ip"=>"7", "full-text"=>"11", "pdf"=>"3", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"7"}
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  • {"unique-ip"=>"7", "full-text"=>"34", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"9"}
  • {"unique-ip"=>"7", "full-text"=>"50", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"2", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2017", "month"=>"10"}
  • {"unique-ip"=>"13", "full-text"=>"45", "pdf"=>"4", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"8", "cited-by"=>"0", "year"=>"2017", "month"=>"11"}
  • {"unique-ip"=>"3", "full-text"=>"28", "pdf"=>"1", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"5", "cited-by"=>"0", "year"=>"2017", "month"=>"12"}
  • {"unique-ip"=>"7", "full-text"=>"23", "pdf"=>"2", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"1", "supp-data"=>"1", "cited-by"=>"0", "year"=>"2018", "month"=>"1"}
  • {"unique-ip"=>"1", "full-text"=>"1", "pdf"=>"0", "abstract"=>"0", "scanned-summary"=>"0", "scanned-page-browse"=>"0", "figure"=>"0", "supp-data"=>"0", "cited-by"=>"0", "year"=>"2018", "month"=>"2"}
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Relative Metric

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