title: Argonaute-2 enhances suppression of human cytomegalovirus replication by polycistronic short hairpin RNAs targeting UL46, UL70 and UL122.
abstract: BACKGROUND
  Human cytomegalovirus (HCMV) is a common human pathogen that causes significant morbidity and mortality. The efficacy of anti-HCMV drugs such as ganciclovir, foscarnet and cidofovir is limited because of drug toxicities and frequent development of resistance. Here, we report an alternative anti-HCMV method using RNA silencing.
  
  
  METHODS
  Combinatorial use of second-generation short hairpin RNAs (shRNA-miRs) targeting various transcripts of HCMV and an RNA-silencing endonuclease Argonaute-2 (Ago2) expression vector were applied to inhibit replication of HCMV AD169. Normal human fetal lung MRC-5 fibroblasts were transfected with pSM30-shRNA-miRs harbouring single or multiple shRNA-miR cassettes with or without Ago2 and then infected with HCMV AD169. Production of small interfering RNA (siRNA) was quantified by reverse transcription PCR. Virus secretion was evaluated by plaque reduction assays.
  
  
  RESULTS
  The use of shRNA-miRs targeting a single HCMV gene suppressed HCMV AD169 viral titres by 50-70%. Polycistronic shRNA-miRs targeting UL46+UL122 and UL70+UL46+UL122 reached nearly 80% of inhibition. Coexpression of Ago2 with shRNA-miRs targeting UL46+UL122 and UL70+UL46+UL122 achieved a 95% reduction in viral maturation.
  
  
  CONCLUSIONS
  Coexpression of Ago2 with shRNA-miRs enhanced the production of mature siRNAs and increased the efficiency of RNA silencing in the suppression of HCMV replication. This strategy may be universally applied to RNA interference-based therapies.
authors.affiliation: Department of Pediatrics, National Taiwan University Hospital, National Taiwan University College of Medicine, Taipei, Taiwan.
authors.forename: Pei-Lan
authors.initials: PL
authors.lastname: Shao
authors.forename: Meng-You
authors.initials: MY
authors.lastname: Lu
authors.forename: Yi-Jen
authors.initials: YJ
authors.lastname: Liau
authors.forename: Miau-Fen
authors.initials: MF
authors.lastname: Chao
authors.forename: Luan-Yin
authors.initials: LY
authors.lastname: Chang
authors.forename: Chun-Yi
authors.initials: CY
authors.lastname: Lu
authors.forename: Chuan-Liang
authors.initials: CL
authors.lastname: Kao
authors.forename: Shu-Yuan
authors.initials: SY
authors.lastname: Chang
authors.forename: Ya-Hui
authors.initials: YH
authors.lastname: Chi
authors.forename: Li-Min
authors.initials: LM
authors.lastname: Huang
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D003587:Cytomegalovirus / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development; D003586:Cytomegalovirus Infections / Q000188:drug therapy* / Q000235:genetics; D005347:Fibroblasts / Q000378:metabolism; D005796:Genes / Q000502:physiology; D005822:Genetic Vectors; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D058990:Molecular Targeted Therapy; D010957:Plasmids; D034741:RNA, Small Interfering / Q000032:analysis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication / Q000187:drug effects* / Q000235:genetics
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; C069186:IE2 protein, Cytomegalovirus; D017874:Immediate-Early Proteins; D035683:MicroRNAs; D034741:RNA, Small Interfering; D015534:Trans-Activators; C446319:UL47 protein, cytomegalovirus; D014764:Viral Proteins