501. Argonaute-2 enhances suppression of human cytomegalovirus replication by polycistronic short hairpin RNAs targeting UL46, UL70 and UL122.
- Author
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Shao PL, Lu MY, Liau YJ, Chao MF, Chang LY, Lu CY, Kao CL, Chang SY, Chi YH, and Huang LM
- Subjects
- Argonaute Proteins metabolism, Cell Line, Cytomegalovirus drug effects, Cytomegalovirus growth & development, Cytomegalovirus Infections genetics, Fibroblasts metabolism, Genes physiology, Genetic Vectors, Humans, Immediate-Early Proteins genetics, Immediate-Early Proteins metabolism, MicroRNAs analysis, MicroRNAs genetics, MicroRNAs isolation & purification, Plasmids, RNA, Small Interfering analysis, RNA, Small Interfering genetics, RNA, Small Interfering isolation & purification, Trans-Activators genetics, Trans-Activators metabolism, Viral Proteins genetics, Viral Proteins metabolism, Virus Replication genetics, Argonaute Proteins genetics, Cytomegalovirus genetics, Cytomegalovirus Infections drug therapy, MicroRNAs metabolism, Molecular Targeted Therapy, RNA, Small Interfering metabolism, Virus Replication drug effects
- 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.
- Published
- 2011
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