Argonaute-2 enhances suppression of human cytomegalovirus replication by polycistronic short hairpin RNAs targeting UL46, UL70 and UL122
Journal
Antiviral Therapy
Journal Volume
16
Journal Issue
5
Pages
741-749
Date Issued
2011
Author(s)
Lu M.-Y.
Liau Y.-J.
Chao M.-F.
Chi Y.-H.
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-shRNAmiRs 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. ?2011 International Medical Press.
SDGs
Other Subjects
argonaute 2 protein; plasmid vector; short hairpin RNA; small interfering RNA; virus enzyme; antiviral therapy; article; drug targeting; gene silencing; genetic transfection; human; human cell; Human cytomegalovirus; lung fibroblast; nucleotide sequence; priority journal; protein expression; protein function; protein targeting; RNA interference; RNA synthesis; UL122 gene; UL46 gene; UL70 gene; virus capsid; virus gene; virus inhibition; virus plaque; virus release; virus replication; Argonaute Proteins; Cell Line; Cytomegalovirus; Cytomegalovirus Infections; Fibroblasts; Genes; Genetic Vectors; Humans; Immediate-Early Proteins; MicroRNAs; Molecular Targeted Therapy; Plasmids; RNA, Small Interfering; Trans-Activators; Viral Proteins; Virus Replication
Type
journal article