Inhibition of coxsackievirus B3 and related enteroviruses by antiviral short interfering RNA pools produced using φ6 RNA-dependent RNA polymerase

Abstract

Coxsackievirus B3 (CBV3) is a member of the human enterovirus B species and a common human pathogen. Even though much is known about the enteroviral life cycle, no specific drugs are available to treat enterovirus infections. RNA interference (RNAi) has evolved to be an important tool for antiviral experimental therapies and gene function studies. We describe here a novel approach for RNAi against CBVs by using a short interfering (siRNA) pool covering 3.5 kb of CBV3 genomic sequence. The RNA-dependent RNA polymerase (RdRP) of bacteriophage w6 was used to synthesize long double-stranded RNA (dsRNA) from a cloned region (nt 3837-7399) of the CBV3 genome. The dsRNA was cleaved using Dicer, purified and introduced to cells by transfection. The siRNA pool synthesized using the w6 RdRP (w6-siRNAs) was considerably more effective than single-site siRNAs. The w6-siRNA pool also inhibited replication of other enterovirus B species, such as coxsackievirus B4 and coxsackievirus A9.

Enzymatically Produced Pools of Canonical and Dicer-Substrate siRNA Molecules Display Comparable Gene Silencing and Antiviral Activities against Herpes Simplex Virus

“…Fractions of 0.5 ml were collected and those containing siRNA molecules were combined, desalted with NAP columns, and concentrated using a SpeedVac concentrator (Thermo Savant). Commercial single-site UL29S siRNA (21-nt) [29] and UL29L DsiRNA (27-nt, Table S1) were purchased from Dharmacon and the eGFPS and GAPDH siRNAs were from Qiagen [13]. The single-site siRNAs did not harbor any modifications.…”

“…In the course of the enzymatic reaction it is possible to synthesize both siRNA [8], [11] and long dsRNA molecules [7], [9]. The latter can be subsequently cleaved in vitro by Dicer enzyme generating a pool of target-specific siRNAs representing sequences along the entire region of interest [7], [9], [12], [13]. Although chemical synthesis of single-site siRNAs is the main approach in current RNAi applications, the pools may better maintain their silencing potency in long term usage, in particular when applied to combat viral infections, by reducing the probability of functional escape mutants [14]–[16].…”

New Trends in the Development of Treatments of Viral Myocarditis

“…Thus, in recent years, the discovery of the occurrence of escape mutants due to siR-NA treatment of HCV, poliovirus and HIV infections [76][77][78] greatly encouraged re-searchers to search for new approaches to counteract drug resistance. One direction is the application of multiple distinct siRNAs or a siRNA pool to target more than one target genes of the virus [79,80]. The other direction is the identification of conserved cisacting replication elements (CRE) [81].…”

Inhibition of Coxsackievirus A24 in permissive Hep2 cells by small interfering RNA (siRNA)

“…Attempts were made to develop anti-CA24 therapeutics, still, no effective medication is available to treat CA24 infections. RNAi tool found very effective against a wide range of viruses both in vitro and in vivo, including Coxsackievirus groups, Dengue virus, Ebola virus, Human Immunodeficiency virus, Hepatitis C virus [8,9,10,11,12]. CA24 is a non-enveloped, positive sense single-stranded RNA virus of ∼7.5 kb in length.…”