Denise Werk

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

Abstract Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80–90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.

Application of small interfering RNAs modified by unlocked nucleic acid (UNA) to inhibit the heart‐pathogenic coxsackievirus B3

This study describes the first application of unlocked nucleic acid (UNA)‐modified small interfering RNAs (siRNAs) directed against a medically relevant target, the coxsackievirus B3. We systematically analyzed the impact of different siRNA modification patterns and observed good compatibility of the introduction of UNA with the maintenance of high antiviral activity. Additionally, the polarity of an siRNA was successfully reversed by modulating the relative stability of the termini with locked nucleic acid (LNA) and UNA as shown in a reporter assay. The potency of the reversed siRNA against the full‐length target was, however, too low to inhibit the infectious virus. Altogether, combined modification of siRNAs with LNA und UNA provides a promising approach to alter and improve properties of an siRNA.

Combination of soluble coxsackievirus-adenovirus receptor and anti-coxsackievirus siRNAs exerts synergistic antiviral activity against coxsackievirus B3

Coxsackievirus B3 (CVB-3) is a major causative agent of chronic heart muscle infections. The present study describes a cell culture system with an ongoing virus infection to evaluate two novel inhibitory strategies, either individually or combined: (1) RNA interference (RNAi) to degrade cytoplasmatic CVB-3 RNA and (2) a vector-delivered soluble variant of the coxsackievirus-adenovirus receptor fused to a human immunoglobulin (sCAR-Fc), which inhibits cellular uptake of CVB-3. Both approaches were capable of inhibiting CVB-3 in persistently infected human myocardial fibroblasts. The antiviral effect of a single treatment lasted for up to one week and could be extended by repeated applications. Each of the single treatments initially reduced the virus titer by approximately 1-log, whereas the combination of both approaches resulted in 4-log inhibition and retained substantial antiviral activity at later time points, when the effect of sCAR-Fc or siRNAs alone had already disappeared. Further analysis revealed that sCAR-Fc protects cells from virus-induced lysis but does not diminish the virus load. Reduction of the virus titer was only achieved with additional destruction of viral RNA by RNAi. Taken together, combination of RNAi and a protein-based antiviral strategy was found to result in a strong synergistic inhibition of an ongoing virus infection.

Antiviral activity of highly potent siRNAs against echovirus 30 and its receptor

RNA interference (RNAi) has been shown to be suitable to inhibit viruses in experimental setups and is considered a promising antiviral strategy that is currently being tested in various clinical trials. The present study provides an approach to design siRNAs with high potency against a virus-specific target gene. In recent years, several outbreaks of aseptic meningitis caused by an echovirus 30 (EV-30) infection have been described. Based on an initial set of 30 in silico designed siRNAs, six siRNAs targeting the 3D RNA-dependent RNA-Polymerase (3D(Pol)) of EV-30 were selected. All but one of them showed high efficiency in both, reporter and virus assays. A second aim of the study was to re-investigate the relevance of the decay-accelerating factor (DAF, also known as CD55) as cellular entry receptor of EV-30 by means of RNAi, a question which had been under debate in previous studies. Knockdown of DAF inhibited drastically infection by EV-30 indicating that DAF plays an important role either as an attachment factor or as a receptor.

Inhibition of coxsackievirus B3 by RNA interference (RNAi)

Here, we demonstrate the potential of RNAi to inhibit a picornavirus, coxsackievirus B3 (CVB3), a major causes of myocardial diseases. Targeting the 3D RNA dependent RNA polymerase (RdRP) with virus specific small interfering RNAs (siRNAs) resulted in a reduction of the virus titre by up to 90%. A detailed analysis of the mechanism of virus silencing with strand-specific siRNAs revealed that only the viral plus-strand can be efficiently targeted in an RNAi approach, whereas the minus-strand is resistant to siRNA-mediated silencing. CVB3 is known to have a high error-rate during replication and can thus be expected to escape RNAi-silencing upon prolonged treatment. We have therefore developed an siRNA double expression vector (SiDEx), which generates two independent siRNAs simultaneously. This vector maintained its silencing capacity against the target RNA with an artificially introduced mutation in a reporter assay, whereas single siRNA-expression vectors lost their capacity to silence their respective target after substitution of a base in the centre of the target site. We will present a recently developed method to drastically facilitate and accelerate the generation of SiDEx vectors. Delivery of the siRNA double expression cassette into neonatal rat cardiomyocytes by an adenovirus-associated virus vector resulted in up to 370 fold reduction of the CVB3 titre. As an alternative approach to cope with the problem of viral escape, we silenced the coxsackievirus-adenovirus receptor on the host cell and achieved a significant reduction of CVB3 propagation.