Interference of ribosomal frameshifting by antisense peptide nucleic acids suppresses SARS coronavirus replication

Abstract

The programmed −1 ribosomal frameshifting (−1 PRF) utilized by eukaryotic RNA viruses plays a crucial role for the controlled, limited synthesis of viral RNA replicase polyproteins required for genome replication. The viral RNA replicase polyproteins of severe acute respiratory syndrome coronavirus (SARS-CoV) are encoded by the two overlapping open reading frames 1a and 1b, which are connected by a −1 PRF signal. We evaluated the antiviral effects of antisense peptide nucleic acids (PNAs) targeting a highly conserved RNA sequence on the – PRF signal. The ribosomal frameshifting was inhibited by the PNA, which bound sequence-specifically a pseudoknot structure in the −1 PRF signal, in cell lines as assessed using a dual luciferase-based reporter plasmid containing the −1 PRF signal. Treatment of cells, which were transfected with a SARS-CoV-replicon expressing firefly luciferase, with the PNA fused to a cell-penetrating peptide (CPP) resulted in suppression of the replication of the SARS-CoV replicon, with a 50% inhibitory concentration of 4.4 μM. There was no induction of type I interferon responses by PNA treatment, suggesting that the effect of PNA is not due to innate immune responses. Our results demonstrate that −1 PRF, critical for SARS-CoV viral replication, can be inhibited by CPP-PNA, providing an effective antisense strategy for blocking −1 PRF signals.

miRNA Therapeutics: Delivery and Biological Activity of Peptide Nucleic Acids Targeting miRNAs

“…This issue is however far from being concluded. Ahn et al evaluated the antiviral effects of antisense PNAs targeting a highly conserved RNA sequence on the programmed-1 ribosomal frameshifting signal [122]. The ribosomal frameshifting was inhibited by the PNA without induction of type I interferon responses, suggesting that the effect of PNA is not due to innate immune responses [122].…”

The Antimicrobial and Antiviral Applications of Cell-Penetrating Peptides

“…Another interesting result was produced by Chaubey et al, who described that a Transportan-PNA molecule, which had previously been shown to display signifi cant antiviral effi ciency (14a), is also a potent virucidal agent, rendering HIV-1 virions pre-treated with the conjugate noninfectious, suggesting a probable prophylactic medical application for their molecule [ 129 ]. PNA-CPPs such as the Tat-FS can also be used to block the frameshift process required for the replication of such viruses as SARS-Coronavirus, making them a viable tool against any viruses that require frameshifting to take place during their replication [ 130 ]. A successful Tat-based application of PNAs has been shown to lead to Japanese encephalitis virus inhibition by competing with RdRp through interactions with its binding sites at 3′ regions of the RNA [ 131 ].…”

Ribosomal Frameshift Signals in Viral Genomes

“…This suggests that disruption of translation initiation is more effective than an RNAi target within the ORF at limiting viral propagation. Even so, PNAs targeting the pseudoknot caused a significant reduction in the replication of a SARS replicon at concentrations in the low μM range validating the selection of the pseudoknot as an antiviral target (Ahn et al, 2011).…”

“…The complexities of coronaviral replication are still being unravelled but it is apparent that www.intechopen.com Viral Genomes -Molecular Structure, Diversity, Gene Expression Mechanisms and Host-Virus Interactions 96 frameshifting is essential for the production of the proteins involved. Altering frameshifting efficiency has been shown to be detrimental for coronavirus replication (Ahn et al, 2011;McDonagh et al, 2011;Plant et al, 2010).…”

“…Nuclease mapping, NMR, crystallography and mass spectrometry has confirmed some of the 3' stimulatory structures but many remain unresolved at the atomic level due to difficulties purifying larger structures. Molecular modeling has been useful in elucidating some of these structures (Ahn et al, 2011). A list of stimulatory elements (with the exception of HIV which is discussed below) is provided in Table 1.…”