Inhibition and escape of SARS-CoV treated with antisense morpholino oligomers

Identification of potential SARS-CoV antiviral compounds has progressed swiftly, thanks in part to the availability of bioinformatic and virus structural data. Antivirals that target the SARS-CoV superfamily 1 helicase and the 3C-related serine proteinase with low micromolar EC50 values have been reported. The papain-related cysteine proteinase may prove to be an unsuitable target, as a coronavirus molecular clone lacking one of the two known cleavage sites for this enzyme displayed only minor growth defects in cell culture. Other confirmed and putative viral enzymes including the polymerase, poly(U)-specific endo-ribonuclease homolog, S-adenosyl-methionine-dependent ribose 2’-O-methyltransferase, and cyclic phosphodiesterase represent plausible anti-SARS targets.Antivirals targeting the interaction of the viral spike protein with the ACE-2 receptor, or with the spike-mediated fusion event, and showing micromolar-scale efficiency in cell culture, have been reported. Several groups have also reported antiviral in vitroefficacy with siRNAs. The antisense agents directed against single-stranded RNA are known to act by two general mechanisms: by causing damage to an RNA strand containing the complementary “target” sequence through priming of endogenous RNase H activity, or by stably binding to and steric interference with targeted RNA function. Phosphorodiamidate morpholino oligomers (PMO) act by the latter mechanism, duplexing to specific RNA sequence by Watson-Crick base pairing and forming a steric block. The most frequently successful targeting strategies for PMO-based gene knockdown involve interfering with translation initiation or masking splice sites. We recently demonstrated antiviral effects in vitro for one peptide-conjugated PMO (P-PMO) complementary to the AUG translation start site region of a murine coronavirus replicase polyprotein. We reasoned that antiviral effects of P-PMO might be improved by choosing conserved RNA sequence elements and secondary structures critical for replication, transcription, and host factor interaction as targets. In this report, we demonstrate that antisense-mediated suppression of viral replication can be achieved by targeting conserved RNA elements required for viral RNA synthesis and translation.