Differential inhibition of intra- and inter-molecular protease cleavages by antiviral compounds

Enteroviruses encode two protease active sites, in the 2A and 3C coding regions. While they target many host proteins, they first need to be excised from the viral polyprotein in which they are embedded. Polyprotein cleavage can occur either intra-molecularly (in cis) or inter-molecularly (in trans). Previous work suggested that antivirals targeting intra-molecular cleavages could generate inhibitory precursors that can suppress the outgrowth of drug-resistant variants. Therefore, we wanted to evaluate enteroviral cleavage patterns to identify such obligate intra-molecular cleavages as a method of drug target selection. Using translation extracts, we show that cis cleavage of the 2A protease N-terminal junction is conserved across three enterovirus clades, while the mechanism for the N-terminal junction of 3C varies, with enterovirus D68 3C cleavage occurring in cis and poliovirus 3C cleavage occurring in trans. Antiviral agents targeting proteases are generally identified via their ability to block the cleavage of artificial peptide substrates. Here, we show that antivirals identified for their ability to block inter-molecular cleavage can sometimes block intra-molecular cleavage of the protease from its polyprotein but with widely varying efficacy. Additionally, we demonstrate that for three enteroviral species, genomes defective in 2A protease activity suppress the growth of wild-type virus in mixed populations, supporting the hypothesis that preventing intra-molecular cleavage at the VP1 center dot 2A junction can create dominantly inhibitory precursors. These data argue that, to reduce the likelihood of drug resistance, protease-targeted antivirals should be evaluated for their ability to block intra-molecular polyprotein cleavages in addition to inter-molecular cleavage of other substrates.IMPORTANCEMost protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants. Most protease-targeted antiviral development evaluates the ability of small molecules to inhibit the cleavage of artificial substrates. However, before they can cleave any other substrates, viral proteases need to cleave themselves out of the viral polyprotein in which they have been translated. This can occur either intra- or inter-molecularly. Whether this process occurs intra- or inter-molecularly has implications for the potential for precursors to accumulate and for the effectiveness of antiviral drugs. We argue that evaluating candidate antivirals for their ability to block these cleavages is vital to drug development because the buildup of uncleaved precursors can be inhibitory to the virus and potentially suppress the selection of drug-resistant variants.

Automated summary

Enteroviruses encode protease active sites that can cleave viral polyprotein either intra- or inter-molecularly. This study evaluates the cleavage patterns and effects of antiviral agents on enteroviral cleavage. The results show that targeting intra-molecular cleavage can inhibit drug-resistant variants and that the efficacy of antiviral drugs varies in blocking intra-molecular cleavage. Additionally, defective 2A protease activity can suppress the growth of wild-type virus in enteroviral species.