Proteolytic cleavage of host proteins by the Group IV viral proteases of Venezuelan equine encephalitis virus and Zika virus

The alphaviral nonstructural protein 2 (nsP2) cysteine proteases (EC 3.4.22.-) are essential for the proteolytic processing of the nonstructural (ns) polyprotein and are validated drug targets. A common secondary role of these proteases is to antagonize the effects of interferon (IFN). After delineating the cleavage site motif of the Venezuelan equine encephalitis virus (VEEV) nsP2 cysteine protease, we searched the human genome to identify host protein substrates. Here we identify a new host substrate of the VEEV nsP2 protease, human TRIM14, a component of the mitochondria] antiviral-signaling protein (MAVS) signalosome. Short stretches of homologous host-pathogen protein sequences (SSHHPS) are present in the nonstructural polyprotein and TRIM14. A 25-residue cyan-yellow fluorescent protein TRIM14 substrate was cleaved in vitro by the VEEV nsP2 protease and the cleavage site was confirmed by tandem mass spectrometry. A TRIM14 cleavage product also was found in VEEV-infected cell lysates. At least ten other Group IV ( + )ssRNA viral proteases have been shown to cleave host proteins involved in generating the innate immune responses against viruses, suggesting that the integration of these short host protein sequences into the viral protease cleavage sites may represent an embedded mechanism of IFN antagonism. This interference mechanism shows several parallels with those of CRISPR/Cas9 and RNAi/RISC, but with a protease recognizing a protein sequence common to both the host and pathogen. The short host sequences embedded within the viral genome appear to be analogous to the short phage sequences found in a host's CRISPR spacer sequences. To test this algorithm, we applied it to another Group IV virus, Zika virus (ZIKV), and identified cleavage sites within human SFRP1 (secreted frizzled related protein 1), a retinal G, alpha subunit, NT5M, and Forkhead box protein G1 (FOXG1) in vitro. Proteolytic cleavage of these proteins suggests a possible link between the protease and the virus-induced phenotype of ZIKV. The algorithm may have value for selecting cell lines and animal models that recapitulate virus-induced phenotypes, predicting host-range and susceptibility, selecting oncolytic viruses, identifying biomarkers, and de-risking live virus vaccines. Inhibitors of the proteases that utilize this mechanism may both inhibit viral replication and alleviate suppression of the innate immune responses.