RESUMO
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 mitochondrial 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 Gs 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.
Assuntos
Cisteína Proteases/metabolismo , Vírus da Encefalite Equina Venezuelana/enzimologia , Proteínas Virais/metabolismo , Zika virus/enzimologia , 5'-Nucleotidase/metabolismo , Linhagem Celular , Inibidores de Cisteína Proteinase/farmacologia , Vírus da Encefalite Equina Venezuelana/patogenicidade , Encefalomielite Equina Venezuelana/virologia , Fatores de Transcrição Forkhead/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteólise , Replicação Viral/efeitos dos fármacos , Zika virus/patogenicidade , Infecção por Zika virus/virologiaRESUMO
The Venezuelan equine encephalitis virus (VEEV) nonstructural protein 2 (nsP2) cysteine protease (EC 3.4.22.-) is essential for viral replication and is involved in the cytopathic effects (CPE) of the virus. The VEEV nsP2 protease is a member of MEROPS Clan CN and characteristically contains a papain-like protease linked to an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. The protease contains an alternative active site motif, (475)NVCWAK(480), which differs from papain's (CGS(25)CWAFS), and the enzyme lacks a transition state-stabilizing residue homologous to Gln-19 in papain. To understand the roles of conserved residues in catalysis, we determined the structure of the free enzyme and the first structure of an inhibitor-bound alphaviral protease. The peptide-like E64d inhibitor was found to bind beneath a ß-hairpin at the interface of the SAM MTase and protease domains. His-546 adopted a conformation that differed from that found in the free enzyme; one or both of the conformers may assist in leaving group departure of either the amine or Cys thiolate during the catalytic cycle. Interestingly, E64c (200 µM), the carboxylic acid form of the E64d ester, did not inhibit the nsP2 protease. To identify key residues involved in substrate binding, a number of mutants were analyzed. Mutation of the motif residue, N475A, led to a 24-fold reduction in kcat/Km, and the conformation of this residue did not change after inhibition. N475 forms a hydrogen bond with R662 in the SAM MTase domain, and the R662A and R662K mutations both led to 16-fold decreases in kcat/Km. N475 forms the base of the P1 binding site and likely orients the substrate for nucleophilic attack or plays a role in product release. An Asn homologous to N475 is similarly found in coronaviral papain-like proteases (PLpro) of the Severe Acute Respiratory Syndrome (SARS) virus and Middle East Respiratory Syndrome (MERS) virus. Mutation of another motif residue, K480A, led to a 9-fold decrease in kcat and kcat/Km. K480 likely enhances the nucleophilicity of the Cys. Consistent with our substrate-bound models, the SAM MTase domain K706A mutation increased Km 4.5-fold to 500 µM. Within the ß-hairpin, the N545A mutation slightly but not significantly increased kcat and Km. The structures and identified active site residues may facilitate the discovery of protease inhibitors with antiviral activity.