The leader proteinase (Lpro) of the foot-and-mouth disease virus inhibits the host innate immune response by at least three different mechanisms. The most well-characterised of these is the prevention of the synthesis of cytokines such as interferons immediately after infection, brought about by specific proteolytic cleavage of the eukaryotic initiation factor 4G. This prevents the recruitment of capped cellular mRNA; however, the viral RNA can be translated under these conditions. The two other mechanisms are the induction of NF-κB cleavage and the deubiquitination of immune signalling molecules. This review focuses on the structure-function relationships in Lpro responsible for these widely divergent activities.
Translation directed by several picornavirus IRES elements can usually take place after cleavage of eIF4G by picornavirus proteases 2Apro or Lpro. The hepatitis A virus (HAV) IRES is thought to be an exception to this rule because it requires intact eIF4F complex for translation. In line with previous results we report that poliovirus (PV) 2Apro strongly blocks protein synthesis directed by HAV IRES. However, in contrast to previous findings we now demonstrate that eIF4G cleavage by foot-and-mouth disease virus (FMDV) Lpro strongly stimulates HAV IRES-driven translation. Thus, this is the first observation that 2Apro and Lpro exhibit opposite effects to what was previously thought to be the case in HAV IRES. This effect has been observed both in hamster BHK and human hepatoma Huh7 cells. In addition, this stimulation of translation is also observed in cell free systems after addition of purified Lpro. Notably, in presence of this FMDV protease, translation directed by HAV IRES takes place when eIF2α has been inactivated by phosphorylation. Our present findings clearly demonstrate that protein synthesis directed by HAV IRES can occur when eIF4G has been cleaved and after inactivation of eIF2. Therefore, translation directed by HAV IRES without intact eIF4G and active eIF2 is similar to that observed with other picornavirus IRESs.
Hippuristanol (Hipp) is a natural product that selectively inhibits protein synthesis by targeting eukaryotic initiation factor (eIF) 4A, a DEAD-box RNA helicase required for ribosome recruitment to mRNA templates. Hipp binds to the carboxyl-terminal domain of eIF4A, locks it in a closed conformation, and inhibits its RNA binding. The dependencies of mRNAs for eIF4A during initiation is contingent on the degree of secondary structure within their 5′ leader region. Interest in targeting eIF4A therapeutically in cancer and viral-infected settings stems from the dependencies that certain cellular (e.g. pro-oncogenic, pro-survival) and viral mRNAs show towards eIF4A. Using a CRISPR/Cas9-based variomics screen, we identify functional EIF4A1 Hipp-resistant alleles, which in turn allowed us to link the translation-inhibitory and cytotoxic properties of Hipp to eIF4A1 target engagement. Genome-wide translational profiling in the absence or presence of Hipp were undertaken and our validation studies provided insight into the structure-activity relationships of eIF4A-dependent mRNAs. We find that mRNA 5′ leader length, overall secondary structure and cytosine content are defining features of Hipp-dependent mRNAs.
Translation of foot-and-mouth disease virus RNA initiates at one of two start codons leading to the synthesis of two forms of leader proteinase Lpro (Labpro and Lbpro). These forms free themselves from the viral polyprotein by intra- and intermolecular self-processing and subsequently cleave the cellular eukaryotic initiation factor (eIF) 4G. During infection, Lbpro removes six residues from its own C-terminus, generating sLbpro. We present the structure of sLbpro bound to the inhibitor E64-R-P-NH2, illustrating how sLbpro can cleave between Lys/Gly and Gly/Arg pairs. In intermolecular cleavage on polyprotein substrates, Lbpro was unaffected by P1 or P1′ substitutions and processed a substrate containing nine eIF4GI cleavage site residues whereas sLbpro failed to cleave the eIF4GI containing substrate and cleaved appreciably more slowly on mutated substrates. Introduction of 70 eIF4GI residues bearing the Lbpro binding site restored cleavage. These data imply that Lbpro and sLbpro may have different functions in infected cells.
The foot-and-mouth disease virus leader proteinase (Lbpro) cleaves itself off the nascent viral polyprotein. NMR studies on the monomeric variant Lbpro L200F provide structural evidence for intramolecular self-processing. 15N-HSQC measurements of Lbpro L200F showed specifically shifted backbone signals in the active and substrate binding sites compared to the monomeric variant sLbpro, lacking six C-terminal residues. This indicates transient intramolecular interactions between the C-terminal extension (CTE) of one molecule and its own active site. Contrastingly, the porcine reproductive and respiratory syndrome virus (PRRSV) leader proteinase nsp1α, with a papain-like fold like Lbpro, stably binds its own CTE. Parts of the β-sheet domains but none of the α-helical domains of Lbpro and nsp1α superimpose; consequently, the α-helical domain of nsp1α is oriented differently relative to its β-sheet domain. This provides a large interaction surface for the CTE with the globular domain, stabilising the intramolecular complex. Consequently, self-processing inactivates nsp1α but not Lbpro.
Biotherapeutics have revolutionized modern medicine by providing medicines that would not have been possible with small molecules. With respect to cancer therapies, this represents the current sector of the pharmaceutical industry having the largest therapeutic impact, as exemplified by the development of recombinant antibodies and cell-based therapies. In cancer, one of the most common regulatory alterations is the perturbation of translational control. Among these, changes in eukaryotic initiation factor 4F (eIF4F) are associated with tumor initiation, progression, and drug resistance in a number of settings. This, coupled with the fact that systemic suppression of eIF4F appears well tolerated, indicates that therapeutic agents targeting eIF4F hold much therapeutic potential. Here, we discuss opportunities offered by biologicals for this purpose.
Highlights d CRISPR/Cas9 engineering harnessed to generate a targetagnostic phenotypic screen d GFP tracing of c-MYC expression in multiple myeloma cells d Cardiac glycosides and cytoskeletal disruptors are potent MYC expression inhibitors
SMARCA4 (BRG1) and SMARCA2 (BRM) are the two paralogous ATPases of the SWI/SNF chromatin remodeling complexes frequently inactivated in cancers. Cells deficient in either ATPase have been shown to depend on the remaining counterpart for survival. Contrary to this paralog synthetic lethality, concomitant loss of SMARCA4/2 occurs in a subset of cancers associated with very poor outcomes. Here, we uncover that SMARCA4/2-loss represses expression of the glucose transporter GLUT1, causing reduced glucose uptake and glycolysis accompanied with increased dependency on oxidative phosphorylation (OXPHOS); adapting to this, these SMARCA4/2-deficient cells rely on elevated SLC38A2, an amino acid transporter, to increase glutamine import for fueling OXPHOS. Consequently, SMARCA4/2-deficient cells and tumors are highly sensitive to inhibitors targeting OXPHOS or glutamine metabolism. Furthermore, supplementation of alanine, also imported by SLC38A2, restricts glutamine uptake through competition and selectively induces death in SMARCA4/2-deficient cancer cells. At a clinically relevant dose, alanine supplementation synergizes with OXPHOS inhibition or conventional chemotherapy eliciting marked antitumor activity in patient-derived xenografts. Our findings reveal multiple druggable vulnerabilities of SMARCA4/2-loss exploiting a GLUT1/SLC38A2-mediated metabolic shift. Particularly, unlike dietary deprivation approaches, alanine supplementation can be readily applied to current regimens for better treatment of these aggressive cancers.
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