SARS-CoV-2 viral protein Nsp2 stimulates translation under normal and hypoxic conditions.

When viruses like SARS-CoV-2 infect cells, they reprogram the repertoire of cellular and viral transcripts that are being translated to optimize their strategy of replication, often targeting host translation initiation factors, particularly eIF4F complex consisting of eIF4E, eIF4G and eIF4A. A proteomic analysis of SARS-CoV-2/human proteins interaction revealed viral Nsp2 and initiation factor eIF4E2, but a role of Nsp2 in regulating translation is still controversial. HEK293T cells stably expressing Nsp2 were tested for protein synthesis rates of synthetic and endogenous mRNAs known to be translated via cap- or IRES-dependent mechanism under normal and hypoxic conditions. Both cap- and IRES-dependent translation were increased in Nsp2-expressing cells under normal and hypoxic conditions, especially mRNAs that require high levels of eIF4F. This could be exploited by the virus to maintain high translation rates of both viral and cellular proteins, particularly in hypoxic conditions as may arise in SARS-CoV-2 patients with poor lung functioning.

A cautionary note on the use of cap analogue affinity resins.

All cellular cytoplasmic mRNAs are capped at their 5' ends with an m7GpppN group. Several proteins that mediate cap function have been identified by cap affinity purification, enabling their characterization in a number of biological processes. Among these, eukaryotic initiation factor (eIF) 4E is the best characterized and plays a critical role in regulating ribosome recruitment to mRNAs during translation initiation. Cap affinity chromatography is often used to identify eIF4E-interacting proteins, which could play critical roles in molding the eIF4E-interactome and impacting on eIF4E-directed translation. Here we address how improper implementation of this technology can lead to false conclusions and provide recommendations to ensure correct interpretation of data obtained by this approach.

Evolutionary changes in the Leishmania eIF4F complex involve variations in the eIF4E-eIF4G interactions.

Translation initiation in eukaryotes is mediated by assembly of the eIF4F complex over the m(7)GTP cap structure at the 5'-end of mRNAs. This requires an interaction between eIF4E and eIF4G, two eIF4F subunits. The Leishmania orthologs of eIF4E are structurally diverged from their higher eukaryote counterparts, since they have evolved to bind the unique trypanosomatid cap-4 structure. Here, we characterize a key eIF4G candidate from Leishmania parasites (LeishIF4G-3) that contains a conserved MIF4G domain. LeishIF4G-3 was found to coelute with the parasite eIF4F subunits from an m(7)GTP-Sepharose column and to bind directly to LeishIF4E. In higher eukaryotes the eIF4E-eIF4G interaction is based on a conserved peptide signature [Y(X(4))Lphi], where X is any amino acid and Phi is a hydrophobic residue. A parallel eIF4E-binding peptide was identified in LeishIF4G-3 (20-YPGFSLDE-27). However, the binding motif varies extensively: in addition to Y20 and L25, binding strictly requires the presence of F23, whereas the hydrophobic amino acid (Phi) is dispensable. The LeishIF4E-LeishIF4G-3 interaction was also confirmed by nuclear magnetic resonance (NMR) studies. In view of these diversities, the characterization of the parasite eIF4E-eIF4G interaction may not only serve as a novel target for inhibiting Leishmaniasis but also provide important insight for future drug discovery.

Crystallization and preliminary X-ray analysis of eukaryotic initiation factor 4E from Pisum sativum.

Crystals of an N-terminally truncated 20 kDa fragment of Pisum sativum eIF4E (DeltaN-eIF4E) were grown by vapour diffusion. X-ray data were recorded to a resolution of 2.2 A from a single crystal in-house. Indexing was consistent with primitive monoclinic symmetry and solvent-content estimations suggested that between four and nine copies of the eIF4E fragment were possible per crystallographic asymmetric unit. eIF4E is an essential component of the eukaryotic translation machinery and recent studies have shown that point mutations of plant eIF4Es can confer resistance to potyvirus infection.

Phosphorylation of translation initiation factor eIFiso4E promotes translation through enhanced binding to potyvirus VPg.

Interactions of phosphorylated eIFiso4E binding to VPg as a function of temperature and ionic strength were assessed employing fluorescence spectroscopic. Phosphorylation increased the binding affinity ∼3.5-fold between VPg and eIFiso4E under equilibrium conditions. Binding affinity of VPg for eIFiso4Ep correlates with the ability to enhance in vitro protein synthesis. Addition of VPg and eIFiso4Ep together to Dep WGE enhances the translation for both uncapped and capped mRNA. However, capped mRNA translation was inhibited with addition of eIFiso4Ep alone in dep WGE, suggesting that phosphorylation prevents the cap binding and favours the VPg binding to promotes translation. Temperature dependence showed that the phosphorylated form of the eIFiso4E is preferred for complex formation. A van't Hoff analysis reveals that eIFiso4Ep binding to VPg was enthalpy driven (ΔH = -43.9 ± 0.3 kJ.mol-1) and entropy-opposed (ΔS = -4.3 ± 0.1 J.mol-1K-1). Phosphorylation increased the enthalpic contributions ∼33% for eIFiso4Ep-VPg complex. The thermodynamic values and ionic strength dependence of binding data suggesting that phosphorylation increased hydrogen-bonding and decreased hydrophobic interactions, which leads to more stable complex formation and favour efficient viral translation. Overall these data correlate well with the observed translational data and provide more detailed information on the translational strategy of potyviruses.

Characterization of human malaria parasite Plasmodium falciparum eIF4E homologue and mRNA 5' cap status.

The mRNA 5' cap is an essential structural feature for translation of eukaryotic mRNA. Translation is initiated by recognition of the cap by the translation initiation factor eIF4E. To further our understanding of mRNA translation in the human malaria parasite Plasmodium falciparum, we have investigated the parasite eIF4E and its interaction with capped mRNA. We have purified P. falciparum eIF4E as a recombinant protein and demonstrated that it has canonical mRNA cap binding activity. We used this protein to purify P. falciparum capped mRNAs from total parasite RNA. Microarray analysis comparing total and eIF4E-purified capped mRNAs shows that 34 features were more than twofold under-represented in the purified RNA sample, including 19 features representative of nuclear transcripts. The putatively uncapped nuclear transcripts may represent a class of mRNAs targeted for storage and cap removal.

Use of the novel technique of analytical ultracentrifugation with fluorescence detection system identifies a 77S monosomal translation complex.

A fundamental problem in proteomics is the identification of protein complexes and their components. We have used analytical ultracentrifugation with a fluorescence detection system (AU-FDS) to precisely and rapidly identify translation complexes in the yeast Saccharomyces cerevisiae. Following a one-step affinity purification of either poly(A)-binding protein (PAB1) or the large ribosomal subunit protein RPL25A in conjunction with GFP-tagged yeast proteins/RNAs, we have detected a 77S translation complex that contains the 80S ribosome, mRNA, and components of the closed-loop structure, eIF4E, eIF4G, and PAB1. This 77S structure, not readily observed previously, is consistent with the monosomal translation complex. The 77S complex abundance decreased with translational defects and following the stress of glucose deprivation that causes translational stoppage. By quantitating the abundance of the 77S complex in response to different stress conditions that block translation initiation, we observed that the stress of glucose deprivation affected translation initiation primarily by operating through a pathway involving the mRNA cap binding protein eIF4E whereas amino acid deprivation, as previously known, acted through the 43S complex. High salt conditions (1M KCl) and robust heat shock acted at other steps. The presumed sites of translational blockage caused by these stresses coincided with the types of stress granules, if any, which are subsequently formed.

Development of biochemical assays for the identification of eIF4E-specific inhibitors.

Control of mRNA translation plays a critical role in cell growth, proliferation, and differentiation and is tightly regulated by AKT and RAS oncogenic pathways. A key player in the regulation of this process is the mRNA 5' cap-binding protein, eukaryotic translation initiation factor 4E (eIF4E). eIF4E contributes to malignancy by selectively enabling the translation of a limited pool of mRNAs that generally encode key proteins involved in cell cycle progression, angiogenesis, and metastasis. Several data indicate that the inhibition of eIF4E in tumor cell lines and xenograft models impairs tumor growth and induces apoptosis; eIF4E, therefore, can be considered a valuable target for cancer therapy. Targeting the cap-binding pocket of eIF4E should represent a way to inhibit all the eIF4E cellular functions. We present here the development and validation of different biochemical assays based on fluorescence polarization and surface plasmon resonance techniques. These assays could support high-throughput screening, further refinement, and characterization of eIF4E inhibitors, as well as selectivity assessment against CBP80/CBP20, the other major cap-binding complex of eukaryotic cells, overall providing a robust roadmap for development of eIF4E-specific inhibitors.

Bisphosphonate mRNA cap analog attached to Sepharose for affinity chromatography of decapping enzymes.

m(7)GTP-Sepharose is routinely used for cap binding protein isolation. Here we present the synthesis of a new affinity resin containing a mononucleotide cap analog resistant to hydrolysis by DcpS. The resin has been designed in order to identify and purify Arabidopsis thaliana DcpS and other pyrophosphatases. The binding efficiency of the new resin to eIF4E protein was compared with standard m(7)GTP-Sepharose. The utility of non-hydrolysable resin was demonstrated on yeast extract.

After fertilization of sea urchin eggs, eIF4G is post-translationally modified and associated with the cap-binding protein eIF4E.

Release of eukaryotic initiation factor 4E (eIF4E) from its translational repressor eIF4E-binding protein (4E-BP) is a crucial event for the first mitotic division following fertilization of sea urchin eggs. Finding partners of eIF4E following fertilization is crucial to understand how eIF4E functions during this physiological process. The isolation and characterization of cDNA encoding Sphaerechinus granularis eIF4G (SgIF4G) are reported. mRNA of SgIF4G is present as a single 8.5-kb transcript in unfertilized eggs, suggesting that only one ortholog exists in echinoderms. The longest open reading frame predicts a sequence of 5235 nucleotides encoding a deduced polypeptide of 1745 amino acids with a predicted molecular mass of 192 kDa. Among highly conserved domains, SgIF4G protein possesses motifs that correspond to the poly(A) binding protein and eIF4E protein-binding sites. A specific polyclonal antibody was produced and used to characterize the SgIF4G protein in unfertilized and fertilized eggs by SDS-PAGE and western blotting. Multiple differentially migrating bands representing isoforms of sea urchin eIF4G are present in unfertilized eggs. Fertilization triggers modifications of the SgIF4G isoforms and rapid formation of the SgIF4G-eIF4E complex. Whereas rapamycin inhibits the formation of the SgIF4G-eIF4E complex, modification of these SgIF4G isoforms occurs independently from the rapamycin-sensitive pathway. Microinjection of a peptide corresponding to the eIF4E-binding site derived from the sequence of SgIF4G into unfertilized eggs affects the first mitotic division of sea urchin embryos. Association of SgIF4G with eIF4E is a crucial event for the onset of the first mitotic division following fertilization, suggesting that cap-dependent translation is highly regulated during this process. This hypothesis is strengthened by the evidence that microinjection of the cap analog m(7)GDP into unfertilized eggs inhibits the first mitotic division.