There are two major components of Escherichia coli ribosomes directly involved in selection and binding of mRNA during initiation of protein synthesis-the highly conserved 39 end of 16S rRNA (aSD) complementary to the ShineDalgarno (SD) domain of mRNA, and the ribosomal protein S1. A contribution of the SD-aSD and S1-mRNA interactions to translation yield in vivo has been evaluated in a genetic system developed to compare efficiencies of various ribosome-binding sites (RBS) in driving b-galactosidase synthesis from the single-copy (chromosomal) lacZ gene. The in vivo experiments have been supplemented by in vitro toeprinting and gel-mobility shift assays. A shortening of a potential SD-aSD duplex from 10 to 8 and to 6 bp increased the b-galactosidase yield (four-and sixfold, respectively) suggesting that an extended SD-aSD duplex adversely affects translation, most likely due to its redundant stability causing ribosome stalling at the initiation step. Translation yields were significantly increased upon insertion of the A/U-rich S1 binding targets upstream of the SD region, but the longest SD remained relatively less efficient. In contrast to complete 30S ribosomes, the S1-depleted 30S particles have been able to form an extended SD-aSD duplex, but not the true ternary initiation complex. Taken together, the in vivo and in vitro data allow us to conclude that S1 plays two roles in translation initiation: It forms an essential part of the mRNA-binding track even when mRNA bears a long SD sequence, and through the binding to the 59 untranslated region, it can ensure a substantial enhancing effect on translation.

The worldwide SARS-CoV-2 outbreak poses a serious challenge to human societies and economies. SARS-CoV-2 proteins orchestrate complex pathogenic mechanisms that underlie COVID-19 disease. Thus, understanding how viral polypeptides rewire host protein networks enables better-founded therapeutic research. In complement to existing proteomic studies, in this study we define the first proximal interaction network of SARS-CoV-2 proteins, at the whole proteome level in human cells. Applying a proximity-dependent biotinylation (BioID)-based approach greatly expanded the current knowledge by detecting interactions within poorly soluble compartments, transient, and/or of weak affinity in living cells. Our BioID study was complemented by a stringent filtering and uncovered 2,128 unique cellular targets (1,717 not previously associated with SARS-CoV-1 or 2 proteins) connected to the N- and C-ter BioID-tagged 28 SARS-CoV-2 proteins by a total of 5,415 (5,236 new) proximal interactions. In order to facilitate data exploitation, an innovative interactive 3D web interface was developed to allow customized analysis and exploration of the landscape of interactions (accessible at http://www.sars-cov-2-interactome.org/). Interestingly, 342 membrane proteins including interferon and interleukin pathways factors, were associated with specific viral proteins. We uncovered ORF7a and ORF7b protein proximal partners that could be related to anosmia and ageusia symptoms. Moreover, comparing proximal interactomes in basal and infection-mimicking conditions (poly(I:C) treatment) allowed us to detect novel links with major antiviral response pathway components, such as ORF9b with MAVS and ISG20; N with PKR and TARB2; NSP2 with RIG-I and STAT1; NSP16 with PARP9-DTX3L. Altogether, our study provides an unprecedented comprehensive resource for understanding how SARS-CoV-2 proteins orchestrate host proteome remodeling and innate immune response evasion, which can inform development of targeted therapeutic strategies.

We have shown previously that when the Escherichia coli chromosomal lacZ gene is put under the control of an extended Shine-Dalgarno (SD) sequence (10 or 6 nucleotides in length), the translation efficiency can be highly variable, depending on the presence of AU-rich targets for ribosomal protein S1 in the mRNA leader. Here, the same strains have been used to examine the question of how strong ribosome binding to extended SD sequences affects the stability of lacZ mRNAs translated with different efficiencies. The steady-state concentration of the lacZ transcripts has been found to vary over a broad range, directly correlating with translation efficiency but not with the SD duplex stability. The observed strain-to-strain variations in lacZ mRNA level became far less marked in the presence of the rne-1 mutation, which partially inactivates RNase E. Together, the results show that (i) an SD sequence, even one that is very long, cannot stabilize the lacZ mRNA in E. coli if translation is inefficient; (ii) inefficiently translated lacZ transcripts are sensitive to RNase E; and (iii) AU-rich elements inserted upstream of a long SD sequence enhance translation and stabilize mRNA, despite the fact that they constitute potential RNase E sites. These data strongly support the idea that the lacZ mRNA in E. coli can be stabilized only by translating, and not by stalling, ribosomes.As a rule, prokaryotic mRNAs are unstable, with half-lives in the minute range. In Escherichia coli, pathways of mRNA decay are complex and include an initial rate-limiting endonucleolytic cleavage (generally mediated by RNase E) followed by 3Ј35Ј exonucleolytic degradation of the cleavage products (reviewed in references 6, 10, 19, and 34). For several mRNAs, decay pathways have been studied in detail, allowing molecular models to be formulated (4-6, 11, 13, 28). Several lines of evidence suggest that translating ribosomes usually protect the mRNA from endonucleolytic attacks (10). Much of the available information has been derived from experiments with lacZ translational fusions. In this case, mRNA translation and degradation are intimately correlated. In particular, mutations in the ribosome binding site (RBS) which reduce translation initiation efficiency, and hence efficiency of the overall translation, accelerate mRNA degradation (14,22,38). However, despite all efforts, the mechanism of the lacZ mRNA decay still remains poorly characterized.Although in many cases translation efficiency positively correlates with stability of the lacZ mRNA in E. coli, the question of whether merely the ribosome binding to the RBS is able to stabilize the whole transcript remains equivocal. On one hand, it has been suggested that a ribosome bound to a strong ShineDalgarno (SD) sequence, but not translation per se, could be the main stability factor for the lacZ mRNA (37). This would imply that E. coli mRNAs can be protected from degradation by the same mechanism as in bacilli, where ribosome stalling within 5Ј untranslated regions can protect kilobases of the...

The RIG-I-like receptors (RLRs) play a major role in sensing RNA virus infection to initiate and modulate antiviral immunity. They interact with particular viral RNAs, most of them being still unknown. To decipher the viral RNA signature on RLRs during viral infection, we tagged RLRs (RIG-I, MDA5, LGP2) and applied tagged protein affinity purification followed by next-generation sequencing (NGS) of associated RNA molecules. Two viruses with negative- and positive-sense RNA genome were used: measles (MV) and chikungunya (CHIKV). NGS analysis revealed that distinct regions of MV genome were specifically recognized by distinct RLRs: RIG-I recognized defective interfering genomes, whereas MDA5 and LGP2 specifically bound MV nucleoprotein-coding region. During CHIKV infection, RIG-I associated specifically to the 3’ untranslated region of viral genome. This study provides the first comparative view of the viral RNA ligands for RIG-I, MDA5 and LGP2 in the presence of infection.DOI: http://dx.doi.org/10.7554/eLife.11275.001

The flavivirus genus comprises major human pathogens, such as Dengue (DENV) and Zika (ZIKV) viruses. RIG-I and MDA5 are key cytoplasmic pathogen recognition receptors that are implicated in detecting viral RNAs. Here, we show that RNAs that co-purified with RIG-I during DENV infection are immuno-stimulatory, whereas RNAs bound to MDA5 are not. An affinity purification method combined with next-generation sequencing (NGS) revealed that the 5' region of the DENV genome is recognized by RIG-I. No DENV RNA was bound to MDA5. In vitro production of fragments of the DENV genome confirmed the NGS data and revealed that the 5' end of the genome, when bearing 5'-triphosphates, is the RIG-I ligand. The 5' region of the ZIKV genome is also a RIG-I agonist. We propose that RIG-I binds to the highly structured and conserved 5' region of flavivirus nascent transcripts before capping and that this mechanism leads to interferon secretion by infected cells.

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of interactions with host cell components to achieve replication and spreading. Ideally, these virus-host protein interactions should be mapped directly in infected cell culture, but such a high standard is often difficult to reach when using conventional approaches. We thus developed a new strategy based on recombinant viruses expressing tagged viral proteins to capture both direct and indirect physical binding partners during infection. As a proof of concept, we engineered a recombinant measles virus (MV) expressing one of its virulence factors, the MV-V protein, with a One-STrEP amino-terminal tag. This allowed virus-host protein complex analysis directly from infected cells by combining modified tandem affinity chromatography and mass spectrometry analysis. Using this approach, we established a prosperous list of 245 cellular proteins interacting either directly or indirectly with MV-V, and including four of the nine already known partners of this viral factor. These interactions were highly specific of MV-V because they were not recovered when the nucleoprotein MV-N, instead of MV-V, was tagged. Besides key components of the antiviral response, cellular proteins from mitochondria, ribosomes, endoplasmic reticulum, protein phosphatase 2A, and histone deacetylase complex were identified for the first time as prominent targets of MV-V and the critical role of the later protein family in MV replication was addressed. Most interestingly, MV-V showed some preferential attachment to essential proteins in the human interactome network, as assessed by centrality and interconnectivity measures. Furthermore, the list of MV-V interactors also showed a massive enrichment for well-known targets of other viruses. Altogether, this clearly supports our approach based on reverse genetics of viruses combined with high-throughput proteomics to probe the interaction network that viruses establish in infected cells.

The glycine-alanine repeat (GAr) sequence of the Epstein-Barr virus-encoded EBNA-1 prevents presentation of antigenic peptides to major histocompatibility complex class I molecules. This has been attributed to its capacity to suppress mRNA translation in cis. However, the underlying mechanism of this function remains largely unknown. Here, we have further investigated the effect of the GAr as a regulator of mRNA translation. Introduction of silent mutations in each codon of a 30-amino-acid GAr sequence does not significantly affect the translation-inhibitory capacity, whereas minimal alterations in the amino acid composition have strong effects, which underscores the observation that the amino acid sequence and not the mRNA sequence mediates GAr-dependent translation suppression. The capacity of the GAr to repress translation is dose and position dependent and leads to a relative accumulation of preinitiation complexes on the mRNA. Taken together with the surprising observation that fusion of the 5 untranslated region (UTR) of the c-myc mRNA to the 5 UTR of GAr-carrying mRNAs specifically inactivates the effect of the GAr, these results indicate that the GAr targets components of the translation initiation process. We propose a model in which the nascent GAr peptide delays the assembly of the initiation complex on its own mRNA.Epstein-Barr Virus (EBV) nuclear antigen 1 (EBNA-1) and latency-associated nuclear antigen 1 (LANA-1), from Kaposi's sarcoma-associated herpesvirus (KSHV), are major latency proteins of these two gammaherpesviruses that are essential for maintaining viral episomes in infected cells (21,22). Independent studies suggest that both proteins have evolved mechanisms to remain largely invisible to the immune system, which could otherwise eliminate latently infected cells (8,9,19,25). These mechanisms act in cis and are mediated via an internal repeat region. In the case of EBNA-1 this region consists of an N-terminal glycine-alanine repeat (GAr), and for LANA-1 the region consists of a glutamine-glutamate-aspartate central repeat (QED-CR). Although the two domains do not share amino acid homology, both retard their own synthesis to reduce the production of defective ribosomal products that can be processed for the major histocompatibility complex (MHC) class I-restricted antigen presentation pathway (23, 24), highlighting the importance of translation control in regulating MHC class I-restricted antigen presentation. To compensate for their low rates of synthesis, both proteins also have slow turnover rates (4, 8).Regulation of translation for most prokaryotic and eukaryotic mRNAs occurs at the level of initiation, but there are examples where regulation of protein synthesis depends on the elongation stage (17). The two main types of translation initiation are the classic cap-dependent and the less frequent cap-independent translation mechanisms (5,7,11,14,16). In the former, the preinitiation complex is formed around the cap structure in the 5Ј untranslated region (UTR) of the message, whereas i...

The retinoic acid–inducible gene I (RIG-I)–like receptors (RLRs) RIG-I, MDA5, and LGP2 stimulate inflammatory and antiviral responses by sensing nonself RNA molecules produced during viral replication. Here, we investigated how LGP2 regulates the RIG-I– and MDA5-dependent induction of type I interferon (IFN) signaling and showed that LGP2 interacted with different components of the RNA-silencing machinery. We identified a direct protein-protein interaction between LGP2 and the IFN-inducible, double-stranded RNA binding protein PACT. The LGP2-PACT interaction was mediated by the regulatory C-terminal domain of LGP2 and was necessary for inhibiting RIG-I–dependent responses and for amplifying MDA5-dependent responses. We described a point mutation within LGP2 that disrupted the LGP2-PACT interaction and led to the loss of LGP2-mediated regulation of RIG-I and MDA5 signaling. These results suggest a model in which the LGP2-PACT interaction regulates the inflammatory responses mediated by RIG-I and MDA5 and enables the cellular RNA-silencing machinery to coordinate with the innate immune response.