High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Institut Pasteur, CNRS UMR3569

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 3' end of 16S rRNA (aSD) complementary to the Shine-Dalgarno (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 beta-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 beta-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 5' untranslated region, it can ensure a substantial enhancing effect on translation.

Protein S1 counteracts the inhibitory effect of the extended Shine-Dalgarno sequence on translation.

The translation initiation region (TIR) of the Escherichia coli rpsA mRNA coding for ribosomal protein S1 is characterized by a remarkable efficiency in driving protein synthesis despite the absence of the canonical Shine-Dalgarno element, and by a strong and specific autogenous repression in the presence of free S1 in trans. The efficient and autoregulated E.coli rpsA TIR comprises not less than 90 nt upstream of the translation start and can be unambiguously folded into three irregular hairpins (HI, HII and HIII) separated by A/U-rich single-stranded regions (ss1 and ss2). Phylogenetic comparison revealed that this specific fold is highly conserved in the gamma-subdivision of proteobacteria (but not in other subdivisions), except for the Pseudomonas group. To test phylogenetic predictions experimentally, we have generated rpsA'-'lacZ translational fusions by inserting the rpsA TIRs from various gamma-proteobacteria in-frame with the E.coli chromosomal lacZ gene. Measurements of their translation efficiency and negative regulation by excess protein S1 in trans have shown that only those rpsA TIRs which share the structural features with that of E.coli can govern efficient and regulated translation. We conclude that the E.coli-like mechanism for controlling the efficiency of protein S1 synthesis evolved after divergence of Pseudomona.

A key role for the mRNA leader structure in translational control of ribosomal protein S1 synthesis in gamma-proteobacteria.

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.

AU-rich sequences within 5' untranslated leaders enhance translation and stabilize mRNA in Escherichia coli.

The case for mRNA 5' and 3' end cross talk during translation in a eukaryotic cell.

It is now well established that Hepatitis C Virus (HCV) translation is driven by an Internal Ribosome Entry Site (IRES) resulting in cap-independent translation. Such a mechanism usually occurs with the help of IRES Associated Factors (ITAFs). Moreover, an important translational feature is likely conserved from the model of classical mRNA circularisation (5'-3' cross-talk), involving the HCV RNA highly structured 3' extremity called the 3'X region. This could bind several cellular factors and modulate the translation efficacy, at least in Rabbit Reticulocyte Lysate (RRL). In particular, polypyrimidine-binding proteins have been proposed to be potential HCV ITAFs, such as Polypyrimidine Tract Binding protein (PTB). However, contradictions still exist as to the role of PTB: its ability to bind both the HCV IRES and the 3'X region leads to the hypothesis that it could positively modulate IRES-driven translation in the presence of the X structure. Results of translational and PTB-binding studies of X mutant sequences led us to discredit PTB as protagonist of 3'X region stimulation on HCV IRES-driven translation. Moreover, competition assays of X RNA in trans on IRES-driven translation demonstrate the involvement of at least two stimulating factors and led to the conclusion that this mechanism is more complex than initially thought. Although we did not identify these factors, it is no longer doubtful that there is effectively a stimulating functional interaction between the HCV IRES and the 3'X region in RRL.

Evidence that PTB does not stimulate HCV IRES-driven translation.

Viral proteins are frequently multifunctional to accommodate the high density of information encoded in viral genomes. Matrix (M) protein of negative-stranded RNA viruses such as Rhabdoviridae is one such example. Its primary function is virus assembly/budding but it is also involved in the switch from viral transcription to replication and the concomitant down regulation of host gene expression. In this study we undertook a search for potential rabies virus (RV) M protein's cellular partners. In a yeast two-hybrid screen the eIF3h subunit was identified as an M-interacting cellular factor, and the interaction was validated by co-immunoprecipitation and surface plasmon resonance assays. Upon expression in mammalian cell cultures, RV M protein was localized in early small ribosomal subunit fractions. Further, M protein added in trans inhibited in vitro translation on mRNA encompassing classical (Kozak-like) 5'-UTRs. Interestingly, translation of hepatitis C virus IRES-containing mRNA, which recruits eIF3 via a different noncanonical mechanism, was unaffected. Together, the data suggest that, as a complement to its functions in virus assembly/budding and regulation of viral transcription, RV M protein plays a role in inhibiting translation in virus-infected cells through a protein-protein interaction with the cellular translation machinery.

Rabies virus matrix protein interplay with eIF3, new insights into rabies virus pathogenesis.

Prot&eacute;ines virales sont fr&eacute;quemment multifonctionnels pour tenir compte de la forte densit&eacute; des informations cod&eacute;es dans le g&eacute;nome viral. Prot&eacute;ine de la matrice (M) de virus &agrave; ARN n&eacute;gatif brin comme Rhabdoviridae en est un exemple. Sa fonction principale est de virus Assembl&eacute;e/en herbe, mais il est &eacute;galement impliqu&eacute; dans le passage de la transcription virale &agrave; la r&eacute;plication et le concomitante dans la r&eacute;gulation de l&#39;expression des g&egrave;nes h&ocirc;tes. Dans cette &eacute;tude, nous avons entrepris une recherche de partenaires cellulaires potentiels la rage virus (RV) M de cette prot&eacute;ine. Dans un &eacute;cran de deux-hybride de levure que la sous-unit&eacute; d&#39;eIF3h a &eacute;t&eacute; identifi&eacute;e comme un facteur cellulaire interagissant avec M et l&#39;interaction a &eacute;t&eacute; valid&eacute; par co-immunopr&eacute;cipitation et surface plasmon resonance dosages. Une expression dans des cultures de cellules de mammif&egrave;res, prot&eacute;ine RV M a &eacute;t&eacute; localis&eacute;e dans les fractions de petite sous-unit&eacute; ribosomique pr&eacute;coce. En outre, prot&eacute;ine M ajout&eacute; dans la trans inhib&eacute;e traduction in vitro sur l&#39;ARNm englobant classique (type Kozak) 5&#39;-UTR. Fait int&eacute;ressant, translation of hepatitis C virus ARNm contenant de l&#39;IRES, qui recrute eIF3 via un autre m&eacute;canisme non canonique, n&#39;est pas affect&eacute;e. Ensemble, les donn&eacute;es semblent indiquer que, en compl&eacute;ment de ses fonctions en virus Assembl&eacute;e/en herbe et r&eacute;gulation de la transcription virale, la prot&eacute;ine RV M joue un r&ocirc;le dans l&#39;inhibition de la traduction dans les cellules infect&eacute;es par le virus &agrave; une interaction de prot&eacute;ine-prot&eacute;ine avec la machinerie de traduction cellulaire.

La rage virus matrice prot&eacute;ique interaction avec eIF3, nouvelles perspectives sur la pathog&eacute;nie de virus de la rage.

Le proteine virali sono frequentemente multifunzionale per ospitare l&#39;alta densit&agrave; di informazioni codificate nel genoma virale. Proteine di matrice (M) del virus RNA filamento negativo come Rhabdoviridae sono un esempio. La sua funzione primaria &egrave; virus assembly/in erba, ma &egrave; anche coinvolto nello switch da trascrizione virale per la replica e la concomitante gi&ugrave; regolazione dell&#39;espressione genica di host. In questo studio abbiamo intrapreso una ricerca di cellulare partner potenziali rabies virus (RV) M della proteina. In uno schermo di due-ibrido del lievito che la subunit&agrave; eIF3h &egrave; stata identificata come un fattore cellulare interagendo M e l&#39;interazione &egrave; stato convalidato da dosaggi di risonanza plasmonica co-immunoprecipitazione e superficie. Su espressione in colture di cellule di mammifero, RV M proteina &egrave; stata localizzata nelle prime frazioni di piccola subunit&agrave; ribosomiale. Ulteriormente, proteina M aggiunta in trans inibito traduzione in vitro su mRNA che comprende classici (come Kozak) 5&#39;-UTRs. &Egrave; interessante notare che, traduzione del virus dell&#39;epatite C mRNA contenente IRES, che recluta eIF3 tramite un meccanismo diverso canonica, era inalterata. Insieme, i dati suggeriscono che, come un complemento alle sue funzioni nel virus assembly/in erba e regolazione della trascrizione virale, RV M proteina svolge un ruolo nell&#39;inibire traduzione in cellule infettate da virus attraverso un&#39;interazione proteina-proteina con il macchinario di traduzione cellulare.

Rabbia virus matrix protein interazione con eIF3, nuove intuizioni patogenesi virus di rabbia.

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.

mRNA translation regulation by the Gly-Ala repeat of Epstein-Barr virus nuclear antigen 1.

Regulation of protein synthesis by viruses occurs at all levels of translation. Even prior to protein synthesis itself, the accessibility of the various open reading frames contained in the viral genome is precisely controlled. Eukaryotic viruses resort to a vast array of strategies to divert the translation machinery in their favor, in particular, at initiation of translation. These strategies are not only designed to circumvent strategies common to cell protein synthesis in eukaryotes, but as revealed more recently, they also aim at modifying or damaging cell factors, the virus having the capacity to multiply in the absence of these factors. In addition to unraveling mechanisms that may constitute new targets in view of controlling virus diseases, viruses constitute incomparably useful tools to gain in-depth knowledge on a multitude of cell pathways.

Virus versus host cell translation love and hate stories.

A number of paramyxoviruses are responsible for acute respiratory infections in children, elderly and immuno-compromised individuals, resulting in airway inflammation and exacerbation of chronic diseases like asthma. To understand the molecular pathogenesis of these infections, we searched for cellular targets of the virulence protein C of human parainfluenza virus type 3 (hPIV3-C). We found that hPIV3-C interacts directly through its C-terminal domain with STAT1 and GRB2, whereas C proteins from measles or Nipah viruses failed to do so. Binding to STAT1 explains the previously reported capacity of hPIV3-C to block type I interferon signaling, but the interaction with GRB2 was unexpected. This adaptor protein bridges Epidermal Growth Factor (EGF) receptor to MAPK/ERK pathway, a signaling cascade recently found to be involved in airway inflammatory response. We report that either hPIV3 infection or transient expression of hPIV3-C both increase cellular response to EGF, as assessed by Elk1 transactivation and phosphorylation levels of ERK1/2, 40S ribosomal subunit protein S6 and translation initiation factor 4E (eIF4E). Furthermore, inhibition of MAPK/ERK pathway with U0126 prevented viral protein expression in infected cells. Altogether, our data provide molecular basis to explain the role of hPIV3-C as a virulence factor and determinant of pathogenesis and demonstrate that Paramyxoviridae have evolved a single virulence factor to block type I interferon signaling and to boost simultaneous cellular response to growth factors.

Differential regulation of type I interferon and epidermal growth factor pathways by a human Respirovirus virulence factor.

Un certain nombre de paramyxovirus est responsable des infections respiratoires aigu&euml;s chez les enfants, personnes &acirc;g&eacute;es et immunod&eacute;prim&eacute;es, ayant pour r&eacute;sultat l&#39;inflammation des voies respiratoires et l&#39;exacerbation des maladies chroniques comme l&#39;asthme. Pour comprendre la pathogen&egrave;se mol&eacute;culaire de ces infections, nous avons cherch&eacute; des cibles cellulaires de la prot&eacute;ine de virulence C humain virus parainfluenza de type 3 (hPIV3-C). Nous avons trouv&eacute; que hPIV3-C interagit directement par l&#39;interm&eacute;diaire de son domaine C-terminal avec STAT1 et GRB2, tandis que les prot&eacute;ines C de rougeole ou de virus de Nipah a &eacute;chou&eacute; &agrave; le faire. Liaison &agrave; STAT1 explique la capacit&eacute; pr&eacute;c&eacute;demment d&eacute;clar&eacute;e de hPIV3-C pour bloquer les signaux de l&#39;interf&eacute;ron de type I, mais l&#39;interaction avec GRB2 n&#39;&eacute;tait pas pr&eacute;vue. Cet adaptateur ponts facteur de croissance &eacute;pidermique (EGF) r&eacute;cepteur prot&eacute;ique &agrave; voie de MAPK/ERK, une cascade de signalisation trouv&eacute;e r&eacute;cemment d&#39;&ecirc;tre impliqu&eacute;s dans la r&eacute;ponse inflammatoire des voies respiratoires. Nous rapport que soit hPIV3 infection ou transitoire expression de C-hPIV3 les deux augmente la r&eacute;ponse cellulaire &agrave; l&#39;EGF, tel qu&#39;&eacute;valu&eacute; par Elk1 niveaux transactivation et la phosphorylation de ERK1/2, prot&eacute;ine de sous-unit&eacute; ribosomique 40 s S6 et translation initiation factor 4E (eIF4E). De plus, l&#39;inhibition de la voie MAPK/ERK avec U0126 emp&ecirc;ch&eacute; expression de la prot&eacute;ine virale dans les cellules infect&eacute;es. Au total, nos donn&eacute;es fournissent une base mol&eacute;culaire pour expliquer le r&ocirc;le de hPIV3-C comme un facteur de virulence et le d&eacute;terminant de la pathogen&egrave;se et d&eacute;montrer que l&#39;&eacute;volution des Paramyxoviridae un facteur de virulence unique pour bloquer j&#39;ai tapez interf&eacute;ron signalisation et pour stimuler une r&eacute;ponse cellulaire simultan&eacute;e aux facteurs de croissance.

R&eacute;gulation diff&eacute;rentielle de type I interf&eacute;ron et facteur de croissance &eacute;pidermique voies par un facteur de virulence Respirovirus humain.

Un certo numero di aviari &egrave; responsabile di infezioni respiratorie acute in bambini, gli anziani e gli individui immunocompromessi, con conseguente infiammazione delle vie aeree ed esacerbazione di malattie croniche come l&#39;asma. Per comprendere la patogenesi molecolare di queste infezioni, abbiamo cercato per bersagli cellulari della proteina C virulenza del virus parainfluenzale umano tipo 3 (hPIV3-C). Abbiamo trovato che hPIV3-C interagisce direttamente attraverso il suo dominio C-terminale con STAT1 e GRB2, considerando che le proteine C da morbillo o virus Nipah non &egrave; riuscito a farlo. Associazione di STAT1 spiega la capacit&agrave; precedentemente riportata di hPIV3-C blocco di tipo interferone di segnalazione, ma l&#39;interazione con GRB2 &egrave; stato inaspettato. Questo adattatore della proteina ponti Epidermal Growth Factor (EGF) recettore per via MAPK/Erk, una cascata di segnalazione recentemente trovata ad per essere coinvolti nella risposta infiammatoria delle vie aeree. Abbiamo relazione che espressione di infezione o transitori o hPIV3 di hPIV3-C sia aumenta la risposta cellulare all&#39;EGF, come valutato da livelli di Elk1 transactivation e fosforilazione di ERK1/2, proteina di 40S subunit&agrave; ribosomiale S6 e traduzione iniziazione fattore 4E il (eIF4E). Inoltre, l&#39;inibizione della via MAPK/Erk con U0126 impedito l&#39;espressione della proteina virale nelle cellule infettate. Complessivamente, i nostri dati forniscono le basi molecolari per spiegare il ruolo di hPIV3-C come un fattore di virulenza e determinante della patogenesi e dimostrare che Paramyxoviridae hanno evoluto un fattore di virulenza singolo blocco di tipo interferone segnalazione e Spinta simultanea risposta cellulare a fattori di crescita.

Regolamento differenziale di tipo interferone e fattore di crescita epidermico percorsi da un fattore di virulenza Respirovirus umano.

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.

Proteomic analysis of virus-host interactions in an infectious context using recombinant viruses.

RNA viruses exhibit small-sized genomes encoding few proteins, but still establish complex networks of protein-protein and RNA-protein interactions within a cell to achieve efficient replication and spreading. Deciphering these interactions is essential to reach a comprehensive understanding of the viral infection process. To study RNA-protein complexes directly in infected cells, we developed a new approach based on recombinant viruses expressing tagged viral proteins that were purified together with their specific RNA partners. High-throughput sequencing was then used to identify these RNA molecules. As a proof of principle, this method was applied to measles virus nucleoprotein (MV-N). It revealed that in addition to full-length genomes, MV-N specifically interacted with a unique population of 5' copy-back defective interfering RNA genomes that we characterized. Such RNA molecules were able to induce strong activation of interferon-stimulated response element promoter preferentially via the cytoplasmic pattern recognition receptor RIG-I protein, demonstrating their biological functionality. Thus, this method provides a new platform to explore biologically active RNA-protein networks that viruses establish within infected cells.

Anastassia Komarova

Unité de Génomique Virale et Vaccination,
Virology Department,
Unité de Génomique Virale et Vaccination, Virology Department

Institut Pasteur, CNRS UMR3569

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Anastassia Komarova

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Unité de Génomique Virale et Vaccination,Virology Department,Unité de Génomique Virale et Vaccination, Virology Department

Institut Pasteur, CNRS UMR3569

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

Unité de Génomique Virale et Vaccination,
Virology Department,
Unité de Génomique Virale et Vaccination, Virology Department