Anastassia V. Komarova

mRNA Translation Regulation by the Gly-Ala Repeat of Epstein-Barr Virus Nuclear Antigen 1

ABSTRACT 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.

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 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.

Comparative analysis of viral RNA signatures on different RIG-I-like receptors

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

Evidence that PTB does not stimulate HCV IRES-driven translation

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.

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

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.

Identification of RNA partners of viral proteins in infected cells

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.

Virus versus host cell translation: love and hate stories.

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. Abstract 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.

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 because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

Nonencapsidated 5′ Copy-Back Defective Interfering Genomes Produced by Recombinant Measles Viruses Are Recognized by RIG-I and LGP2 but Not MDA5

ABSTRACT Attenuated measles virus (MV) is one of the most effective and safe vaccines available, making it an attractive candidate vector for preventing other infectious diseases. Yet the great capacity of this vaccine still needs to be understood at the molecular level. MV vaccine strains have different type I interferon (IFN)-inducing abilities that partially depend on the presence of 5′ copy-back defective interfering genomes (DI-RNAs). DI-RNAs are pathogen-associated molecular patterns recognized by RIG-I-like receptors (RLRs) (RIG-I, MDA5, and LGP2) that activate innate immune signaling and shape the adaptive immune response. In this study, we characterized the DI-RNAs produced by various modified recombinant MVs (rMVs), including vaccine candidates, as well as wild-type MV. All tested rMVs produced 5′ copy-back DI-RNAs that were different in length and nucleotide sequence but still respected the so-called “rule of six.” We correlated the presence of DI-RNAs with a larger stimulation of the IFN-β pathway and compared their immunostimulatory potentials. Importantly, we revealed that encapsidation of DI-RNA molecules within the MV nucleocapsid abolished their immunoactive properties. Furthermore, we identified specific interactions of DI-RNAs with both RIG-I and LGP2 but not MDA5. Our results suggest that DI-RNAs produced by rMV vaccine candidates may indeed strengthen their efficiency by triggering RLR signaling. IMPORTANCE Having been administered to hundreds of millions of children, the live attenuated measles virus (MV) vaccine is the safest and most widely used human vaccine, providing high protection with long-term memory. Additionally, recombinant MVs carrying heterologous antigens are promising vectors for new vaccines. The great capacity of this vaccine still needs to be elucidated at the molecular level. Here we document that recombinant MVs produce defective interfering genomes that have high immunostimulatory properties via their binding to RIG-I and LGP2 proteins, both of which are cytosolic nonself RNA sensors of innate immunity. Defective interfering genome production during viral replication should be considered of great importance due to the immunostimulatory properties of these genomes as intrinsic adjuvants produced by the vector that increase recognition by the innate immune system.