Cytosolic 5'-triphosphate ended viral leader transcript of measles virus as activator of the RIG I-mediated interferon response.

International audienceBACKGROUND: Double stranded RNA (dsRNA) is widely accepted as an RNA motif recognized as a danger signal by the cellular sentries. However, the biology of non-segmented negative strand RNA viruses, or Mononegavirales, is hardly compatible with the production of such dsRNA. METHODOLOGY AND PRINCIPAL FINDINGS: During measles virus infection, the IFN-beta gene transcription was found to be paralleled by the virus transcription, but not by the virus replication. Since the expression of every individual viral mRNA failed to activate the IFN-beta gene, we postulated the involvement of the leader RNA, which is a small not capped and not polyadenylated RNA firstly transcribed by Mononegavirales. The measles virus leader RNA, synthesized both in vitro and in vivo, was efficient in inducing the IFN-beta expression, provided that it was delivered into the cytosol as a 5'-trisphosphate ended RNA. The use of a human cell line expressing a debilitated RIG-I molecule, together with overexpression studies of wild type RIG-I, showed that the IFN-beta induction by virus infection or by leader RNA required RIG-I to be functional. RIG-I binds to leader RNA independently from being 5-trisphosphate ended; while a point mutant, Q299A, predicted to establish contacts with the RNA, fails to bind to leader RNA. Since the 5'-triphosphate is required for optimal RIG-I activation but not for leader RNA binding, our data support that RIG-I is activated upon recognition of the 5'-triphosphate RNA end. CONCLUSIONS/SIGNIFICANCE: RIG-I is proposed to recognize Mononegavirales transcription, which occurs in the cytosol, while scanning cytosolic RNAs, and to trigger an IFN response when encountering a free 5'-triphosphate RNA resulting from a mislocated transcription activity, which is therefore considered as the hallmark of a foreign invader

Dynamiques des activités polymérasiques du virus de la rougeole et induction de l'immunité innée

La réplication du virus de la rougeole (VR) est entièrement cytosolique et met en jeu une ARN-polymérase ARN-dépendante (RdRp) travaillant sur une matrice génomique nucléoprotéine-ARN de polarité négative. Cette RdRp réalise la transcription et la maturation des ARNm viraux (addition d'une coiffe en 5', d'une queue poly-A en 3') correspondant aux 6 gènes N, P, M, F, H, L. Elle assure également la réplication en antigénomes de polarité positive et encapsidés par la nucléoprotéine. Ces derniers, à leur tour, servent de matrice pour la synthèse de génomes également encapsidés. J'ai mis au point la quantification des ARNs du virus de la rougeole (6 ARNm, génome, antigénome) par reverse transcription et PCR quantitative en temps réel en utilisant la technologie SYBR green associée au Light Cycler (Roche). Cet outil m'a permis d'analyser la cinétique des processus régulant les différentes activités de l'ARN-polymérase ARN-dépendante du virus de la rougeole, depuis la libération de la nucléocapside dans le cytoplasme, jusqu'à l'amplification du matériel viral dans la cellule infectée à l'étape tardive de l'infection. J'ai pu estimer pour la première fois la vitesse d'élongation d'une polymérase virale in vivo. J'ai également construit le premier modèle dynamique de l'accumulation des différents ARNs viraux au cours d'une infection virale et la régulation du passage transcription-réplication par la quantité de nucléoprotéine néosynthétisée disponible. En tirant parti du modèle ainsi établi, j'ai pu caractériser l'élément viral qui déclenche l'un des effecteurs de l'immunité innée dans la cellule infectée, la réponse interféron, et définir le senseur cellulaire capable de reconnaître cette molécule comme un "signal danger". Dans un autre volet de ce travail, réalisé avec Hélène Valentin, je me suis intéressé à l'amplification de cette réponse interféron et en particulier au rôle des protéines d'enveloppe du virus de la rougeole. L'infection morbilleuse est caractérisée par la formation de cellules multinucléées géantes (syncytia). Nous proposons la fusion comme mécanisme permettant une amplification de la réponse interféron indépendamment des voies classiques autocrines et paracrines associées aux récepteurs IFNARs et discutons l'implication d'un tel phénomène dans le cycle infectieux du virus au sein de l'organisme. Un second travail réalisé sur les glycoprotéines d'enveloppe m'a permis de montrer l'activation d'un autre effecteur de l'immunité innée, le complément, par la protéine de fusion. Cette activation augmente le ciblage des particules virales et des cellules infectées vers les cellules immunocompétentes et pourrait expliquer pourquoi le virus de la rougeole est un excellent immunogène malgré son activité fortement immunosuppressiveLYON1-BU.Sciences (692662101) / SudocSudocFranceF

Dynamics of Viral RNA Synthesis during Measles Virus Infection

We propose a reference model of the kinetics of a viral RNA-dependent RNA polymerase (vRdRp) activities and its regulation during infection of eucaryotic cells. After measles virus infects a cell, mRNAs from all genes immediately start to accumulate linearly over the first 5 to 6 h and then exponentially until ∼24 h. The change from a linear to an exponential accumulation correlates with de novo synthesis of vRdRp from the incoming template. Expression of the virus nucleoprotein (N) prior to infection shifts the balance in favor of replication. Conversely, inhibition of protein synthesis by cycloheximide favors the latter. The in vivo elongation speed of the viral polymerase is ∼3 nucleotides/s. A similar profile with fivefold-slower kinetics can be obtained using a recombinant virus expressing a structurally altered polymerase. Finally, virions contain only encapsidated genomic, antigenomic, and 5′-end abortive replication fragment RNAs

A rapid and specific real time RT-PCR assay for diagnosis of Toscana virus infection.

International audienceTo scan a virus (TOSV) belongs to the Phlebovirus genus within the Bunyaviridae family. TOSV is an arbovirus transmitted by sandflies. In Mediterranean countries, TOSV is one of the major viral pathogens involved in aseptic meningitis and meningoencephalitis. Development and assessment of a new sensitive and specific real-time RT-PCR assay for TOSV diagnosis. TOSV-specific primers and probe targeting the S-segment of the genome were designed, based on recent TOSV sequences available in public databases. Sensitivity was assessed using 10-fold serial dilutions of a RNA transcript and serial dilutions of TOSV strains isolated from infected human beings. Specificity was determined by testing RNA extracts from closely related Phleboviruses. The assay was then used for TOSV infection diagnosis in 971 clinical samples and for TOSV detection in 2000 sandflies. The real-time RT-PCR assay exhibited a sensitivity of under 257 copies per reaction for the RNA transcripts and 0.0056 and 0.014 TCID50 of Italian and Spanish TOSV genotypes per reaction, respectively. No other close Phleboviruses were detected. TOSV was identified in 17 clinical samples and in 3 sandflies. The assay described is a rapid, robust and reliable real-time RT-PCR test for accurate diagnosis of human TOSV infection as well as for the surveillance of TOSV in vector populations

Cell–cell interactions via non-covalent click chemistry

International audienceMetabolic glycoengineering with unnatural sugars became a valuable tool for introducing recognition markers on the cell membranes via bioorthogonal chemistry. By using this strategy, we functionalized the surface of tumor and T cells using complementary artificial markers based on both β-cyclodextrins (β-CDs) and adamantyl trimers, respectively. Once tied on cell surfaces, the artificial markers induced cell–cell adhesion through non-covalent click chemistry. These unnatural interactions between A459 lung tumor cells and Jurkat T cells triggered the activation of natural killer (NK) cells thanks to the increased production of interleukin-2 (IL-2) in the vicinity of cancer cells, leading ultimately to their cytolysis. The ready-to-use surface markers designed in this study can be easily inserted on the membrane of a wide range of cells previously submitted to metabolic glycoengineering, thereby offering a simple way to investigate and manipulate intercellular interactions

Real-Time Reverse-Transcription Loop-Mediated Isothermal Amplification for Rapid Detection of Rift Valley Fever Virus▿

The development and validation of a one-step, single-tube, real-time accelerated reverse-transcription loop-mediated isothermal amplification (RT-LAMP) for the detection of the L RNA segment of Rift Valley fever virus (RVFV) are described. The assay was performed at a constant temperature (63°C), with a real-time follow-up using a LightCycler and a double-stranded-DNA-intercalating fluorochrome. The assay is highly sensitive and comparable to real-time RT-PCR, with a detection limit of ∼10 RNA copies per assay. However, the RT-LAMP assay is much faster than traditional RT-PCR and generates results in <30 min for most diluted samples. The specificity of the primers was established using other, related arboviruses as well as virus-containing and virus-free sera. The RT-LAMP assay reported here is thus a valuable tool for the rapid detection of RVFV in field diagnostic laboratories

A β-Cyclodextrin-Albumin Conjugate for Enhancing Therapeutic Efficacy of Cytotoxic Drugs

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