[Rift Valley fever virus and the amazing NSs protein]. / Le virus de la fièvre de la vallée du Rift et son étonnante protéine NSs.

Rift Valley Fever Virus (RVFV) is an emerging zoonotic pathogen transmitted to humans and livestock through mosquito bites, which was first isolated in Kenya in 1930. The virus is classified by the WHO among the pathogens for which there is an urgent need to develop research, diagnostics, and therapies. However, the efforts developed to control the virus remain limited, and the virus is not well characterized. In this article, we will introduce RVFV and then focus on its virulence factor, the nonstructural protein NSs. We will mainly discuss the ability of this viral protein to form amyloid-like fibrils and its implication in the neurotoxicity associated with RVFV infection.

TITLE: Le virus de la fièvre de la vallée du Rift et son étonnante protéine NSs. ABSTRACT: Le virus de la fièvre de la vallée du Rift (VFVR) est un agent pathogène transmis à l'homme et au bétail par la piqûre de moustiques. Ce virus, découvert au Kenya en 1930, est considéré par l'Organisation mondiale de la santé comme présentant un risque important de provoquer de vastes épidémies. Les moyens dédiés à la lutte contre le VFVR restent toutefois particulièrement limités et le virus est mal connu. Dans cette Synthèse, nous nous attacherons à présenter ce virus avant de nous intéresser plus spécifiquement à son facteur de virulence, la protéine NSs. Nous discuterons la capacité de cette protéine virale à former des fibrilles de type amyloïde et son implication dans la neurotoxicité du virus chez les animaux infectés.

NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice.

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.

Novel Toscana Virus Reverse Genetics System Establishes NSs as an Antagonist of Type I Interferon Responses.

The sand fly-borne Toscana virus (TOSV) is the major cause of human meningoencephalitis in the Mediterranean basin during the summer season. In this work, we have developed a T7 RNA polymerase-driven reverse genetics system to recover infectious particles of a lineage B strain of TOSV. The viral protein pattern and growth properties of the rescued virus (rTOSV) were found to be similar to those of the corresponding wild-type (wt) virus. Using this system, we genetically engineered a TOSV mutant lacking expression of the non-structural protein NSs (rTOSVɸNSs). Unlike rTOSV and the wt virus, rTOSVɸNSs was unable to (i) suppress interferon (IFN)-b messenger RNA induction; and (ii) grow efficiently in cells producing IFN-b. Together, our results highlight the importance of NSs for TOSV in evading the IFN response and provide a comprehensive toolbox to investigate the TOSV life cycle in mammalian and insect host cells, including several novel polyclonal antibodies.

[Cell biology of phlebovirus entry]. / Entrée cellulaire des phlébovirus chez l'hôte mammifère.

Phleboviruses constitute a large group of arthropod-borne viruses (arboviruses), mainly transmitted to their hosts by sandflies and ticks, occasionally by mosquitoes. These viruses have a worldwide distribution and many cause serious diseases - often fatal - in both domestic animals and humans. The global warming, the apparent wide distribution of arthropod reservoirs, and the increasing number of outbreaks show that phleboviruses must be taken seriously as emerging disease agents. This review proposes to focus on the early steps of phlebovirus infection, from virus binding to penetration into the cytosol. We address the most recent knowledge and advances in the entry of these viruses into vertebrate host cells, including virus receptors, cellular factors, endocytic pathways, and fusion.

Deciphering Virus Entry with Fluorescently Labeled Viral Particles.

To infect host cells, viruses have to gain access to the intracellular compartment. The infection process starts with the attachment of viruses to the cell surface. Then a complex series of events, highly dynamic, tightly intricate, and often hard to investigate, follows. This includes virus displacement at the plasma membrane, binding to receptors, signaling, internalization, and release of the viral genome and material into the cytosol. In the past decades, the emergence of sensitive, accurate fluorescence-based technologies has opened new perspectives of investigations in the field. Visualization of single viral particles in fixed and living cells as well as quantification of each virus entry step has been made possible. Here we describe the procedure to fluorescently label viral particles. We also illustrate how to use this powerful tool to decipher the entry of viruses with the most recent fluorescence-based techniques such as high-speed confocal and total internal reflection microscopy, flow cytometry, and fluorimetry.

DDX3 suppresses type I interferons and favors viral replication during Arenavirus infection.

Several arenaviruses cause hemorrhagic fever (HF) diseases that are associated with high morbidity and mortality in humans. Accordingly, HF arenaviruses have been listed as top-priority emerging diseases for which countermeasures are urgently needed. Because arenavirus nucleoprotein (NP) plays critical roles in both virus multiplication and immune-evasion, we used an unbiased proteomic approach to identify NP-interacting proteins in human cells. DDX3, a DEAD-box ATP-dependent-RNA-helicase, interacted with NP in both NP-transfected and virus-infected cells. Importantly, DDX3 deficiency compromised the propagation of both Old and New World arenaviruses, including the HF arenaviruses Lassa and Junin viruses. The DDX3 role in promoting arenavirus multiplication associated with both a previously un-recognized DDX3 inhibitory role in type I interferon production in arenavirus infected cells and a positive DDX3 effect on arenavirus RNA synthesis that was dependent on its ATPase and Helicase activities. Our results uncover novel mechanisms used by arenaviruses to exploit the host machinery and subvert immunity, singling out DDX3 as a potential host target for developing new therapies against highly pathogenic arenaviruses.

Differential Use of the C-Type Lectins L-SIGN and DC-SIGN for Phlebovirus Endocytosis.

Bunyaviruses represent a growing threat to humans and livestock globally. The receptors, cellular factors and endocytic pathways used by these emerging pathogens to infect cells remain largely unidentified and poorly characterized. DC-SIGN is a C-type lectin highly expressed on dermal dendritic cells that has been found to act as an authentic entry receptor for many phleboviruses (Bunyaviridae), including Rift Valley fever virus (RVFV), Toscana virus (TOSV) and Uukuniemi virus (UUKV). We found that these phleboviruses can exploit another C-type lectin, L-SIGN, for infection. L-SIGN shares 77% sequence homology with DC-SIGN and is expressed on liver sinusoidal endothelial cells. L-SIGN is required for UUKV binding but not for virus internalization. An endocytosis-defective mutant of L-SIGN was still able to mediate virus uptake and infection, indicating that L-SIGN acts as an attachment receptor for phleboviruses rather than an endocytic receptor. Our results point out a fundamental difference in the use of the C-type lectins L-SIGN and DC-SIGN by UUKV to enter cells, although both proteins are closely related in terms of molecular structure and biological function. This study sheds new light on the molecular mechanisms by which phleboviruses target the liver and also highlights the added complexity in virus-receptor interactions beyond attachment.

CD28 Deficiency Enhances Type I IFN Production by Murine Plasmacytoid Dendritic Cells.

Type I IFNs (IFN-I) are key innate mediators that create a profound antiviral state and orchestrate the activation of almost all immune cells. Plasmacytoid dendritic cells (pDCs) are the most powerful IFN-I-producing cells and play important roles during viral infections, cancer, and autoimmune diseases. By comparing gene expression profiles of murine pDCs and conventional DCs, we found that CD28, a prototypic T cell stimulatory receptor, was highly expressed in pDCs. Strikingly, CD28 acted as a negative regulator of pDC IFN-I production upon TLR stimulation but did not affect pDC survival or maturation. Importantly, cell-intrinsic CD28 expression restrained pDC (and systemic) IFN-I production during in vivo RNA and DNA viral infections, limiting antiviral responses and enhancing viral growth early after exposure. Finally, CD28 also downregulated IFN-I response upon skin injury. Our study identified a new pDC regulatory mechanism by which the same CD28 molecule that promotes stimulation in most cells that express it is co-opted to negatively regulate pDC IFN-I production and limit innate responses.

Characterization of wild-type and alternate transcription termination signals in the Rift Valley fever virus genome.

Rift Valley fever (RVF) is a mosquito-borne zoonotic disease caused by a phlebovirus of the family Bunyaviridae, which affects humans and ruminants in Africa and the Middle East. RFV virus (RVFV) possesses a single-stranded tripartite RNA genome of negative/ambisense polarity. The S segment utilizes the ambisense strategy and codes for two proteins, the N nucleoprotein and the nonstructural NSs protein, in opposite orientations. The two open reading frames (ORFs) are separated by an intergenic region (IGR) highly conserved among strains and containing a motif, 5'-GCUGC-3', present on the genome and antigenome, which was shown previously to play a role in transcription termination (C. G. Albarino, B. H. Bird, and S. T. Nichol, J. Virol. 81:5246-5256, 2007; T. Ikegami, S. Won, C. J. Peters, and S. Makino, J. Virol. 81:8421-8438, 2007). Here, we created recombinant RVFVs with mutations or deletions in the IGR and showed that the substitution of the motif sequence by a series of five A's inactivated transcription termination at the wild-type site but allowed the transcriptase to recognize another site with the consensus sequence present in the opposite ORF. Similar situations were observed for mutants in which the motif was still present in the IGR but located close to the stop codon of the translated ORF, supporting a model in which transcription is coupled to translation and translocating ribosomes abrogate transcription termination. Our data also showed that the signal tolerated some sequence variations, since mutation into 5'-GCAGC-3' was functional, and 5'-GUAGC-3' is likely the signal for the termination of the 3' end of the L mRNA.

A SAP30 complex inhibits IFN-beta expression in Rift Valley fever virus infected cells.

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon beta (IFN-beta) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-beta gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-beta promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-beta promoter chromatin structure.