A SAP30 Complex Inhibits IFN-β 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 β (IFN-β) 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-β 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-β 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-β promoter chromatin structure

Rift Valley fever phlebovirus NSs protein core domain structure suggests molecular basis for nuclear filaments

Rift Valley fever phlebovirus (RVFV) is a clinically and economically important pathogen increasingly likely to cause widespread epidemics. RVFV virulence depends on the interferon antagonist non-structural protein (NSs), which remains poorly characterized. We identified a stable core domain of RVFV NSs (residues 83-248), and solved its crystal structure, a novel all-helical fold organized into highly ordered fibrils. A hallmark of RVFV pathology is NSs filament formation in infected cell nuclei. Recombinant virus encoding the NSs core domain induced intranuclear filaments, suggesting it contains all essential determinants for nuclear translocation and filament formation. Mutations of key crystal fibril interface residues in viruses encoding full-length NSs completely abrogated intranuclear filament formation in infected cells. We propose the fibrillar arrangement of the NSs core domain in crystals reveals the molecular basis of assembly of this key virulence factor in cell nuclei. Our findings have important implications for fundamental understanding of RVFV virulence.Publisher PDFPeer reviewe

Restriction of Rift Valley Fever Virus Virulence in Mosquito Cells

Arboviruses are maintained in a natural cycle that requires blood-sucking arthropod and vertebrate hosts. Arboviruses are believed to persistently infect their arthropod host without overt pathology and cause acute infection with viremia in their vertebrate host. We have focused on elucidating how a specific arbovirus, Rift Valley fever (RVF) virus, causes cytopathic effect in cells derived from vertebrates and non-cytopathic infection in cells derived from arthropods. We demonstrate that the vertebrate virulence factor, NSs, is functional in arthropod cells but is expressed at significantly lower levels in infected arthropod versus infected vertebrate cells

Inversing the natural hydrogen bonding rule to selectively amplify GC-rich ADAR-edited RNAs

DNA complementarity is expressed by way of three hydrogen bonds for a G:C base pair and two for A:T. As a result, careful control of the denaturation temperature of PCR allows selective amplification of AT-rich alleles. Yet for the same reason, the converse is not possible, selective amplification of GC-rich alleles. Inosine (I) hydrogen bonds to cytosine by two hydrogen bonds while diaminopurine (D) forms three hydrogen bonds with thymine. By substituting dATP by dDTP and dGTP by dITP in a PCR reaction, DNA is obtained in which the natural hydrogen bonding rule is inversed. When PCR is performed at limiting denaturation temperatures, it is possible to recover GC-rich viral genomes and inverted Alu elements embedded in cellular mRNAs resulting from editing by dsRNA dependent host cell adenosine deaminases. The editing of Alu elements in cellular mRNAs was strongly enhanced by type I interferon induction indicating a novel link mRNA metabolism and innate immunity

Association of the interferon-β gene with pericentromeric heterochromatin is dynamically regulated during virus infection through a YY1-dependent mechanism

Nuclear architecture as well as gene nuclear positioning can modulate gene expression. In this work, we have analyzed the nuclear position of the interferon-β (IFN-β) locus, responsible for the establishment of the innate antiviral response, with respect to pericentromeric heterochromatin (PCH) in correlation with virus-induced IFN-β gene expression. Experiments were carried out in two different cell types either non-infected (NI) or during the time course of three different viral infections. In NI cells, we showed a monoallelic IFN-β promoter association with PCH that strongly decreased after viral infection. Dissociation of the IFN-β locus away from these repressive regions preceded strong promoter transcriptional activation and was reversible within 12 h after infection. No dissociation was observed after infection with a virus that abnormally maintained the IFN-β gene in a repressed state. Dissociation induced after virus infection specifically targeted the IFN-β locus without affecting the general structure and nuclear distribution of PCH clusters. Using cell lines stably transfected with wild-type or mutated IFN-β promoters, we identified the proximal region of the IFN-β promoter containing YY1 DNA-binding sites as the region regulating IFN-β promoter association with PCH before as well as during virus infection

Host Alternation Is Necessary to Maintain the Genome Stability of Rift Valley Fever Virus

Arthropod-borne viruses are transmitted among vertebrate hosts by insect vectors. Unusually, Rift Valley fever virus (RVFV) can also be transmitted by direct contacts of animals/humans with infectious tissues. What are the molecular mechanisms and evolutionary events leading to adopt one mode of transmission rather than the other? Viral replication is implied to be different in a vertebrate host and an invertebrate host. The alternating host cycle tends to limit virus evolution by adopting a compromise fitness level for replication in both hosts. To test this hypothesis, we used a cell culture model system to study the evolution of RVFV. We found that freeing RVFV from alternating replication in mammalian and mosquito cells led to large deletions in the NSs gene carrying the virulence factor. Resulting NSs-truncated viruses were able to protect mice from a challenge with a virulent RVFV. Thus, in nature, virulence is likely maintained by continuous alternating passages between vertebrates and insects. Thereby, depending on the mode of transmission adopted, the evolution of RVFV will be of major importance to predict the outcome of outbreaks

Expression of recombinant Araraquara Hantavirus nucleoprotein in insect cells and its use as an antigen for immunodetection compared to the same antigen expressed in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Antigens for Hantavirus serological tests have been produced using DNA recombinant technology for more than twenty years. Several different strategies have been used for that purpose. All of them avoid the risks and difficulties involved in multiplying Hantavirus in the laboratory. In Brazil, the Araraquara virus is one of the main causes of Hantavirus Cardio-Pulmonary Syndrome (HCPS).</p> <p>Methods</p> <p>In this investigation, we report the expression of the N protein of the Araraquara Hantavirus in a Baculovirus Expression System, the use of this protein in IgM and IgG ELISA and comparison with the same antigen generated in <it>E. coli</it>.</p> <p>Results</p> <p>The protein obtained, and purified in a nickel column, was effectively recognized by antibodies from confirmed HCPS patients. Comparison of the baculovirus generated antigen with the N protein produced in <it>E. coli </it>showed that both were equally effective in terms of sensitivity and specificity.</p> <p>Conclusions</p> <p>Our results therefore indicate that either of these proteins can be used in serological tests in Brazil.</p

Quantitative analysis of particles, genomes and infectious particles in supernatants of haemorrhagic fever virus cell cultures

Information on the replication of viral haemorrhagic fever viruses is not readily available and has never been analysed in a comparative approach. Here, we compared the cell culture growth characteristics of haemorrhagic fever viruses (HFV), of the Arenaviridae, Filoviridae, Bunyaviridae, and Flavivridae virus families by performing quantitative analysis of cell culture supernatants by (i) electron microscopy for the quantification of virus particles, (ii) quantitative real time PCR for the quantification of genomes, and (iii) determination of focus forming units by coating fluorescent antibodies to infected cell monolayers for the quantification of virus infectivity

Tissue Tropism and Target Cells of NSs-Deleted Rift Valley Fever Virus in Live Immunodeficient Mice

Rift Valley fever, caused by a member of the Bunyaviridae family, has spread during recent years to most sub-Saharan African countries, in Egypt and in the Arabian peninsula. The virus can be transmitted by insect vectors or by direct contacts with infectious tissues. The analysis of virus replication and dissemination in laboratory animals has been hampered by the need to euthanize sufficient numbers of animals and to assay appropriate organs at various time points after infection to evaluate the viral replication. By following the bioluminescence and fluorescence of Rift Valley fever viruses expressing light reporters, we were able to track the real-time dissemination of the viruses in live immunodeficient mice. We showed that the first infected organs were the thymus, spleen and liver, but the liver rapidly became the main location of viral replication. Phagocytes also appeared as important targets, and their systemic depletion by use of clodronate liposomes decreased the number of viruses in the blood, delayed the viral dissemination and prolonged the survival of the infected mice

Infection and Transmission of Rift Valley Fever Viruses Lacking the NSs and/or NSm Genes in Mosquitoes: Potential Role for NSm in Mosquito Infection

Rift Valley fever virus is transmitted mainly by mosquitoes and causes disease in humans and animals throughout Africa and the Arabian Peninsula. The impact of disease is large in terms of human illness and mortality, and economic impact on the livestock industry. For these reasons, and because there is a risk of this virus spreading to Europe and North America, it is important to develop a vaccine that is stable, safe and effective in preventing infection. Potential vaccine viruses have been developed through deletion of two genes (NSs and NSm) affecting virus virulence. Because this virus is normally transmitted by mosquitoes we must determine the effects of the deletions in these vaccine viruses on their ability to infect and be transmitted by mosquitoes. An optimal vaccine virus would not infect or be transmitted. The viruses were tested in two mosquito species: Aedes aegypti and Culex quinquefasciatus. Deletion of the NSm gene reduced infection of Ae. aegypti mosquitoes indicating a role for the NSm protein in mosquito infection. The virus with deletion of both NSs and NSm genes was the best vaccine candidate since it did not infect Ae. aegypti and showed reduced infection and transmission rates in Cx. quinquefasciatus