Characterization of RVFV Nucleocapsid Protein Binding Sites on RNA by iCLIP-seq.

The nucleocapsid protein (N) in Rift Valley fever virus is an RNA-binding protein that functions in viral transcription, replication, and packaging. In this chapter, the method for studying protein-RNA interactions in context of viral infection using individual nucleotide resolution, cross-linking, immunoprecipitation, and sequencing (iCLIP-seq) is explained. The method is useful for identifying the interactions between both host and viral RNAs with N and can identify RNA motifs that interact with the protein of interest.

HSP90 is part of a protein complex with the L polymerase of Rift Valley fever phlebovirus and prevents its degradation by the proteasome during the viral genome replication/transcription stage.

The mosquito-borne Rift Valley fever virus (RVFV) from the Phenuiviridae family is a single-stranded RNA virus that causes the re-emerging zoonotic disease Rift Valley fever (RVF). Classified as a Category A agent by the NIH, RVFV infection can cause debilitating disease or death in humans and lead to devastating economic impacts by causing abortion storms in pregnant cattle. In a previous study, we showed that the host chaperone protein HSP90 is an RVFV-associated host factor that plays a critical role post viral entry, during the active phase of viral genome replication/transcription. In this study, we have elucidated the molecular mechanisms behind the regulatory effect of HSP90 during infection with RVFV. Our results demonstrate that during the early infection phase, host HSP90 associates with the viral RNA-dependent RNA polymerase (L protein) and prevents its degradation through the proteasome, resulting in increased viral replication.

An LIR motif in the Rift Valley fever virus NSs protein is critical for the interaction with LC3 family members and inhibition of autophagy.

Rift Valley fever virus (RVFV) is a viral zoonosis that causes severe disease in ruminants and humans. The nonstructural small (NSs) protein is the primary virulence factor of RVFV that suppresses the host's antiviral innate immune response. Bioinformatic analysis and AlphaFold structural modeling identified four putative LC3-interacting regions (LIR) motifs (NSs 1-4) in the RVFV NSs protein, which suggest that NSs interacts with the host LC3-family proteins. Using, isothermal titration calorimetry, X-ray crystallography, co-immunoprecipitation, and co-localization experiments, the C-terminal LIR motif (NSs4) was confirmed to interact with all six human LC3 proteins. Phenylalanine at position 261 (F261) within NSs4 was found to be critical for the interaction of NSs with LC3, retention of LC3 in the nucleus, as well as the inhibition of autophagy in RVFV infected cells. These results provide mechanistic insights into the ability of RVFV to overcome antiviral autophagy through the interaction of NSs with LC3 proteins.

Alternative Splicing of RIOK3 Engages the Noncanonical NFκB Pathway during Rift Valley Fever Virus Infection.

Although the noncanonical NFκB pathway was originally identified as a cellular pathway contributing to lymphoid organogenesis, in the past 20 years, its involvement in innate immunity has become more appreciated. In particular, the noncanonical NFκB pathway has been found to be activated and even exploited by some RNA viruses during infection. Intriguingly, activation of this pathway has been shown to have a role in disrupting transcription of type 1 interferon (IFN), suggesting a rationale for why this response could be co-opted by some viruses. Rift Valley fever virus (RVFV) is a trisegmented ambisense RNA virus that poses a considerable threat to domestic livestock and human health. Previously, we showed the atypical kinase RIOK3 is important for mounting an IFN response to RVFV infection of human epithelial cells, and shortly following infection with RVFV (MP12 strain), RIOK3 mRNA is alternatively spliced to its X2 isoform that encodes a truncated RIOK3 protein. Alternative splicing of RIOK3 mRNA has an inhibitory effect on the IFN response but also stimulates an NFκB-mediated inflammatory response. Here, we demonstrate alternative splicing of RIOK3 mRNA is associated with activation of the noncanonical NFκB pathway and suggest this pathway is co-opted by RVFV (MP12) to enhance viral success during infection.

Low-density lipoprotein receptor-related protein 1 (LRP1) as an auxiliary host factor for RNA viruses.

Viruses with an RNA genome are often the cause of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that are resistant to Rift Valley fever virus (RVFV). This screen returned the low-density lipoprotein receptor-related protein 1 (LRP1) as a top hit, a plasma membrane protein involved in a wide variety of cell activities. Inactivation of LRP1 in human cells reduced RVFV RNA levels already at the attachment and entry stages of infection. Moreover, the role of LRP1 in promoting RVFV infection was dependent on physiological levels of cholesterol and on endocytosis. In the human cell line HuH-7, LRP1 also promoted early infection stages of sandfly fever Sicilian virus and La Crosse virus, but had a minor effect on late infection by vesicular stomatitis virus, whereas encephalomyocarditis virus was entirely LRP1-independent. Moreover, siRNA experiments in human Calu-3 cells demonstrated that also SARS-CoV-2 infection benefitted from LRP1. Thus, we identified LRP1 as a host factor that supports infection by a spectrum of RNA viruses.

Rift Valley fever virus Gn V5-epitope tagged virus enables identification of UBR4 as a Gn interacting protein that facilitates Rift Valley fever virus production.

Rift Valley fever virus (RVFV) is an arbovirus that was first reported in the Rift Valley of Kenya which causes significant disease in humans and livestock. RVFV is a tri-segmented, negative-sense RNA virus consisting of a L, M, and S segments with the M segment encoding the glycoproteins Gn and Gc. Host factors that interact with Gn are largely unknown. To this end, two viruses containing an epitope tag (V5) on the Gn protein in position 105 or 229 (V5Gn105 and V5Gn229) were generated using the RVFV MP-12 vaccine strain as a backbone. The V5-tag insertion minimally impacted Gn functionality as measured by replication kinetics, Gn localization, and antibody neutralization assays. A proteomics-based approach was used to identify novel Gn-binding host proteins, including the E3 ubiquitin-protein ligase, UBR4. Depletion of UBR4 resulted in a significant decrease in RVFV titers and a reduction in viral RNA production.

Visualizing the ribonucleoprotein content of single bunyavirus virions reveals more efficient genome packaging in the arthropod host.

Bunyaviruses have a genome that is divided over multiple segments. Genome segmentation complicates the generation of progeny virus, since each newly formed virus particle should preferably contain a full set of genome segments in order to disseminate efficiently within and between hosts. Here, we combine immunofluorescence and fluorescence in situ hybridization techniques to simultaneously visualize bunyavirus progeny virions and their genomic content at single-molecule resolution in the context of singly infected cells. Using Rift Valley fever virus and Schmallenberg virus as prototype tri-segmented bunyaviruses, we show that bunyavirus genome packaging is influenced by the intracellular viral genome content of individual cells, which results in greatly variable packaging efficiencies within a cell population. We further show that bunyavirus genome packaging is more efficient in insect cells compared to mammalian cells and provide new insights on the possibility that incomplete particles may contribute to bunyavirus spread as well.

Reproducing the Rift Valley fever virus mosquito-lamb-mosquito transmission cycle.

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus that is pathogenic to ruminants and humans. The virus is endemic to Africa and the Arabian Peninsula where outbreaks are characterized by abortion storms and mortality of newborns, particularly in sheep herds. Vector competence experiments in laboratory settings have suggested that over 50 mosquito species are capable of transmitting RVFV. Transmission of mosquito-borne viruses in the field is however influenced by numerous factors, including population densities, blood feeding behavior, extrinsic incubation period, longevity of vectors, and viremia levels in vertebrate hosts. Animal models to study these important aspects of RVFV transmission are currently lacking. In the present work, RVFV was transmitted to European (Texel-swifter cross-breed) lambs by laboratory-reared Aedes aegypti mosquitoes that were infected either by membrane feeding on a virus-spiked blood meal or by feeding on lambs that developed viremia after intravenous inoculation of RVFV. Feeding of mosquitoes on viremic lambs resulted in strikingly higher infection rates as compared to membrane feeding. Subsequent transmission of RVFV from lamb to lamb by infected mosquitoes was highly efficient in both models. The animal models described here can be used to study mosquito-mediated transmission of RVFV among the major natural target species and to evaluate the efficacy of vaccines against mosquito-mediated RVFV infection.

The NSs Protein Encoded by the Virulent Strain of Rift Valley Fever Virus Targets the Expression of Abl2 and the Actin Cytoskeleton of the Host, Affecting Cell Mobility, Cell Shape, and Cell-Cell Adhesion.

Rift Valley fever virus (RVFV) is a highly pathogenic zoonotic arbovirus endemic in many African countries and the Arabian Peninsula. Animal infections cause high rates of mortality and abortion among sheep, goats, and cattle. In humans, an estimated 1 to 2% of RVFV infections result in severe disease (encephalitis, hepatitis, or retinitis) with a high rate of lethality when associated with hemorrhagic fever. The RVFV NSs protein, which is the main virulence factor, counteracts the host innate antiviral response to favor viral replication and spread. However, the mechanisms underlying RVFV-induced cytopathic effects and the role of NSs in these alterations remain for the most part unknown. In this work, we have analyzed the effects of NSs expression on the actin cytoskeleton while conducting infections with the NSs-expressing virulent (ZH548) and attenuated (MP12) strains of RVFV and the non-NSs-expressing avirulent (ZH548ΔNSs) strain, as well as after the ectopic expression of NSs. In macrophages, fibroblasts, and hepatocytes, NSs expression prevented the upregulation of Abl2 (a major regulator of the actin cytoskeleton) expression otherwise induced by avirulent infections and identified here as part of the antiviral response. The presence of NSs was also linked to an increased mobility of ZH548-infected cells compared to ZH548ΔNSs-infected fibroblasts and to strong changes in cell morphology in nonmigrating hepatocytes, with reduction of lamellipodia, cell spreading, and dissolution of adherens junctions reminiscent of the ZH548-induced cytopathic effects observed in vivo Finally, we show evidence of the presence of NSs within long actin-rich structures associated with NSs dissemination from NSs-expressing toward non-NSs-expressing cells.IMPORTANCE Rift Valley fever virus (RVFV) is a dangerous human and animal pathogen that was ranked by the World Health Organization in 2018 as among the eight pathogens of most concern for being likely to cause wide epidemics in the near future and for which there are no, or insufficient, countermeasures. The focus of this work is to address the question of the mechanisms underlying RVFV-induced cytopathic effects that participate in RVFV pathogenicity. We demonstrate here that RVFV targets cell adhesion and the actin cytoskeleton at the transcriptional and cellular level, affecting cell mobility and inducing cell shape collapse, along with distortion of cell-cell adhesion. All these effects may participate in RVFV-induced pathogenicity, facilitate virulent RVFV dissemination, and thus constitute interesting potential targets for future development of antiviral therapeutic strategies that, in the case of RVFV, as with several other emerging arboviruses, are presently lacking.

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.

A DNA Vaccine Delivery Platform Based on Elastin-Like Recombinamer Nanosystems for Rift Valley Fever Virus.

This work analyzes the immunogenicity of six genetically engineered constructs based on elastin-like recombinamers (ELRs) fused to the Gn glycoprotein from Rift Valley fever virus (RVFV). Upon transfection, all constructs showed no effect on cell viability. While fusion constructs including ELR blocks containing hydrophobic amino acids (alanine or isoleucine) did not increase the expression of viral Gn in eukaryotic cells, glutamic acid- or valine-rich fusion proteins showed enhanced expression levels compared with the constructs encoding the viral antigen alone. However, in vivo DNA plasmid immunization assays determined that the more hydrophobic constructs reduced viremia levels after RVFV challenge to a higher extent than glutamic- or valine-rich encoding plasmids and were better inducers of cellular immunity as judged by in vitro restimulation experiments. Although the Gn-ELR fusion constructs did not surpass the protective efficacy of a plasmid vaccine expressing nonfused Gn, our results warrant further experiments directed to take advantage of the immunomodulatory potential of ELR biomaterials for improving vaccines against infectious diseases.

Elongin C Contributes to RNA Polymerase II Degradation by the Interferon Antagonist NSs of La Crosse Orthobunyavirus.

Mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) causes up to 100 annual cases of severe meningoencephalitis in children and young adults in the United States. A major virulence factor of LACV is the nonstructural protein NSs, which inhibits host cell mRNA synthesis to prevent the induction of antiviral type I interferons (IFN-α/ß). To achieve this host transcriptional shutoff, LACV NSs drives the proteasomal degradation of RPB1, the large subunit of mammalian RNA polymerase II. Here, we show that NSs acts in a surprisingly rapid manner, as RPB1 degradation was commencing already at 1 h postinfection. The RPB1 degradation was partially dependent on the cellular E3 ubiquitin ligase subunit Elongin C. Consequently, removal of Elongin C, but also of the subunits Elongin A or B by siRNA transfection partially rescued general RNAP II transcription and IFN-beta mRNA synthesis from the blockade by NSs. In line with these results, LACV NSs was found to trigger the redistribution of Elongin C out of nucleolar speckles, which, however, is an epiphenomenon rather than part of the NSs mechanism. Our study also shows that the molecular phenotype of LACV NSs is different from RNA polymerase II inhibitors like α-amanitin or Rift Valley fever virus NSs, indicating that LACV is unique in involving the Elongin complex to shut off host transcription and IFN response.IMPORTANCE The mosquito-borne La Crosse virus (LACV; genus Orthobunyavirus, family Peribunyaviridae, order Bunyavirales) is prevalent in the United States and can cause severe childhood meningoencephalitis. Its main virulence factor, the nonstructural protein NSs, is a strong inhibitor of the antiviral type I interferon (IFN) system. NSs acts by imposing a global host mRNA synthesis shutoff, mediated by NSs-driven proteasomal degradation of the RPB1 subunit of RNA polymerase II. Here, we show that RPB1 degradation commences as early as 1 h postinfection, and identify the E3 ubiquitin ligase subunit Elongin C (and its binding partners Elongins A and B) as an NSs cofactor involved in RPB1 degradation and in suppression of global as well as IFN-related mRNA synthesis.

Microencapsulated plasmids expressing Gn and Gc glycoproteins of Rift Valley Fever virus enhance humoral immune response in mice.

OBJECTIVES: The aim of the current study was to develop biodegradable alginate (ALG)/poly-L-lysine (PLL) microcapsules (MC) with entrapped plasmids expressing Gn and Gc glycoproteins of Rift Valley Fever virus (RVFV) and to evaluate the humoral immune response in mice. RESULTS: Expressing phRVF/Gn and phRVF/Gc plasmids which encode full-sized Gn and Gc glycoproteins and contain signal fusion protein F sequences of human parainfluenza (HPIV-1) were constructed. To protect the plasmids from cleavage by extracellular nucleases, they were entrapped into multilayer ALG/PLL microcapsules by layer-by-layer technique. To study the efficacy of humoral immune response, both native and microencapsulated plasmids were injected intramuscular into BALB/c mice. The humoral response in the mice immunized with free plasmids was characterized by virus-neutralizing antibody induction (with titres 1:4 to 1:8), while the injection of microencapsulated plasmids allowed to increase the titre level (from 1:16 to 1:32). CONCLUSION: The plasmids microencapsulated in biodegradable MC could be promising for development of DNA vaccines against RVFV.

NSs Filament Formation Is Important but Not Sufficient for RVFV Virulence In Vivo.

Rift Valley fever virus (RVFV) is a mosquito-borne phlebovirus that represents as a serious health threat to both domestic animals and humans. The viral protein NSs is the key virulence factor of RVFV, and has been proposed that NSs nuclear filament formation is critical for its virulence. However, the detailed mechanisms are currently unclear. Here, we generated a T7 RNA polymerase-driven RVFV reverse genetics system based on a strain imported into China (BJ01). Several NSs mutations (T1, T3 and T4) were introduced into the system for investigating the correlation between NSs filament formation and virulence in vivo. The NSs T1 mutant showed distinct NSs filament in the nuclei of infected cells, the T3 mutant diffusively localized in the cytoplasm and the T4 mutant showed fragmented nuclear filament formation. Infection of BALB/c mice with these NSs mutant viruses revealed that the in vivo virulence was severely compromised for all three NSs mutants, including the T1 mutant. This suggests that NSs filament formation is not directly correlated with RVFV virulence in vivo. Results from this study not only shed new light on the virulence mechanism of RVFV NSs but also provided tools for future in-depth investigations of RVFV pathogenesis and anti-RVFV drug screening.

Structure of a functional cap-binding domain in Rift Valley fever virus L protein.

Rift Valley fever virus (RVFV) belongs to the family of Phenuiviridae within the order of Bunyavirales. The virus may cause fatal disease both in livestock and humans, and therefore, is of great economical and public health relevance. In analogy to the influenza virus polymerase complex, the bunyavirus L protein is assumed to bind to and cleave off cap structures of cellular mRNAs to prime viral transcription. However, even though the presence of an endonuclease in the N-terminal domain of the L protein has been demonstrated for several bunyaviruses, there is no evidence for a cap-binding site within the L protein. We solved the structure of a C-terminal 117 amino acid-long domain of the RVFV L protein by X-ray crystallography. The overall fold of the domain shows high similarity to influenza virus PB2 cap-binding domain and the putative non-functional cap-binding domain of reptarenaviruses. Upon co-crystallization with m7GTP, we detected the cap-analogue bound between two aromatic side chains as it has been described for other cap-binding proteins. We observed weak but specific interaction with m7GTP rather than GTP in vitro using isothermal titration calorimetry. The importance of m7GTP-binding residues for viral transcription was validated using a RVFV minigenome system. In summary, we provide structural and functional evidence for a cap-binding site located within the L protein of a virus from the Bunyavirales order.

Transcriptome profiling in Rift Valley fever virus infected cells reveals modified transcriptional and alternative splicing programs.

Rift Valley fever virus (RVFV) is a negative-sense RNA virus belonging to the Phenuiviridae family that infects both domestic livestock and humans. The NIAID has designated RVFV as a Category A priority emerging pathogen due to the devastating public health outcomes associated with epidemic outbreaks. However, there is no licensed treatment or vaccine approved for human use. Therefore it is of great interest to understand RVFV pathogenesis in infected hosts in order to facilitate creation of targeted therapies and treatment options. Here we provide insight into the host-pathogen interface in human HEK293 cells during RVFV MP-12 strain infection using high-throughput mRNA sequencing technology. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes showed robust innate immune and cytokine-mediated inflammatory pathway activation as well as alterations in pathways associated with fatty acid metabolism and extracellular matrix receptor signaling. We also analyzed the promoter regions of DEGs for patterns in transcription factor binding sites, and found several that are known to act synergistically to impact apoptosis, immunity, metabolism, and cell growth and differentiation. Lastly, we noted dramatic changes in host alternative splicing patterns in genes associated with mRNA decay and surveillance, RNA transport, and DNA repair. This study has improved our understanding of RVFV pathogenesis and has provided novel insight into pathways and signaling modules important for RVFV diagnostics and therapeutic development.

Expression of Rift Valley fever virus N-protein in Nicotiana benthamiana for use as a diagnostic antigen.

BACKGROUND: Rift Valley fever virus (RVFV), the causative agent of Rift Valley fever, is an enveloped single-stranded negative-sense RNA virus in the genus Phlebovirus, family Bunyaviridae. The virus is spread by infected mosquitoes and affects ruminants and humans, causing abortion storms in pregnant ruminants, high neonatal mortality in animals, and morbidity and occasional fatalities in humans. The disease is endemic in parts of Africa and the Arabian Peninsula, but is described as emerging due to the wide range of mosquitoes that could spread the disease into non-endemic regions. There are different tests for determining whether animals are infected with or have been exposed to RVFV. The most common serological test is antibody ELISA, which detects host immunoglobulins M or G produced specifically in response to infection with RVFV. The presence of antibodies to RVFV nucleocapsid protein (N-protein) is among the best indicators of RVFV exposure in animals. This work describes an investigation of the feasibility of producing a recombinant N-protein in Nicotiana benthamiana and using it in an ELISA. RESULTS: The human-codon optimised RVFV N-protein was successfully expressed in N. benthamiana via Agrobacterium-mediated infiltration of leaves. The recombinant protein was detected as monomers and dimers with maximum protein yields calculated to be 500-558 mg/kg of fresh plant leaves. The identity of the protein was confirmed by liquid chromatography-mass spectrometry (LC-MS) resulting in 87.35% coverage, with 264 unique peptides. Transmission electron microscopy revealed that the protein forms ring structures of ~ 10 nm in diameter. Preliminary data revealed that the protein could successfully differentiate between sera of RVFV-infected sheep and from sera of those not infected with the virus. CONCLUSIONS: To the best of our knowledge this is the first study demonstrating the successful production of RVFV N-protein as a diagnostic reagent by Agrobacterium-mediated transient heterologous expression in N. benthamiana. Preliminary testing of the antigen showed its ability to distinguish RVFV-positive animal sera from RVFV negative animal sera when used in an enzyme linked immunosorbent assay (ELISA). The cost-effective, scalable and simple production method has great potential for use in developing countries where rapid diagnosis of RVFV is necessary.

NSs protein of severe fever with thrombocytopenia syndrome virus suppresses interferon production through different mechanism than Rift Valley fever virus.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly identified Phlebovirus that causes severe fever with thrombocytopenia syndrome. Our study demonstrated that SFTSV NSs functioned as IFN antagonist mainly by suppressing TBK1/IKKε-IRF3 signaling pathway. NSs interacted with and relocalized TANK-binding kinase 1 (TBK1) into NSs-induced cytoplasmic structures and this interaction could effectively inhibit downstream phosphorylation and dimerization of interferon regulatory factor 3 (IRF3), resulting in the suppression of antiviral signaling and IFN induction. Functional sites of SFTSV NSs binding with TBK1 were then studied and results showed that NSs had lost their IFN-inhibiting activity after deleting the 25 amino acids in N-terminal. Furthermore, the mechanism of Rift Valley fever virus (RVFV) NSs blocking IFN-ß response were also investigated. Preliminary results showed that RVFV NSs proteins could neither interact nor co-localize with TBK1 in cytoplasm, but suppressed its expression levels, phosphorylation and dimerization of IRF3 in the subsequent steps, resulting in inhibition of the IFN-ß production. Altogether, our data demonstrated the probable mechanism used by SFTSV to inhibit IFN responses which was different from RVFV and pointed toward a novel mechanism for RVFV suppressing IFN responses.

Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope.

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4-5 reveals that helices α6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.

Rift Valley fever virus NSs protein functions and the similarity to other bunyavirus NSs proteins.

Rift Valley fever is a mosquito-borne zoonotic disease that affects both ruminants and humans. The nonstructural (NS) protein, which is a major virulence factor for Rift Valley fever virus (RVFV), is encoded on the S-segment. Through the cullin 1-Skp1-Fbox E3 ligase complex, the NSs protein promotes the degradation of at least two host proteins, the TFIIH p62 and the PKR proteins. NSs protein bridges the Fbox protein with subsequent substrates, and facilitates the transfer of ubiquitin. The SAP30-YY1 complex also bridges the NSs protein with chromatin DNA, affecting cohesion and segregation of chromatin DNA as well as the activation of interferon-ß promoter. The presence of NSs filaments in the nucleus induces DNA damage responses and causes cell-cycle arrest, p53 activation, and apoptosis. Despite the fact that NSs proteins have poor amino acid similarity among bunyaviruses, the strategy utilized to hijack host cells are similar. This review will provide and summarize an update of recent findings pertaining to the biological functions of the NSs protein of RVFV as well as the differences from those of other bunyaviruses.