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PubMed1999

The carboxy-terminal acidic domain of Rift Valley Fever virus NSs protein is essential for the formation of filamentous structures but not for the nuclear localization of the protein.

The ambisense S segment of Rift Valley fever (RVF) virus (a phlebovirus in the Bunyaviridae family) codes for two proteins: the viral complementary-sense RNA for the N nucleoprotein and the genomic-sense RNA for the nonstructural protein NSs. Except for the fact that the NSs protein is phosphorylated and forms filamentous structures in the nuclei of infected cells (R. Swanepoel and N. K. Blackburn, J. Gen. Virol. 34:557-561, 1977), its role is poorly understood, especially since the replication cycle of all these viruses takes place in the cytoplasm. To investigate the mechanisms involved in filament formation, we expressed NSs in mammalian cells via a recombinant Semliki Forest virus and demonstrated that the protein alone was able to form structures similar to those observed in RVF virus-infected cells, indicating that the presence of other RVF virus proteins is not required for filament formation. The yeast two-hybrid system was used to show that the protein interacts with itself and to map the interacting domains. Various deletion and substitution mutants were constructed, and the mutant proteins were analyzed by immunoprecipitation, Western blotting and immunofluorescence. These experiments indicated that the 10 to 17 amino acids of the carboxy-terminal domain were involved in self-association of the protein and that deletion of this acidic carboxy-terminal domain prevents the protein from forming filaments but does not affect its nuclear localization. The role of two phosphorylation sites present in this domain was also investigated, but they were not found to have a major influence on the formation of the nuclear filament.

Amino Acid Sequence↗

PubMed2000

Peptide-derived broad-reacting antisera against tospovirus NSs-protein.

Sequence alignments of tospovirus species of serogroup I to IV revealed a stretch of 24 amino acids at the C terminus of the non-structural protein NSs with a highly conserved sequence. Based on this sequence the 24 amino acids peptide YFLSKTLEVLPKNLQTMSYLDSIQC was synthesized and used to raise antisera in two rabbits. The specificity of the antisera against NSs from infected plants was confirmed with Western blots and by immunogold labelling and electron microscopy. These antisera detected tospovirus isolates of serogroup I to III in antigen-coated plate ELISA and Western blots but failed to detect isolates of serogroup IV. Epitope scanning using overlapping octopeptides composing the peptide suggested that the antisera contained antibodies against two different epitopes. Strongly reacting peptides were found at the C-terminus of the original peptide sequence when probing with one of the antisera. In this part the sequence was homologous to serogroup I, II and III, with all deviations from serogroup IV located here. Additional octopeptides, based on this region, synthesized with sequence modifications back to the serogroup IV sequence in all possible combinations, had low reactivity. However two of the modified peptides with partly restored serogroup IV sequences revealed promising reactivity and could be suitable to raise an antiserum with broader reactivity, including serogroup IV.

Amino Acid Sequence↗

PubMed1987

Identification of the N and NSS proteins coded by the ambisense S RNA of Punta Toro phlebovirus using monospecific antisera raised to baculovirus expressed N and NSS proteins.

An essentially complete DNA copy of the ambisense S RNA species of Punta Toro (PT) phlebovirus (T. Ihara, H. Akashi, and D.H.L. Bishop, 1984, Virology 136, 293-306) has been inserted in either orientation into Autographa californica nuclear polyhedrosis baculovirus (AcNPV) in lieu of the 5' coding region of the AcNPV polyhedrin gene (G.E. Smith, M.D. Summers, and M.J. Fraser, 1983, Mol. Cell. Biol. 3, 2156-2165). The two types of recombinant viruses were used to infect Spodoptera frugiperda cells and the expressed PT viral proteins characterized. Recombinant AcNPV having the S DNA in one orientation expressed PT virus N protein in amounts estimated to represent some 50% of the infected cell extracts, whereas recombinants with the S DNA in the other orientation expressed the putative PT virus NSS protein in lower quantities. Antisera that were monospecific with respect to each of the two PT proteins virus were raised in mice using the corresponding S. frugiperda infected cell extracts and were employed to identify N and NSS proteins in PT virus-infected Vero cells.

Animals↗

PubMed2025

Efficient Expression of Oropouche Virus Nonstructural Proteins NSs and NSm.

Oropouche fever, a mosquito- or midge-borne emerging zoonotic disease endemic to South and Central America, manifests as a dengue-like acute febrile illness with occasional occurrences of meningitis or meningoencephalitis. The causative agent, Oropouche virus (OROV), belongs to the genus Orthobunyavirus within the family Peribunyaviridae. Its tripartite negative-sense RNA genome comprises small (S), medium (M), and large (L) segments, encoding structural N, Gn/Gc, and L proteins, respectively. Additionally, the S- and M-segments encode nonstructural proteins: NSs and NSm, which may act as virulence factors. OROV NSs functions as an interferon antagonist with an unknown mechanism, while the roles of OROV NSm remain elusive. This chapter introduces efficient expression systems for OROV NSm and NSs proteins. Validating the presence of a signal peptide at the N-terminus of NSm protein is essential for its expression. Furthermore, expressing OROV NSs protein independently of an RNA polymerase II promoter is crucial to prevent restricted gene expression, potentially caused by NSs inhibiting cellular RNA polymerase II, as observed in closely related bunyavirus NSs proteins. These protein expression strategies offer insights into the molecular characterization of OROV NSm and NSs proteins, facilitating a deeper understanding of their virulence mechanisms.

Viral Nonstructural Proteins↗

PubMed2005

Rift valley fever virus nonstructural protein NSs promotes viral RNA replication and transcription in a minigenome system.

Rift Valley fever virus (RVFV), which belongs to the genus Phlebovirus, family Bunyaviridae, has a tripartite negative-strand genome (S, M, and L segments) and is an important mosquito-borne pathogen for domestic animals and humans. We established an RVFV T7 RNA polymerase-driven minigenome system in which T7 RNA polymerase from an expression plasmid drove expression of RNA transcripts for viral proteins and minigenome RNA transcripts carrying a reporter gene between both termini of the M RNA segment in 293T cells. Like other viruses of the Bunyaviridae family, replication and transcription of the RVFV minigenome required expression of viral N and L proteins. Unexpectedly, the coexpression of an RVFV nonstructural protein, NSs, with N and L proteins resulted in a significant enhancement of minigenome RNA replication. Coexpression of NSs protein with N and L proteins also enhanced minigenome mRNA transcription in the cells expressing viral-sense minigenome RNA transcripts. NSs protein expression increased the RNA replication of minigenomes that originated from S and L RNA segments. Enhancement of minigenome RNA synthesis by NSs protein occurred in cells lacking alpha/beta interferon (IFN-alpha/beta) genes, indicating that the effect of NSs protein on minigenome RNA replication was unrelated to a putative NSs protein-induced inhibition of IFN-alpha/beta production. Our finding that RVFV NSs protein augmented minigenome RNA synthesis was in sharp contrast to reports that Bunyamwera virus (genus Bunyavirus) NSs protein inhibits viral minigenome RNA synthesis, suggesting that RVFV NSs protein and Bunyamwera virus NSs protein have distinctly different biological roles in viral RNA synthesis.

Animals↗

PubMed1999

The Rift Valley fever virus nonstructural protein NSs is phosphorylated at serine residues located in casein kinase II consensus motifs in the carboxy-terminus.

The S segment of Rift Valley fever virus (Bunyaviridae, Phlebovirus) codes for two proteins, the nucleoprotein N and the nonstructural protein NSs. The NSs protein is a phosphoprotein of unknown function that is localized in the cytoplasm and the nuclei of infected cells where it forms filamentous structures. To characterize further the protein expressed in VC10 cells infected with the MP12 strain, we analyzed its phosphorylation states and showed that phosphorylated forms were found in both compartments. Cytoplasmic and nuclear NSs were phosphorylated only at serine residues. Phosphopeptide mapping and molecular analysis of mutants obtained by site-directed mutagenesis allowed us to map the major phosphorylation sites of nuclear and cytoplasmic forms of NSs to serine residues 252 and 256, located at the carboxy-terminus in consensus sequences for casein kinase II. A similar map was obtained when the protein was purified from mosquito cells infected with MP12. In addition, we showed that the purified unphosphorylated NSs protein expressed from pET-NSs plasmid in a coupled transcription-translation reaction containing Escherichia coli S30 extracts did not possess autophosphorylation activity but was phosphorylated in vitro after incubation with recombinant casein kinase II.

Amino Acid Motifs↗

PubMed2002

Identification of a novel RNA silencing suppressor, NSs protein of Tomato spotted wilt virus.

RNA silencing or post-transcriptional gene silencing (PTGS) in plants is known as a defense system against virus infection. Several plant viruses have been shown to encode an RNA silencing suppressor. Using a green fluorescent protein-based transient suppression assay, we show that NSs protein of Tomato spotted wilt virus (TSWV) has RNA silencing suppressor activity. TSWV NSs protein suppressed sense transgene-induced PTGS but did not suppress inverted repeat transgene-induced PTGS. TSWV NSs protein is the first RNA silencing suppressor identified in negative-strand RNA viruses.

Gene Expression Regulation, Plant↗

PubMed2001

The Bunyamwera virus nonstructural protein NSs inhibits viral RNA synthesis in a minireplicon system.

The small (S) genomic segment of Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) encodes the nucleocapsid protein, N, and a nonstructural protein, NSs, in overlapping reading frames. In order to elucidate the function of NSs, we established a plasmid-based minireplicon system using mammalian cells that express large amounts of T7 RNA polymerase. Expression of N, the viral polymerase protein (L), and a minireplicon containing a reporter gene was sufficient to reconstitute functional virus nucleocapsids. Coexpression of NSs, however, led to a dose-dependent decrease in reporter activity without affecting expression of controls. The inhibition could not be reversed by overexpression of N, L or the minireplicon, indicating that the NSs effect was not caused by a reduction in virus gene expression. The NSs proteins of two other members of the Bunyavirus genus, Guaroa virus and Lumbo virus, were also inhibitory in our system. The intracellular localisation of Bunyamwera virus NSs was investigated and found to be predominantly cytoplasmic, but intranuclear inclusion was also detected. Taken together, these data suggest that, in mammalian cells, the bunyavirus NSs protein controls the activity of the viral polymerase by a highly conserved mechanism.

Amino Acid Sequence↗

PubMed2006

Interaction of Bunyamwera Orthobunyavirus NSs protein with mediator protein MED8: a mechanism for inhibiting the interferon response.

The NSs protein of Bunyamwera virus (Bunyaviridae) is an antiapoptotic interferon antagonist involved in silencing host protein expression by interfering with mRNA synthesis. Here, we show that the ability to inhibit both host transcription and the interferon response is linked to interaction of NSs with the MED8 component of Mediator, a protein complex necessary for mRNA production. The interacting domain on NSs was mapped to the C-terminal region, which contains amino acids conserved among orthobunyavirus NSs proteins. A recombinant virus in which the interacting domain in NSs was deleted had strongly reduced ability to inhibit host protein expression and was unable to inhibit the interferon response. This study provides further information on the mechanisms by which bunyavirus nonstructural proteins are involved in pathogenesis.

Amino Acid Sequence↗

PubMed1992

Association of the nonstructural protein NSs of Uukuniemi virus with the 40S ribosomal subunit.

The small RNA segment (S segment) of Uukuniemi (UUK) virus encodes two proteins, the nucleocapsid protein (N) and a nonstructural protein (NSs), by an ambisense strategy. The function of NSs has not been elucidated for any of the bunyaviruses expressing this protein. We have now expressed the N and NSs proteins in Sf9 insect cells by using the baculovirus expression system. High yields of both proteins were obtained. A monospecific antibody was raised against gel-purified NSs and used to study the synthesis and localization of the protein in UUK virus-infected BHK21 cells. While the N protein was detected as early as 4 h postinfection (p.i.), NSs was identified only after 8 h p.i. Both proteins were still synthesized at high levels at 24 h p.i. The half-life of NSs was about 1.5 h, while that of the N protein was several hours. Sucrose gradient fractionation of [35S]methionine-labeled detergent-solubilized extracts of infected BHK21 cells indicated that NSs was firmly associated with the 40S ribosomal subunit. This association took place shortly after translation and was partially resistant to 1 M NaCl. NSs expressed by using the T7 vaccinia virus expression system, as well as in vitro-translated NSs, was also associated with the 40S subunit. In contrast, in vitro-translated N protein was found on top of the gradient. Immunolocalization of NSs, in UUK virus-infected cells, by using an affinity-purified antibody showed a granular cytoplasmic staining. A very similar pattern was seen for cells expressing NSs from a cDNA copy by using a vaccinia virus expression system. No staining was observed in the nuclei in either case. Furthermore, NSs was found neither in virions nor in nucleocapsids isolated from infected cells. In vivo labeling with 32Pi indicated that NSs is not phosphorylated. The possible function of NSs is discussed in light of these results.

Animals↗

PubMed2003

Bunyamwera virus nonstructural protein NSs counteracts interferon regulatory factor 3-mediated induction of early cell death.

The genome of Bunyamwera virus (BUN; family Bunyaviridae, genus Orthobunyavirus) consists of three segments of negative-sense RNA. The smallest segment, S, encodes two proteins, the nonstructural protein NSs, which is nonessential for viral replication and transcription, and the nucleocapsid protein N. Although a precise role in the replication cycle has yet to be attributed to NSs, it has been shown that NSs inhibits the induction of alpha/beta interferon, suggesting that it plays a part in counteracting the host antiviral defense. A defense mechanism to limit viral spread is programmed cell death by apoptosis. Here we show that a recombinant BUN that does not express NSs (BUNdelNSs) induces apoptotic cell death more rapidly than wild-type virus. Screening for apoptosis pathways revealed that the proapoptotic transcription factor interferon regulatory factor 3 (IRF-3) was activated by both wild-type BUN and BUNdelNSs infection, but only wild-type BUN was able to suppress signaling downstream of IRF-3. Studies with a BUN minireplicon system showed that active replication induced an IRF-3-dependent promoter, which was suppressed by the NSs protein. In a cell line (P2.1) defective in double-stranded RNA signaling due to low levels of IRF-3, induction of apoptosis was similar for wild-type BUN and BUNdelNSs. These data suggest that the BUN NSs protein can delay cell death in the early stages of BUN infection by inhibiting IRF-3-mediated apoptosis.

Annexin A5↗

PubMed2001

Bunyamwera bunyavirus nonstructural protein NSs is a nonessential gene product that contributes to viral pathogenesis.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB/c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-beta promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.

Aedes↗

PubMed2005

La Crosse virus nonstructural protein NSs counteracts the effects of short interfering RNA.

Through a process known as RNA interference (RNAi), double-stranded short interfering RNAs (siRNAs) silence gene expression in a sequence-specific manner. Recently, several viral proteins, including the nonstructural protein NSs of tomato spotted wilt virus (a plant-infecting bunyavirus), the interferon antagonist protein NS1 of influenza virus, and the E3L protein of vaccinia virus, have been shown to function as suppressors of RNAi, presumably as a counterdefense against cellular mechanisms that decrease viral production. La Crosse virus (LACV), a member of the California serogroup of orthobunyaviruses, has a trisegmented negative-stranded genome comprised of large (L), medium (M), and small (S) segments. To develop a strategy for segment-specific inhibition of transcription, we designed 13 synthetic siRNAs targeting specific RNA segments of the LACV genome that decreased LACV replication and antigen expression in mammalian (293T) and insect (C6/36) cells. Furthermore, NSs, a LACV nonstructural protein, markedly inhibited RNAi directed both against an LACV M segment construct and against a host gene (glyeraldehyde-3-phosphate dehydrogenase), suggesting a possible role for this viral protein in the suppression of RNA silencing. Segment-specific siRNAs will be useful as a tool to analyze LACV transcription and replication and to obtain recombinant viruses. Additionally, NSs will help us to identify molecular pathways involved in RNAi and further define its role in the innate immune system.

Animals↗

PubMed1983

Bunyavirus nucleoprotein, N, and a non-structural protein, NSS, are coded by overlapping reading frames in the S RNA.

It has been shown previously, by sequence analysis of the S RNA segment of snowshoe hare (SSH) bunyavirus, that two overlapping open reading frames in the viral complementary sequence code for proteins with molecular weights of 26.8 X 10(3) and 10.5 X 10(3) respectively. In addition to the viral nucleocapsid (N) protein, which is coded by the S RNA, analyses of parental and reassortant bunyavirus-infected cell extracts have shown that the viral S RNA and M RNA species each code for non-structural proteins (NSS and NSM, respectively). In the present report, in vitro translation analyses of the S mRNA species recovered from virus-infected cells indicate that a single size class of mRNA directs the synthesis of N and NSS. Compositional analyses of selected tryptic peptides of N and NSS have provided proof that N is the product of the first open reading frame, and NSS the product of the second.

Bunyaviridae↗

PubMed2002

Bunyamwera bunyavirus nonstructural protein NSs counteracts the induction of alpha/beta interferon.

Production of alpha/beta interferons (IFN-alpha/beta) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-alpha/beta, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-kappaB and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-alpha/beta system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-alpha/beta in order to increase the virulence of Bunyamwera virus.

Animals↗

PubMed2001

Genetic evidence for an interferon-antagonistic function of rift valley fever virus nonstructural protein NSs.

Rift Valley fever virus (RVFV), a phlebovirus of the family Bunyaviridae, is a major public health threat in Egypt and sub-Saharan Africa. The viral and host cellular factors that contribute to RVFV virulence and pathogenicity are still poorly understood. All pathogenic RVFV strains direct the synthesis of a nonstructural phosphoprotein (NSs) that is encoded by the smallest (S) segment of the tripartite genome and has an undefined accessory function. In this report, we show that MP12 and clone 13, two attenuated RVFV strains with mutations in the NSs gene, were highly virulent in IFNAR(-/-) mice lacking the alpha/beta interferon (IFN-alpha/beta) receptor but remained attenuated in IFN-gamma receptor-deficient mice. Both attenuated strains proved to be excellent inducers of early IFN-alpha/beta production. In contrast, the virulent strain ZH548 failed to induce detectable amounts of IFN-alpha/beta and replicated extensively in both IFN-competent and IFN-deficient mice. Clone 13 has a defective NSs gene with a large in-frame deletion. This defect in the NSs gene results in expression of a truncated protein which is rapidly degraded. To investigate whether the presence of the wild-type NSs gene correlated with inhibition of IFN-alpha/beta production, we infected susceptible IFNAR(-/-) mice with S gene reassortant viruses. When the S segment of ZH548 was replaced by that of clone 13, the resulting reassortants became strong IFN inducers. When the defective S segment of clone 13 was exchanged with the wild-type S segment of ZH548, the reassortant virus lost the capacity to stimulate IFN-alpha/beta production. These results demonstrate that the ability of RVFV to inhibit IFN-alpha/beta production correlates with viral virulence and suggest that the accessory protein NSs is an IFN antagonist.

Animals↗

PubMed1991

The nonstructural protein (NSs) encoded by the ambisense S RNA segment of tomato spotted wilt virus is associated with fibrous structures in infected plant cells.

The open reading frame located in the viral strand of the ambisense S RNA of tomato spotted wilt virus (TSWV), was cloned into transfer vector pAc33DZ1 and inserted downstream of the polyhedrin promoter in the Autographa californica nuclear polyhedrosis virus genome. Recombinant baculoviruses were obtained that showed a high-level expression of a 52.4-kDa protein corresponding to the inserted TSWV gene. The viral protein thus produced was purified and injected into rabbits to raise antibodies. Western immunoblot analyses of extracts from TSWV-infected plants demonstrated that the 52.4-kDa TSWV-specific polypeptide represents a nonstructural protein (denoted NSs), being absent in purified virus particles. Immunogold labeling of tissue sections of TSWV-infected Nicotiana rustica plants showed that this protein was, depending on the virus isolate, either found dispersed throughout the cytoplasm or associated with fibers which appeared as elongated flexible filaments or paracrystalline rods.

Animals↗

PubMed2004

NSs protein of Rift Valley fever virus blocks interferon production by inhibiting host gene transcription.

Rift Valley fever virus (RVFV) is an important cause of epizootics and epidemics in Africa and a potential agent of bioterrorism. A better understanding of the factors that govern RVFV virulence and pathogenicity is required, given the urgent need for antiviral therapies and safe vaccines. We have previously shown that RVFV strains with mutations in the NSs gene are excellent inducers of alpha/beta interferon (IFN-alpha/beta) and are highly attenuated in mice. Here, we demonstrate that NSs is sufficient to block IFN-beta gene expression at the transcriptional level. In cells transiently expressing NSs, IFN-beta transcripts were not inducible by viral infection or by transfection of poly(I:C). NSs with anti-IFN activity accumulated in the nucleus. In contrast, mutant forms of NSs that had lost their IFN-inhibiting activity remained in the cytoplasm, indicating that nuclear localization plays a role. IFN synthesis is regulated by specific transcription factors, including interferon regulatory factor (IRF-3), NF-kappaB, and AP-1. In the presence of NSs, IRF-3 was still activated and moved to the nucleus. Likewise, NF-kappaB and AP-1 were activated normally, as shown in electrophoretic mobility shift assays. Moreover, NSs was found to inhibit transcriptional activity of a constitutive promoter, in agreement with recent findings showing that NSs targets the basal cellular transcription factor TFIIH. The present results suggest that NSs, unlike other viral IFN antagonists, does not inhibit IFN-specific transcription factors but blocks IFN gene expression at a subsequent step.

Amino Acid Sequence↗