NSs is the 11-kDa non-structural accessory protein of Bunyamwera virus (BUNV), the prototype orthobunyavirus. It is translated by alternative initiation from the S-segment mRNA in a reading frame overlapping that of the nucleocapsid protein N, which is why UniProt files this entry under the gene name N; the protein described here is NSs, not the nucleocapsid. NSs is dispensable for replication in cultured cells and in mice but acts as a virulence factor in the mammalian host. Its characterised activity is a block of host RNA polymerase II-dependent transcription. In infected mammalian cells NSs leaves CTD serine 5 phosphorylation and promoter occupancy intact but prevents serine 2 phosphorylation of the polymerase CTD heptapeptide repeat, blocking the transition from initiation to elongation, and it binds the Mediator head-module subunit MED8 through a conserved C-terminal motif (residues 81-93) that is required for the transcriptional block. The consequences are shutoff of host-cell protein synthesis and failure of the infected cell to transcribe the interferon-beta gene, so that NSs-deficient virus becomes a strong inducer of type I interferon and is attenuated in interferon-competent cells and in mouse brain. NSs blocks interferon induction rather than interferon receptor signalling; upstream sensing (IRF-3 and NF-kappaB activation) and the dsRNA-activated effectors PKR and RNase L are unaffected. The transcriptional block is specific to mammalian cells, with no interference with CTD phosphorylation or host shutoff in mosquito cells, consistent with lytic infection of the vertebrate host and persistent infection of the arthropod vector.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0016032 viral process | IEA GO_REF:0000002 | MODIFY | Summary: Root-level InterPro2GO mapping from the Bunya_NSs family signature. True of NSs but uninformative, because the family's defining role is the block of host RNA polymerase II-dependent transcription and the resulting host gene-expression shutoff, both of which are asserted elsewhere in this review. Reason: The gene product is a viral protein acting in the virus-host interaction, so the term is not wrong, but it is a high-level parent that conveys nothing about what NSs does. The InterPro family it comes from (IPR000797 Bunya_NSs) is the orthobunyavirus NSs family whose characterised activity is inhibition of host RNAP II CTD serine 2 phosphorylation and consequent host shutoff [PMID:15150262, PMID:16973571]. Replace with the specific terms the gene already carries on experimental evidence, GO:0039523 for the mechanism and GO:0039657 for the outcome. Proposed replacements: symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity symbiont-mediated suppression of host gene expression Supporting Evidence: PMID:15150262 However, NSs prevented CTD phosphorylation at serine 2, suggesting a block in transition from initiation to elongation. file:BUNYW/N__P16494/N__P16494-uniprot.txt Belongs to the orthobunyavirus NS-S protein family. |
| GO:0019080 viral gene expression | IMP PMID:11209062 Bunyamwera bunyavirus nonstructural protein NSs is a nonesse... | MODIFY | Summary: CACAO row. The cited paper's only observation about viral gene expression is that the NSs-deleted virus makes about ten times MORE nucleocapsid protein than wild type (Fig. 4A, [35S]methionine labelling of BHK cells quantified by PhosphorImager). NSs therefore lowers, rather than supports, the level of viral gene products, so as an involved_in assertion this term has the sign reversed. Reason: This is a direction error rather than a wrong-gene or wrong-paper problem. The assay is on BUNV-infected cells and the protein scored is viral, but deleting NSs increases viral N protein, so NSs cannot be read as contributing to viral gene expression [PMID:11209062]. The correctly signed term for exactly this figure is GO:0046725 negative regulation by virus of viral protein levels in host cell, which the same CACAO submission also entered from the same paper; the two rows as they stand contradict one another. No evidence anywhere in the BUNV NSs literature places NSs in viral transcription or translation, since transcription and replication of BUNV RNA require only N and L [PMID:11209062] and NSs is dispensable for growth. Proposed replacements: negative regulation by virus of viral protein levels in host cell Supporting Evidence: PMID:11209062 Quantification of this gel by using a PhosphorImager indicated that approximately 10 times more N protein was produced in cells infected with BUNdelNSs compared with wt BUN. PMID:11209062 showed that transcription and replication of the RNA required only the N and L proteins, although the possibility of a regulatory role for NSs could not be discounted |
| GO:0039502 symbiont-mediated suppression of host type I interferon-mediated signaling pathway | IDA PMID:15150262 Inhibition of RNA polymerase II phosphorylation by a viral i... | MODIFY | Summary: CACAO row. The cited paper shows that NSs blocks phosphorylation of the RNA polymerase II CTD at serine 2 while serine 5 phosphorylation and RNAP II occupancy of the interferon-beta promoter (chromatin immunoprecipitation) are unaffected, so the interferon-beta gene fails to be transcribed. It does not assay the type I interferon-mediated signaling pathway (interferon binding IFNAR and the downstream JAK-STAT cascade), which is what GO:0039502 denotes. Reason: GO:0039502 is defined as interference with a type I interferon-mediated signaling pathway in the host and sits under GO:0052029 symbiont-mediated suppression of host signal transduction pathway, so it asserts a block downstream of the interferon receptor. What the paper demonstrates is a block of interferon-beta gene transcription, upstream of any interferon being made [PMID:15150262], and the wider literature shows the sensing arm is explicitly NOT blocked, with IRF-3 and NF-kappaB activated normally in infected cells and PKR and RNase L unaffected [PMID:12133999, PMID:12692253]. The claim the evidence supports is suppression of the host innate immune response achieved through the transcription block, so the proposed replacement is GO:0052170; the mechanism is already captured by the gene's GO:0039523 rows. This is an adjacent-branch substitution, not a curator-attribution error, and the paper is unambiguously about this gene product. The companion review of Rift Valley fever virus NSs, the same lesion on host RNA polymerase II with the sensing arm intact, proposes a new term for this step (symbiont-mediated suppression of host type I interferon production) because GO has no symbiont-mediated counterpart of GO:0032480. That term would fit this row equally well and should replace GO:0052170 here if GO creates it; GO:0052170 is used now because it is an existing term the evidence fully supports, and this gene already carries it by other rows. Proposed replacements: symbiont-mediated suppression of host innate immune response Supporting Evidence: PMID:15150262 Furthermore, RNAP II was able to bind to the IFN-beta gene promoter as revealed by chromatin immunoprecipitation analysis, indicating that the initiation of transcription was not disturbed by NSs. PMID:12133999 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. PMID:12692253 NSs is different from other IFN antagonists, since it inhibits dsRNA-dependent IFN induction but has no effect on the dsRNA-activated PKR and RNase L systems. |
| GO:0039502 symbiont-mediated suppression of host type I interferon-mediated signaling pathway | IMP PMID:11209062 Bunyamwera bunyavirus nonstructural protein NSs is a nonesse... | MODIFY | Summary: CACAO row, same term from the reverse-genetics paper. The supporting experiment is an interferon-beta promoter-luciferase reporter in murine BF cells (Fig. 7A). Wild-type BUNV gives background luciferase whereas BUNdelNSs gives an approximately 20-fold induction, with a thymidine kinase promoter control showing the effect is specific. That measures interferon-beta gene induction, not the interferon-mediated signaling pathway. Reason: Same adjacent-branch problem as the IDA row, resolved the same way for consistency. The phenotype is loss of a block on interferon-beta promoter activation [PMID:11209062], and direct measurement in the companion study found neither interferon nor interferon mRNA in wild-type-infected cells while BUNdelNSs was a strong inducer [PMID:12133999], all upstream of IFNAR signalling. GO:0052170 symbiont-mediated suppression of host innate immune response is the supported claim, and the mechanistic term GO:0039523 is annotated separately. The paper is the right paper for this gene product and the knockout evidence is solid, so this is a term-choice correction, not a challenge to the curator's reading. As for the IDA row above, the proposed production term in the companion Rift Valley fever virus NSs review would fit this row better than GO:0052170 and should replace it if GO creates it. Proposed replacements: symbiont-mediated suppression of host innate immune response Supporting Evidence: PMID:11209062 In contrast, there was approximately 20-fold increase in luciferase activity in cells infected with BUNdelNSs, indicating strong activation of the IFN-Ξ² promoter. PMID:11209062 Thus, the loss of NSs changed BUN from a noninducer to an inducer of IFN. PMID:12133999 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. |
| GO:0039523 symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword-based mapping (KW-1104, inhibition of host RNA polymerase II by virus) that agrees with the experimental IMP row for the same term and with UniProt's curated FUNCTION statement. Reason: The keyword rests on the curated UniProt FUNCTION block, which is itself tied to the experimental publication (ECO:0000269 with PubMed:15150262), and the mechanism has since been corroborated by the MED8-interaction study [PMID:16973571] and by a virus whose truncated NSs fails to block RNAP II activity [PMID:20427562]. This is the core function of NSs. Supporting Evidence: file:BUNYW/N__P16494/N__P16494-uniprot.txt Inhibits phosphorylation at serine 2 in the heptapeptide repeat (YSPTSPS) of the CTD of RNA polymerase II, suggesting that the elongation step of transcription and/or 3'-end processing is prevented. PMID:16973571 Wild-type BUNV efficiently inhibited transcription from the CMV promoter, whereas BUNdelNSs2 and BUNNSs-T83 had no major effect on RNA polymerase II activity compared to mock infection |
| GO:0039523 symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity | IMP PMID:15150262 Inhibition of RNA polymerase II phosphorylation by a viral i... | ACCEPT | Summary: CACAO row and the best-matched annotation on this gene product. The cited paper's central result is exactly this term, namely that in BUNV-infected mammalian cells NSs prevents RNA polymerase II CTD serine 2 phosphorylation while serine 5 phosphorylation and promoter binding are normal, so mRNA elongation and 3'-end processing are blocked. Reason: The CACAO term matches the assay at the right granularity, a mechanism-specific term rather than a phenotype read-out, and it is independently corroborated. NSs binds the Mediator head subunit MED8 through residues 81-93, and viruses lacking that domain (BUNNSs-T83) or carrying an N-terminally truncated NSs lose the RNAP II block [PMID:16973571, PMID:20427562]. RNA polymerase II is also destabilised late in wild-type but not NSs-mutant infection [PMID:16973571]. Retained as a core function. Supporting Evidence: PMID:15150262 Here, we demonstrate that NSs targets the RNA polymerase II (RNAP II) complex. PMID:15150262 However, NSs prevented CTD phosphorylation at serine 2, suggesting a block in transition from initiation to elongation. PMID:16973571 The region encompassing amino acids 81 to 93 of NSs was shown to be essential for the interaction. |
| GO:0039657 symbiont-mediated suppression of host gene expression | IEA GO_REF:0000043 | ACCEPT | Summary: Keyword-based mapping (KW-1190, host gene expression shutoff by virus), consistent with the experimental IMP row for the same term. Reason: Host-cell gene-expression shutoff is directly demonstrated by the reduced shutoff of host protein synthesis in NSs-deleted virus [PMID:11209062] and by the loss of reporter-gene suppression in the MED8-interaction mutant [PMID:16973571]. The claim is independent of, rather than entailed by, GO:0039523 in the GO hierarchy (the two are siblings under GO:0039656 symbiont-mediated perturbation of host gene expression), and the 2001 paper notes that shutoff is only partly accounted for by any single pathway, so the broader term is kept rather than collapsed into the transcription term. Supporting Evidence: file:BUNYW/N__P16494/N__P16494-uniprot.txt Inhibition of host transcription machinery leads to shut off of host cell protein synthesis and inhibition of the host innate immune response. PMID:11209062 The reduction, but not the abrogation, of host cell protein shutoff by BUNdelNSs suggests also that more than one pathway may be involved. |
| GO:0039657 symbiont-mediated suppression of host gene expression | IMP PMID:11209062 Bunyamwera bunyavirus nonstructural protein NSs is a nonesse... | ACCEPT | Summary: CACAO row, supported by Fig. 4B of the cited paper. BHK cells infected at 5 pfu/cell and labelled with [35S]methionine show almost complete shutoff of host protein synthesis by 48 h with wild-type BUNV, whereas BUNdelNSs-infected cells retain substantial host synthesis. The term matches the assay. Reason: The annotation is at the right level for the experiment, since a bulk host protein synthesis read-out supports suppression of host gene expression without specifying the step, and the transcription-level mechanism is annotated separately (GO:0039523). Shutoff is cell-type restricted and does not occur in mosquito cells [PMID:11209062], which is a property of the host interaction rather than a reason to doubt the term. Core function. Supporting Evidence: PMID:11209062 At 48 h after infection, host cell protein synthesis was almost completely shut off in wt BUN-infected BHK cells. In contrast, in BUNdelNSs-infected cells, host cell protein synthesis was reduced compared with mock-infected cells but not inhibited to the same extent as in wt-infected cells. PMID:16973571 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. |
| GO:0046725 negative regulation by virus of viral protein levels in host cell | IMP PMID:11209062 Bunyamwera bunyavirus nonstructural protein NSs is a nonesse... | KEEP AS NON CORE | Summary: CACAO row, supported by Fig. 4A of the cited paper. Cells infected with BUNdelNSs produce roughly ten times more nucleocapsid protein than wild-type BUNV-infected cells, so NSs expression lowers the level of a viral protein in the infected cell. The term matches the measurement and is correctly signed. Reason: The observation is real and the term is the right one for it, but down-regulating the virus' own N protein is best read as a consequence of the general NSs-imposed block on host RNAP II-dependent transcription and translation rather than a distinct evolved activity. The paper offers no mechanism specific to viral proteins, and the authors frame NSs as an accessory virulence factor whose production is tightly coupled to N expression, so the row is kept as a peripheral, non-core annotation. It is not removed, because the assay is in BUNV-infected cells, the effect is large, and the full text was read by the curator. Supporting Evidence: PMID:11209062 It was also noticeable that the amount of N protein produced by BUNdelNSs, relative to the G1 protein, was increased compared with wt. PMID:11209062 suggests a selection pressure against viruses in which NSs production is not tightly regulated by being expressed from the same mRNA as N |
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Download this section (compressed HTML)Q: Is host-cell shutoff attributable entirely to the RNA polymerase II serine 2 block, or does NSs also act post-transcriptionally? Shutoff is reduced but not abolished in BUNdelNSs, and the authors explicitly invoke more than one pathway.
Q: Should GO add a term for symbiont-mediated suppression of host type I interferon production, as distinct from the existing signalling terms GO:0039502 and GO:0140886? Several viral proteins, including BUNV and Rift Valley fever virus NSs, block interferon gene transcription without touching IFNAR signalling, and curators appear to reach for the signalling term for lack of an induction term.
Q: What explains the mammalian-versus-mosquito difference, absence of the relevant Mediator or CDK interaction in insect cells, or failure of NSs to reach its target there?
Experiment: Reconstitute the NSs block in vitro with purified Mediator and RNA polymerase II plus P-TEFb/CDK9 and TFIIH, to test directly whether NSs inhibits CTD serine 2 kinase activity or instead prevents kinase recruitment via MED8.
Experiment: Compare Mediator composition and NSs-MED8 binding in mammalian versus Aedes cells (co-immunoprecipitation from both backgrounds, plus complementation of insect cells with the mammalian MED8 orthologue) to explain the host-restricted transcription block.
Experiment: Define the function of the NSs N-terminal 21 residues separately from MED8 binding, using the ambisense-S recombinant viruses that express tagged NSs independently of N, combined with quantitative CTD phospho-profiling (Ser2 and Ser5 ChIP-seq) and host transcriptome measurement.
Experiment: Test whether the reduction of viral N protein levels by NSs is a mechanistically distinct activity or a by-product of global shutoff, by measuring S-segment mRNA and N protein in cells infected with wild-type, BUNdelNSs and BUNNSs-T83 viruses at matched replication levels.
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