Predictable fold switching by the<scp>SARS‐CoV</scp>‐2 protein<scp>ORF9b</scp>

Porter, Lauren L.

doi:10.1002/pro.4097

Cited by 13 publications

(5 citation statements)

References 31 publications

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“…Notably, while purified dimeric Orf9b mainly forms β-strands, monomeric Orf9b displays an overall α-helical structure when in complex with TOM70 [15,16,27]. The ability of Orf9b to switch folds is also indicated by structure-and sequence-based predictions [41]. It is, therefore, tempting to speculate that a portion of Orf9b can undergo dynamic shifts between its dimeric state and its monomeric state.…”

Section: The Mechanisms Of Orf9b-tom70 Interactionmentioning

confidence: 99%

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

Brandherm

Kobaš

Klöhn

et al. 2021

IJMS

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SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b–TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.

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Section: The Mechanisms Of Orf9b-tom70 Interactionmentioning

confidence: 99%

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

Brandherm

Kobaš

Klöhn

et al. 2021

IJMS

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“…This suggests that the competitive binding between ORF9b and HSP90α may not be entirely mutually exclusive, possibly due to HSP90 possessing two binding sites to TOM70 68 and ORF9b being a fold-switching protein. 74 While this deepens our understanding of the regulatory complexities in this scenario, further investigation, particularly through structural analysis, is warranted to elucidate the exact mechanism.…”

Section: Discussionmentioning

confidence: 99%

A Cullin 5-based complex serves as an essential modulator of ORF9b stability in SARS-CoV-2 replication

Zhou,

Chen,

Liu

et al. 2024

Sig Transduct Target Ther

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The ORF9b protein, derived from the nucleocapsid’s open-reading frame in both SARS-CoV and SARS-CoV-2, serves as an accessory protein crucial for viral immune evasion by inhibiting the innate immune response. Despite its significance, the precise regulatory mechanisms underlying its function remain elusive. In the present study, we unveil that the ORF9b protein of SARS-CoV-2, including emerging mutant strains like Delta and Omicron, can undergo ubiquitination at the K67 site and subsequent degradation via the proteasome pathway, despite certain mutations present among these strains. Moreover, our investigation further uncovers the pivotal role of the translocase of the outer mitochondrial membrane 70 (TOM70) as a substrate receptor, bridging ORF9b with heat shock protein 90 alpha (HSP90α) and Cullin 5 (CUL5) to form a complex. Within this complex, CUL5 triggers the ubiquitination and degradation of ORF9b, acting as a host antiviral factor, while HSP90α functions to stabilize it. Notably, treatment with HSP90 inhibitors such as GA or 17-AAG accelerates the degradation of ORF9b, leading to a pronounced inhibition of SARS-CoV-2 replication. Single-cell sequencing data revealed an up-regulation of HSP90α in lung epithelial cells from COVID-19 patients, suggesting a potential mechanism by which SARS-CoV-2 may exploit HSP90α to evade the host immunity. Our study identifies the CUL5-TOM70-HSP90α complex as a critical regulator of ORF9b protein stability, shedding light on the intricate host–virus immune response dynamics and offering promising avenues for drug development against SARS-CoV-2 in clinical settings.

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“…Given the above, and that our focus here is on amyloidogenic proteins, it is of special interest that α-helix-to-β-sheet transitions are a noteworthy feature of such proteins [36], and particularly, for our present purposes, some in SARS-CoV-2 [43], where several proteins are amyloidogenic [44,45]. We note too that some alleles of the fibrinogen Aα chain may produce highly amyloidogenic proteolytic fragments [46], and that fibrinogen can bind to wellestablished amyloids such as Aβ [47][48][49][50].…”

Section: Proteins Of Identical Sequence Can Adopt Alternative Stable ...mentioning

confidence: 99%

Proteomic evidence for amyloidogenic cross-seeding in fibrinaloid microclots

Kell,

Pretorius

2024

Preprint

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In classical amyloidoses, amyloid fibres form through the nucleation and accretion of protein monomers, with protofibrils and fibrils exhibiting a cross-β motif of parallel or antiparallel β-sheets oriented perpendicular to the fibre direction. These protofibrils and fibrils can intertwine to form mature amyloid fibres. Similar phenomena occur in blood from individuals with circulating inflammatory molecules (also those originating from viruses and bacteria). In the presence of inflammagens, pathological clotting can occur, that results in an anomalous amyloid form termed fibrinaloid microclots. Previous proteomic analyses of these microclots have shown the presence of non-fibrin(ogen) proteins, suggesting a more complex mechanism than simple entrapment. We provide evidence against a simple entrapment model, noting that clot pores are too large and centrifugation would have removed weakly bound proteins. Instead, we explore whether co-aggregation into amyloid fibres may involve axial (multiple proteins within the same fibril), lateral (single-protein fibrils contributing to a fibre), or both types of integration. Our analysis of proteomic data from fibrinaloid microclots in different diseases shows no significant overlap with the normal plasma proteome and no correlation between plasma protein abundance and presence in microclots. Notably, abundant plasma proteins like α-2-macroglobulin, fibronectin, and transthyretin are absent from microclots, while less abundant proteins such as adiponectin, periostin, and von Willebrand Factor are well represented. Using bioinformatic tools including AmyloGram and AnuPP, we found that proteins entrapped in fibrinaloid microclots exhibit high amyloidogenic tendencies, suggesting their integration as cross-β elements into amyloid structures. This integration likely contributes to the microclots’ resistance to proteolysis. Our findings underscore the role of cross-seeding in fibrinaloid microclot formation and highlight the need for further investigation into their structural properties and implications in thrombotic and amyloid diseases. These insights provide a foundation for developing novel diagnostic and therapeutic strategies targeting amyloidogenic cross-seeding in blood clotting disorders.

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Predictable fold switching by theSARS‐CoV‐2 proteinORF9b

Cited by 13 publications

References 31 publications

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90

A Cullin 5-based complex serves as an essential modulator of ORF9b stability in SARS-CoV-2 replication

Proteomic evidence for amyloidogenic cross-seeding in fibrinaloid microclots

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