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The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

ABSTRACT: The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed an Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. To exclude that the observed depletion was caused by the antiviral response, we generated a yeast system in which the function of human Tom70 could be recapitulated. Upon expression of Orf9b in these cells, we again observed a specific decline of a subset of mitochondrial proteins and a general reduction of mitochondrial volume. Thus, the SARS-CoV-2 virus is able to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

SUBMITTER: Lenhard S

PROVIDER: S-EPMC10491932 | biostudies-literature | 2023 Oct

REPOSITORIES: biostudies-literature

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The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

Lenhard Svenja S Gerlich Sarah S Khan Azkia A Rödl Saskia S Bökenkamp Jan-Eric JE Peker Esra E Zarges Christine C Faust Janina J Storchova Zuzana Z Räschle Markus M Riemer Jan J Herrmann Johannes M JM

The Journal of cell biology 20230908 10

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the protein Orf9b to target the mitochondrial outer membrane protein Tom70. Tom70 serves as an import receptor for mitochondrial precursors and, independently of this function, is critical for the cellular antiviral response. Previous studies suggested that Orf9b interferes with Tom70-mediated antiviral signaling, but its implication for mitochondrial biogenesis is unknown. In this study, we expressed Orf9 ...[more]

PMID: 37682539

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Project description:Viruses often induce a complex rewiring of cellular physiology to maximize their propagation. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) expresses high amounts of the viral antagonistic protein Orf9b. Increased amounts and mutated versions of Orf9b were found to correlate with improved transmission of more evolved virus forms such as the Alpha and Omicron variants. Orf9b binds to the mitochondrial surface receptor Tom70 which stiffens its highly flexible structure. Tom70 plays a critical role in the antiviral response since it serves as recruiting factor for the mitochondrial antiviral signaling (MAVS) protein. However, the predominant and MAVS-independent role of Tom70 is that of a receptor component of the mitochondrial import system. Whether Orf9b also interferes with this predominant function of Tom70 is unknown. In this study, we expressed Orf9b in human HEK293 cells and observed the Orf9b-mediated depletion of mitochondrial proteins, particularly in respiring cells. The spectrum of depleted mitochondrial proteins was comparable to that of cells in which Tom70 was depleted, consistent with an Orf9b-mediated impairment of Tom70-dependent protein import. To exclude that the observed depletion was caused by the antiviral response triggered by Orf9b expression, we generated a yeast system in which the yeast Tom70 was replaced by a humanized version of Tom70. Upon expression of Orf9b in these cells, we again observed a general but moderate reduction of mitochondrial proteins. Similar to the situation in human cells, a number of mitochondrial proteins were particular sensitive to Orf9b expression, suggesting a rather specific modulation of the mitochondrial import system. In vitro import experiments with semi-intact cells confirmed this Orf9b-mediated impairment of the mitochondrial protein import. Thus, the SARS-CoV-2 virus seems to modulate the mitochondrial proteome by a direct effect of Orf9b on mitochondrial Tom70-dependent protein import.

Project description:Ubiquitin signaling is essential for immunity to restrict pathogen proliferation. Due to its enormous impact on human health and the global economy, intensive efforts have been invested in studying severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its interactions with hosts. However, the role of the ubiquitin network in pathogenicity has not yet been explored. Here, we found that ORF9b of SARS-CoV-2 is ubiquitinated on Lys-4 and Lys-40 by unknown E3 ubiquitin ligases and is degraded by the ubiquitin proteasomal system. Importantly, we identified USP29 as a host factor that prevents ORF9b ubiquitination and subsequent degradation. USP29 interacts with the carboxyl end of ORF9b and removes ubiquitin chains from the protein, thereby inhibiting type I interferon (IFN) induction and NF-κB activation. We also found that ORF9b stabilization by USP29 enhanced the virulence of VSV-eGFP and transcription and replication-competent SARS-CoV-2 virus-like-particles (trVLP). Moreover, we observed that the mRNA level of USP29 in SARS-CoV-2 patients was higher than that in healthy people. Our findings provide important evidence indicating that targeting USP29 may effectively combat SARS-CoV-2 infection. IMPORTANCE Coronavirus disease 2019 (COVID-19) is a current global health threat caused by SARS-CoV-2. The innate immune response such as type I IFN (IFN-I) is the first line of host defense against viral infections, whereas SARS-CoV-2 proteins antagonize IFN-I production through distinct mechanisms. Among them, ORF9b inhibits the canonical IκB kinase alpha (IKKɑ)/β/γ-NF-κB signaling and subsequent IFN production; therefore, discovering the regulation of ORF9b by the host might help develop a novel antiviral strategy. Posttranslational modification of proteins by ubiquitination regulates many biological processes, including viral infections. Here, we report that ORF9b is ubiquitinated and degraded through the proteasome pathway, whereas deubiquitinase USP29 deubiquitinates ORF9b and prevents its degradation, resulting in the enhancement of ORF9b-mediated inhibition of IFN-I and NF-κB activation and the enhancement of virulence of VSV-eGFP and SARS-CoV-2 trVLP.

Dataset Information

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

Publications

The Orf9b protein of SARS-CoV-2 modulates mitochondrial protein biogenesis.

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