A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications

Min, Yuan-Qin; Huang, Mengzhuo; Feng, Kuan; Jia, Yajie; Sun, Xiulian; Ning, Yun‐Jia

doi:10.1016/j.mcpro.2023.100579

Cited by 13 publications

(11 citation statements)

References 60 publications

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“…These include mitochondrial translation, mRNA metabolic process, ribosome biogenesis, regulation of translation, ribonucleoprotein complex assembly, RNA localization, spliceosomal complex assembly, RNA modification, RNA 3’-end processing, among others (Fig 6A-B). Notably, these findings align with the previous interactome analyses of the N proteins from IBV [50], PEDV [51], SARS-CoV-2 [52, 53]. Among the IBV N binding proteins involved in nucleocytoplasmic transport, key players were identified and listed in Fig 6C, including RACK1 (Receptor of activated protein C kinase 1), NXF1 (Nuclear RNA export factor 1), LMNB1 (Lamin B1), KPNA2 (importin α1), PPP1CC (Protein phosphatase PP1γ), RAE1 (mRNA export factor), LBR (Lamin B receptor), SRPK1 (SRSF protein kinase 1), NUPL2 (Nucleoporin-like protein 2, NUP42).…”

Section: Resultssupporting

confidence: 89%

“…These include mitochondrial translation, mRNA metabolic process, ribosome biogenesis, regulation of translation, ribonucleoprotein complex assembly, RNA localization, spliceosomal complex assembly, RNA modification, RNA 3'-end processing, among others (Fig 6A -B). Notably, these findings align with the previous interactome analyses of the N proteins from IBV [50], PEDV [51], SARS-CoV-2 [52,53]. Among the IBV N binding proteins involved in nucleocytoplasmic transport, key To investigate whether the interaction with RACK1 is a common feature among pan-coronaviruses N proteins, Flag-tagged N proteins from various coronaviruses were co-expressed with HA-RACK1 in HEK-293T cells.…”

Section: Pan-coronaviruses N Proteins Interact With Scaffold Protein ...supporting

confidence: 80%

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Coronavirus Nucleocapsid Protein Enhances the binding of p-PKCα to RACK1: Implications for Inhibition of Nucleocytoplasmic Trafficking and Suppression of the Innate Immune Response

Xue,

Chu,

Wang

et al. 2024

Preprint

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The hallmark of coronavirus infection lies in its ability to evade host immune defenses, a process intricately linked to the nuclear entry of transcription factors crucial for initiating the expression of antiviral genes. Central to this evasion strategy is the manipulation of the nucleocytoplasmic trafficking system, which serves as an effective target for the virus to modulate the expression of immune response-related genes. In this investigation, we discovered that infection with the infectious bronchitis virus (IBV) dynamically impedes the nuclear translocation of several transcription factors such as IRF3, STAT1, STAT2, NF-κB p65, and the p38 mitogen-activated protein kinase (MAPK), leading to compromised transcriptional induction of key antiviral genes such as IFNβ, IFITM3, and IL-8. Further examination revealed that during the infection process, components of the nuclear pore complex (NPC), particularly FG-Nups (such as NUP62, NUP153, NUP42, and TPR), undergo cytosolic dispersion from the nuclear envelope; NUP62 undergoes phosphorylation, and NUP42 exhibits a mobility shift in size. These observations suggest a disruption in nucleocytoplasmic trafficking. Screening efforts identified the IBV nucleocapsid protein (N) as the agent responsible for the cytoplasmic distribution of FG-Nups, subsequently hindering the nuclear entry of transcription factors and suppressing the expression of antiviral genes. Interactome analysis further revealed that the IBV N protein interacts with the scaffold protein RACK1, facilitating the recruitment of activated protein kinase C alpha (p-PKCα) to RACK1 and relocating the RACK1-PKCα complex to the cytoplasm. These observations are conserved across pan-coronaviruses N proteins. Concurrently, the presence of both RACK1 and PKCα/β proved essential for the phosphorylation and cytoplasmic dispersion of NUP62, the suppression of antiviral cytokine expression, and efficient virus replication. These findings unveil a novel, highly effective, and evolutionarily conserved mechanism.Author summaryCoronaviruses employ diverse strategies to suppress the host innate immune defense. In this study, we uncovered a novel and highly effective strategy utilized by pan-coronaviruses to inhibit the innate immune response. Specifically, we found that the coronavirus N protein facilitates the binding of p-PKCα to RACK1, leading to the phosphorylation of NUP62 and the cytoplasmic redistribution of multiple FG-Nups. This phenomenon is accompanied by the disruption of nuclear translocation of several innate immune response-related transcription factors and suppression of antiviral/pro-inflammatory genes expression. Our research represents the first elucidation of how the N protein targets and impairs NPC function through the promotion of RACK1-PKCα interaction and NUP62 phosphorylation/disassembly. This discovery unveils a novel mechanism employed by pan-coronaviruses to counteract the host immune response.

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Section: Resultssupporting

confidence: 89%

“…These include mitochondrial translation, mRNA metabolic process, ribosome biogenesis, regulation of translation, ribonucleoprotein complex assembly, RNA localization, spliceosomal complex assembly, RNA modification, RNA 3'-end processing, among others (Fig 6A -B). Notably, these findings align with the previous interactome analyses of the N proteins from IBV [50], PEDV [51], SARS-CoV-2 [52,53]. Among the IBV N binding proteins involved in nucleocytoplasmic transport, key To investigate whether the interaction with RACK1 is a common feature among pan-coronaviruses N proteins, Flag-tagged N proteins from various coronaviruses were co-expressed with HA-RACK1 in HEK-293T cells.…”

Section: Pan-coronaviruses N Proteins Interact With Scaffold Protein ...supporting

confidence: 80%

Coronavirus Nucleocapsid Protein Enhances the binding of p-PKCα to RACK1: Implications for Inhibition of Nucleocytoplasmic Trafficking and Suppression of the Innate Immune Response

Xue,

Chu,

Wang

et al. 2024

Preprint

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“…After synthesizing uridine modified separately at the ribose and nucleobase of the RIBOTAC module, we aim to evaluate their antiviral effectiveness. In the assessment of compound efficacy against SARS-CoV-2, the human hepatocellular carcinoma cell line, Huh7-ACE2, offers a dependable model ( 51 ). This cell line serves as an effective simulation of the infection process of viruses such as SARS-CoV-2 under laboratory conditions.…”

Section: Resultsmentioning

confidence: 99%

Developing nucleoside tailoring strategies against SARS-CoV-2 via ribonuclease targeting chimera

Min,

Xiong,

Shen

et al. 2024

Sci. Adv.

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In response to the urgent need for potent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) therapeutics, this study introduces an innovative nucleoside tailoring strategy leveraging ribonuclease targeting chimeras. By seamlessly integrating ribonuclease L recruiters into nucleosides, we address RNA recognition challenges and effectively inhibit severe acute respiratory syndrome coronavirus 2 replication in human cells. Notably, nucleosides tailored at the ribose 2′-position outperform those modified at the nucleobase. Our in vivo validation using hamster models further bolsters the promise of this nucleoside tailoring approach, positioning it as a valuable asset in the development of innovative antiviral drugs.

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“…The prototypic SARS-CoV-2 WIV04 strain (IVCAS 6.7512) 1 and its Delta (IVCAS 6.7585) and Omicron sublineage BA.4 (IVCAS 6.8980) and BA.5 (IVCAS 6.8981) variants were obtained from the National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, and expanded as described previously 29 . Viral titer was determined by 50% tissue culture infective dose (TCID 50 ) method using cytopathic effect assay (CPE) 29 .…”

Section: Cells Viruses and Animalsmentioning

confidence: 99%

Host protein ARF1 is a proviral factor for SARS-CoV-2 and a candidate broad-spectrum therapeutic target

Ning,

Zhang,

Min

et al. 2024

Preprint

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SARS-CoV-2 and its emerging variants pose continuing threats to public health, necessitating further research into detailed infection mechanisms and broad-spectrum antiviral therapies. SARS-CoV-2 assembles at the ER–Golgi intermediate compartment (ERGIC), where the viral membrane (M) protein highly accumulates to act as the central driver of the viral morphogenesis. However, which host factors are involved in the ERGIC accumulation of M and whether they could be exploited as broad-spectrum antiviral targets remain unclear. Here, we identified an M-interacting host protein ARF1 as a proviral factor that bolsters the propagation of SARS-CoV-2 and its variants in cultured cells and the viral infection and pathogenicity in K18-hACE2 mice. Mechanistically, by interacting with M, ARF1 facilitates M accumulation to the ERGIC and thus M-driven virion production. Consistently, pharmacological ARF1 inhibition by small molecules disrupted ARF1 and M concentration at the ERGIC, blocking virion assembly and propagation. Furthermore, a designed peptide mimicking the M-targeted motif of ARF1 competitively blocked the M-ARF1 interaction, M accumulation at the ERGIC, M-driven virion assembly, and SARS-CoV-2 and its variants’ propagation in vitro. Moreover, the peptidomimetic inhibitor exhibited a therapeutic efficacy against SARS-CoV-2 infection and pathogenicity in vivo. These findings provide critical insights into the basic biology of SARS-CoV-2 propagation and show the potential to develop pan-SARS-CoV-2 therapeutic strategies by targeting ARF1 and/or the ARF1-M interaction interface.

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A New Cellular Interactome of SARS-CoV-2 Nucleocapsid Protein and Its Biological Implications

Cited by 13 publications

References 60 publications

Coronavirus Nucleocapsid Protein Enhances the binding of p-PKCα to RACK1: Implications for Inhibition of Nucleocytoplasmic Trafficking and Suppression of the Innate Immune Response

Coronavirus Nucleocapsid Protein Enhances the binding of p-PKCα to RACK1: Implications for Inhibition of Nucleocytoplasmic Trafficking and Suppression of the Innate Immune Response

Developing nucleoside tailoring strategies against SARS-CoV-2 via ribonuclease targeting chimera

Host protein ARF1 is a proviral factor for SARS-CoV-2 and a candidate broad-spectrum therapeutic target

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