SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication

Liu, Hainan; Bai, Yu; Zhang, Xun; Gao, Ting; Liu, Yue; Li, Entao; Wang, Xuefeng; Cao, Zheng; Zhu, Lin; Dong, Qincai; Hu, Yong; Wang, Guangfei; Song, Caiwei; Niu, Xiayang; Zheng, Tao; Wang, Di; Liu, Zijing; Jin, Yu; Li, Ping; Bian, Xiu‐Wu; Cao, Cheng; Liu, Xuan

doi:10.1128/jvi.00412-22

Cited by 53 publications

(68 citation statements)

References 31 publications

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“…To investigate if inflammatory pathway regulation by the VOCs was associated with viral protein expression, as we found previously for Alpha ( 14 ), we correlated viral protein and RNA levels with pathway module regulation from our integrative computational analysis and ranked viral genes according to their correlation with inflammatory response modules (modules 8, 13, 31, 43, and 53). Expression of N and Orf6, both well-studied innate immune antagonist proteins ( 53 – 55 ), were the most anticorrelated with inflammatory response modules, consistent with the notion that their enhanced expression contributes to the suppression of the host innate immune response across the VOCs (Fig. 6J, S7D).…”

Section: Main Textsupporting

confidence: 78%

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

Bouhaddou

Reuschl

Polacco

et al. 2022

Preprint

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A series of SARS-CoV-2 variants of concern (VOCs) have evolved in humans during the COVID-19 pandemic—Alpha, Beta, Gamma, Delta, and Omicron. Here, we used global proteomic and genomic analyses during infection to understand the molecular responses driving VOC evolution. We discovered VOC-specific differences in viral RNA and protein expression levels, including for N, Orf6, and Orf9b, and pinpointed several viral mutations responsible. An analysis of the host response to VOC infection and comprehensive interrogation of altered virus-host protein-protein interactions revealed conserved and divergent regulation of biological pathways. For example, regulation of host translation was highly conserved, consistent with suppression of VOC replication in mice using the translation inhibitor plitidepsin. Conversely, modulation of the host inflammatory response was most divergent, where we found Alpha and Beta, but not Omicron BA.1, antagonized interferon stimulated genes (ISGs), a phenotype that correlated with differing levels of Orf6. Additionally, Delta more strongly upregulated proinflammatory genes compared to other VOCs. Systematic comparison of Omicron subvariants revealed BA.5 to have evolved enhanced ISG and proinflammatory gene suppression that similarly correlated with Orf6 expression, effects not seen in BA.4 due to a mutation that disrupts the Orf6-nuclear pore interaction. Our findings describe how VOCs have evolved to fine-tune viral protein expression and protein-protein interactions to evade both innate and adaptive immune responses, offering a likely explanation for increased transmission in humans.One sentence summarySystematic proteomic and genomic analyses of SARS-CoV-2 variants of concern reveal how variant-specific mutations alter viral gene expression, virus-host protein complexes, and the host response to infection with applications to therapy and future pandemic preparedness.

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Section: Main Textsupporting

confidence: 78%

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

Bouhaddou

Reuschl

Polacco

et al. 2022

Preprint

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“…Also, while host cell protein synthesis should best be inhibited and kept at a minimum, efficient unperturbed production of viral proteins must be ensured by preventing translational suppression due to activation of the OAS/RNaseL pathway and PKR-dependent induction of the ISR. Recent publications implicated SARS-CoV-2 N protein as an inhibitor of SG formation and an RLR antagonist (33)(34)(35)(60)(61)(62). Here, we confirmed and extended these observations using a convenient procedure to assess PKR-induced ISR activation based on transfection of ISR-inducing plasmid DNA to drive the expression of putative ISR antagonists genetically fused to EGFP.…”

Section: Discussionsupporting

confidence: 78%

“…G3BP1/2-N association through subdomain N1a (39, 50, 51) and N2b, directly via protein-protein interaction and/or indirectly, promoted by RNA binding (70), may well hamper SG assembly and/or thereby impede recruitment and SG-facilitated activation of RLRs and PKR (20-23) already early in infection when levels of N are still low. Indeed, this would be consistent with observations by others (35, 70) that SARS-CoV-2 inhibits induction of type I IFNs by targeting G3BP1 and suppressing SG-associated RLR activation. It has also been proposed that N counteracts the intracellular antiviral response by interfering with innate immune signaling at the level of MAVS and impeding MAVS-RIG-I association (59).…”

Section: Discussionmentioning

confidence: 99%

“…Possibly, such subtleties are missed in over-expression experiments and overshadowed by the robust ISR inhibition by N2b. G3BP1/2-N association through subdomain N1a (39, 50, 51) and N2b, directly via protein-protein interaction and/or indirectly, promoted by RNA binding (70), may well hamper SG assembly and/or thereby impede recruitment and SG-facilitated activation of RLRs and PKR (20-23) already early in infection when levels of N are still low. Indeed, this would be consistent with observations by others (35, 70) that SARS-CoV-2 inhibits induction of type I IFNs by targeting G3BP1 and suppressing SG-associated RLR activation.…”

Section: Discussionmentioning

confidence: 99%

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SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b

Aloise

Schipper

Donselaar

et al. 2022

Preprint

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The nucleocapsid protein N of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) enwraps and condenses the viral genome for packaging but is also an antagonist of the innate antiviral defense. It suppresses the integrated stress response (ISR), purportedly by interacting with stress granule (SG) assembly factors G3BP1 and 2, and inhibits type I interferon responses. To elucidate its mode of action, we systematically deleted and over-expressed distinct regions and domains. We show that N via domain N2b blocks PKR-mediated ISR activation, as measured by suppression of ISR-induced translational arrest and SG formation. N2b mutations that prevent dsRNA binding abrogate these activities also when introduced in the intact N protein. Substitutions reported to block post-translation modifications of N or its interaction with G3BP1/2 did not have a detectable additive effect. In an encephalomyocarditis virus-based infection model, N2b - but not a derivative defective in RNA binding - prevented PKR activation, inhibited β-interferon expression and promoted virus replication. Apparently, SARS-CoV-2 N inhibits innate immunity by sequestering dsRNA to prevent activation of PKR and RIG-I-like receptors. Observations made for the N protein of human coronavirus 229E suggests that this may be a general trait conserved among members of other orthocoronavirus (sub)genera.

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“…N-iORF3, encompassing the C-terminal N2b domain and surrounding linker regions, inhibits the dsRNA-dependent IFN-response (Mears et al, BioRxiv DOI=10.1101/2022.04.20.488895), and overexpression experiments in our laboratory showed that it also blocks the ISR (Aloise et al, BioRxiv DOI=10.1101/2022.09.02.506332). In interactome studies, SARS-CoV-2 N was found to bind to SG assembly factors G3BP1 and G3BP2 via its N-terminal domain N1a, suggesting that it may also directly suppress SG formation 61 , 62 , 63• , 64 , 65 , 66 , 67• .…”

Section: Coronavirus Isr Antagonistsmentioning

confidence: 99%

Move and countermove: the integrated stress response in picorna- and coronavirus-infected cells

Aloise

Schipper

Groot

et al. 2022

Current Opinion in Immunology

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SARS-CoV-2 N Protein Antagonizes Stress Granule Assembly and IFN Production by Interacting with G3BPs to Facilitate Viral Replication

Cited by 53 publications

References 31 publications

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

Global landscape of the host response to SARS-CoV-2 variants reveals viral evolutionary trajectories

SARS-CoV-2 nucleocapsid protein inhibits the PKR-mediated integrated stress response through RNA-binding domain N2b

Move and countermove: the integrated stress response in picorna- and coronavirus-infected cells

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