Single-Molecule Dynamics of SARS-CoV-2 5’ Cap Recognition by Human eIF4F

Hong, Hao; Mg, Guevara; Lin, E. K.; Se, O’Leary

doi:10.1101/2021.05.26.445185

Cited by 6 publications

(8 citation statements)

References 88 publications

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“…In general, it is not known at which stage during scanning individual eIF4 factors might dissociate from the PIC. Selective 40S subunit profiling as well as single-molecule studies of translation initiation ( 27 , 42 , 65–67 ) hold great potential in dissecting the assembly pathways of eIF4s along the initiation process, both for specific mRNAs and transcriptome-wide. Such studies will help pin-point the distinct roles of the individual eIF4 factors and their complexes in the different stages of translation initiation on different mRNAs.…”

Section: Discussionmentioning

confidence: 99%

The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

Krause

Willing

Andreou

et al. 2022

Nucleic Acids Research

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Translation initiation in eukaryotes starts with the recognition of the mRNA 5′-cap by eIF4F, a hetero-trimeric complex of eIF4E, the cap-binding protein, eIF4A, a DEAD-box helicase, and eIF4G, a scaffold protein. eIF4G comprises eIF4E- and eIF4A-binding domains (4E-BD, 4A-BD) and three RNA-binding regions (RNA1–RNA3), and interacts with eIF4A, eIF4E, and with the mRNA. Within the eIF4F complex, the helicase activity of eIF4A is increased. We showed previously that RNA3 of eIF4G is important for the stimulation of the eIF4A conformational cycle and its ATPase and helicase activities. Here, we dissect the interplay between the eIF4G domains and the role of the eIF4E/cap interaction in eIF4A activation. We show that RNA2 leads to an increase in the fraction of eIF4A in the closed state, an increased RNA affinity, and faster RNA unwinding. This stimulatory effect is partially reduced when the 4E-BD is present. eIF4E binding to the 4E-BD then further inhibits the helicase activity and closing of eIF4A, but does not affect the RNA-stimulated ATPase activity of eIF4A. The 5′-cap renders the functional interaction of mRNA with eIF4A less efficient. Overall, the activity of eIF4A at the 5′-cap is thus fine-tuned by a delicately balanced network of stimulatory and inhibitory interactions.

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Section: Discussionmentioning

confidence: 99%

The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

Krause

Willing

Andreou

et al. 2022

Nucleic Acids Research

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“…Dysregulation of this gene pathway may contribute to downstream effects of antigen presentation and sustained T cell activation [75]. Lastly, the 5 cap of SARS CoV-2 is recognized by Eukaryotic initiation factor 4A-1 (EIF4A1) [76] and is known to be significantly increased in the platelet proteome of COVID-19 patients thought to affect viral replication and downstream activation of platelets, resulting in thrombotic microangiopathy and increased mortality [77]. Collectively, the results of our WGCNA analysis identified a substantial network of dysregulated genes with many downstream effects that are linked to clinicopathologic data.…”

Section: Discussionmentioning

confidence: 99%

SARS CoV-2 (Delta Variant) Infection Kinetics and Immunopathogenesis in Domestic Cats

Selvan

Gunasekara

Xiao

et al. 2022

Viruses

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Continued emergence of SARS-CoV-2 variants highlights the critical need for adaptable and translational animal models for acute COVID-19. Limitations to current animal models for SARS CoV-2 (e.g., transgenic mice, non-human primates, ferrets) include subclinical to mild lower respiratory disease, divergence from clinical COVID-19 disease course, and/or the need for host genetic modifications to permit infection. We therefore established a feline model to study COVID-19 disease progression and utilized this model to evaluate infection kinetics and immunopathology of the rapidly circulating Delta variant (B.1.617.2) of SARS-CoV-2. In this study, specific-pathogen-free domestic cats (n = 24) were inoculated intranasally and/or intratracheally with SARS CoV-2 (B.1.617.2). Infected cats developed severe clinical respiratory disease and pulmonary lesions at 4- and 12-days post-infection (dpi), even at 1/10 the dose of previously studied wild-type SARS-CoV-2. Infectious virus was isolated from nasal secretions of delta-variant infected cats in high amounts at multiple timepoints, and viral antigen was co-localized in ACE2-expressing cells of the lungs (pneumocytes, vascular endothelium, peribronchial glandular epithelium) and strongly associated with severe pulmonary inflammation and vasculitis that were more pronounced than in wild-type SARS-CoV-2 infection. RNA sequencing of infected feline lung tissues identified upregulation of multiple gene pathways associated with cytokine receptor interactions, chemokine signaling, and viral protein–cytokine interactions during acute infection with SARS-CoV-2. Weighted correlation network analysis (WGCNA) of differentially expressed genes identified several distinct clusters of dysregulated hub genes that are significantly correlated with both clinical signs and lesions during acute infection. Collectively, the results of these studies help to delineate the role of domestic cats in disease transmission and response to variant emergence, establish a flexible translational model to develop strategies to prevent the spread of SARS-CoV-2, and identify potential targets for downstream therapeutic development.

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“…rather than eIF4E-eIF4G interactions are the main cause for the acceleration eIF4E-cap binding the increased association time of eIF4E during translation initiation, which is consistent with a recent in vivo report suggesting that eIF4G may persist in mRNA and facilitate cap-binding activity 50 . Conversely, another smFRET study found that partial human eIF4G (557-1137) alone slightly affects eIF4E binding kinetics, but the human eIF4F complex substantially changes the eIF4E-cap association 46 . Interestingly, free eIF4A enhances eIF4E cap accessibility via direct eIF4A-RNA contact, which is similar to the role of eIF4G in eIF4E 25 .…”

Section: Smfi Studies In Mrna Circularizationmentioning

confidence: 94%

“…Recognition of eIF4G increases eIF4E's affinity to a cap or cap analog 44,45 . To study the 5' cap-binding kinetics of eIF4E, smFRET was monitored with fluorescently labeled eIF4E in the absence and presence of eIF4G 25,46,47 (Fig. 2a).…”

Section: Smfi Studies In Mrna Circularizationmentioning

confidence: 99%

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Single-molecule visualization of mRNA circularization during translation

et al. 2023

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Translation is mediated by precisely orchestrated sequential interactions among translation initiation components, mRNA, and ribosomes. Biochemical, structural, and genetic techniques have revealed the fundamental mechanism that determines what occurs and when, where and in what order. Most mRNAs are circularized via the eIF4E–eIF4G–PABP interaction, which stabilizes mRNAs and enhances translation by recycling ribosomes. However, studies using single-molecule fluorescence imaging have allowed for the visualization of complex data that opposes the traditional “functional circularization” theory. Here, we briefly introduce single-molecule techniques applied to studies on mRNA circularization and describe the results of in vitro and live-cell imaging. Finally, we discuss relevant insights and questions gained from single-molecule research related to translation.

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Single-Molecule Dynamics of SARS-CoV-2 5’ Cap Recognition by Human eIF4F

Cited by 6 publications

References 88 publications

The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

The domains of yeast eIF4G, eIF4E and the cap fine-tune eIF4A activities through an intricate network of stimulatory and inhibitory effects

SARS CoV-2 (Delta Variant) Infection Kinetics and Immunopathogenesis in Domestic Cats

Single-molecule visualization of mRNA circularization during translation

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