Ubiquitination and degradation of NF90 by Tim-3 inhibits antiviral innate immunity
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Evaluation Summary:
Stress granules are a critical component in the defense against certain viruses. The mechanisms regulating stress granules induced but the NF90 pathway are not well defined. Here, the authors use biochemical, cell-based, and in vivo approaches to make the novel discovery that Tim3 functions as a negative regulator of NF90-mediated antiviral responses.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Abstract
Nuclear Factor 90 (NF90) is a novel virus sensor that serves to initiate antiviral innate immunity by triggering the stress granules (SGs) formation. However, the regulation of the NF90-SGs pathway remain largely unclear. We found that Tim-3, an immune checkpoint inhibitor, promotes the ubiquitination and degradation of NF90 and inhibits NF90-SGs mediated antiviral immunity. Vesicular Stomatitis Virus (VSV) infection induces the up-regulation and activation of Tim-3 in macrophages which in turn recruited the E3 ubiquitin ligase TRIM47 to the zinc finger domain of NF90 and initiated a proteasome-dependent degradation of the NF90 via K48-linked ubiquitination at Lys297. Targeted inactivation of the Tim-3 enhances the NF90 downstream SGs formation by selectively increasing the phosphorylation of PKR and eIF2a, the expression of SGs markers G3BP1 and TIA-1, and protected mice from lethal VSV challenge. These findings provide insights into the crosstalk between Tim-3 and other receptors in antiviral innate immunity and its related clinical significance.
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Evaluation Summary:
Stress granules are a critical component in the defense against certain viruses. The mechanisms regulating stress granules induced but the NF90 pathway are not well defined. Here, the authors use biochemical, cell-based, and in vivo approaches to make the novel discovery that Tim3 functions as a negative regulator of NF90-mediated antiviral responses.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. The reviewers remained anonymous to the authors.)
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Reviewer #1 (Public Review):
In this study, the authors sought to uncover mechanism regulating NF90-induced antiviral pathways. They initially discovered that the checkpoint protein Tim3 was both induced upon viral infection and that loss of Tim3 is protective against the infection. Mechanistically, the authors used biochemical and cell-based approaches to discover that Tim3 binds NF90 and promotes its degradation via ubiquitination mediated by the E3 ligase TRIM47. The authors further assess the functional consequence of Tim3-mediated inhibition of NF90. As NF90 has known roles in stress granule formation, the authors demonstrated that loss of Tim3 expression is associated with increased NF90, which correlated with increased phosphorylation of PKR and eIF2a and induction of stress granule markers. To demonstrate biological relevance, …
Reviewer #1 (Public Review):
In this study, the authors sought to uncover mechanism regulating NF90-induced antiviral pathways. They initially discovered that the checkpoint protein Tim3 was both induced upon viral infection and that loss of Tim3 is protective against the infection. Mechanistically, the authors used biochemical and cell-based approaches to discover that Tim3 binds NF90 and promotes its degradation via ubiquitination mediated by the E3 ligase TRIM47. The authors further assess the functional consequence of Tim3-mediated inhibition of NF90. As NF90 has known roles in stress granule formation, the authors demonstrated that loss of Tim3 expression is associated with increased NF90, which correlated with increased phosphorylation of PKR and eIF2a and induction of stress granule markers. To demonstrate biological relevance, the authors reveal that mice lacking Tim3 are more resistant to VSV infection.
Strengths include diverse experimental approaches, including in vivo work, to identify and characterize the TIM3-TRIM47-NF90 axis. Weaknesses include lack of a few key experiments that will help support what is already a strong study. The impact of the work is fundamental new insight into the pathways regulating an important mechanism of viral control.
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Reviewer #2 (Public Review):
In the present manuscript, the authors provide data on a cross-talk between the immune checkpoint molecule Tim-3 in macrophages and the viral sensor NF90 in the context of VSV infection. In particular, they demonstrate (by using cell lines and primary cells) that VSV infection leads to increased expression and activation of Tim-3, Tim-3 interacts with NF90 via its cytoplasmic tail and enhances NF90 ubiquitination by recruitment of TRIM47. Subsequently, NF90 is degraded and VSV replication is increased. Thus, Tim-3 can mediate viral escape in VSV infection. With this, the authors provide a novel viral escape mechanism involving the previously described immune checkpoint molecule Tim-3 and NF90 in macrophages. Although the authors thoroughly identified key players within this viral escape pathway future …
Reviewer #2 (Public Review):
In the present manuscript, the authors provide data on a cross-talk between the immune checkpoint molecule Tim-3 in macrophages and the viral sensor NF90 in the context of VSV infection. In particular, they demonstrate (by using cell lines and primary cells) that VSV infection leads to increased expression and activation of Tim-3, Tim-3 interacts with NF90 via its cytoplasmic tail and enhances NF90 ubiquitination by recruitment of TRIM47. Subsequently, NF90 is degraded and VSV replication is increased. Thus, Tim-3 can mediate viral escape in VSV infection. With this, the authors provide a novel viral escape mechanism involving the previously described immune checkpoint molecule Tim-3 and NF90 in macrophages. Although the authors thoroughly identified key players within this viral escape pathway future studies are required to comprehensively resolve subsequent steps within this pathway. The manuscript is well written and structured and the data appear reliable.
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