mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism.

Energetic stress compels cells to evolve adaptive mechanisms to adjust their metabolism. Inhibition of mTOR kinase complex 1 (mTORC1) is essential for cell survival during glucose starvation. How mTORC1 controls cell viability during glucose starvation is not well understood. Here we show that the mTORC1 effectors eukaryotic initiation factor 4E binding proteins 1/2 (4EBP1/2) confer protection to mammalian cells and budding yeast under glucose starvation. Mechanistically, 4EBP1/2 promote NADPH homeostasis by preventing NADPH-consuming fatty acid synthesis via translational repression of Acetyl-CoA Carboxylase 1 (ACC1), thereby mitigating oxidative stress. This has important relevance for cancer, as oncogene-transformed cells and glioma cells exploit the 4EBP1/2 regulation of ACC1 expression and redox balance to combat energetic stress, thereby supporting transformation and tumorigenicity in vitro and in vivo. Clinically, high EIF4EBP1 expression is associated with poor outcomes in several cancer types. Our data reveal that the mTORC1-4EBP1/2 axis provokes a metabolic switch essential for survival during glucose starvation which is exploited by transformed and tumor cells.

Methionyl-Methionine Dipeptide Enhances Mammogenesis and Lactogenesis by Suppressing the Expression of a Novel Long Noncoding RNA MGPNCR to Inhibit eIF4B Dephosphorylation.

Previous research has demonstrated that in pregnant mice deficient in l-methionine (Met), the mixture of the dipeptide l-methionyl-l-methionine (Met-Met) with Met was more effective than Met alone in promoting mammogenesis and lactogenesis. This study aimed to investigate the role of a novel long noncoding RNA (lncRNA), named mammary gland proliferation-associated lncRNA (MGPNCR), in these processes. Transcriptomic analysis of mammary tissues from Met-deficient mice, supplemented either with a Met-Met/Met mixture or with Met alone, revealed significantly higher MGPNCR expression in the Met group compared to the mixture group, a finding recapitulated in a mammary epithelial cell model. Our findings suggested that MGPNCR hindered mammogenesis and milk protein synthesis by binding to eukaryotic initiation factor 4B (eIF4B). This interaction promoted the dephosphorylation of eIF4B at serine-422 by enhancing its association with protein phosphatase 2A (PP2A). Our study sheds light on the regulatory mechanisms of lncRNA-mediated dipeptide effects on mammary cell proliferation and milk protein synthesis. These insights underscore the potential benefits of utilizing dipeptides to improve milk protein in animals and potentially in humans.

Comparison of Human Eukaryotic Translation Initiation Factors 5A1 and 5AL1: Identification of Amino Acid Residues Important for EIF5A1 Lysine 50 Hypusination and Its Protein Stability.

The human eukaryotic translation initiation factor 5A (EIF5A) family consists of three members, namely EIF5A1, EIF5A2, and EIF5AL1. Recent studies have shown that the expression of EIF5As is related to many human diseases, such as diabetes, viral infection, central nervous system injury, and cancer. Among them, EIF5A1 plays different functions in various cancers, possibly as a tumor-suppressor or oncogene, while EIF5A2 promotes the occurrence and development of cancer. Yet, the biological function of EIF5AL1 is not being studied so far. Interestingly, although there are only three amino acid (at residues 36, 45, and 109) differences between EIF5A1 and EIF5AL1, we demonstrate that only EIF5A1 can be hypusinated while EIF5AL1 cannot, and EIF5AL1 has a tumor-suppressor-like function by inhibiting cell proliferation and migration. We also show that EIF5AL1 protein turnover is mediated through the proteasomal pathway, and EIF5AL1 protein turnover is much faster than that of EIF5A1, which may explain their differential protein expression level in cells. By engineering single and double mutations on these three amino acids, we pinpoint which of these amino acids are critical for hypusination and protein stability. The data of this work should fill in the gaps in EIF5As research and pave the way for future studies on EIF5AL1.

The eukaryotic translation initiation factor eIF4E reprograms alternative splicing.

Aberrant splicing is typically attributed to splice-factor (SF) mutation and contributes to malignancies including acute myeloid leukemia (AML). Here, we discovered a mutation-independent means to extensively reprogram alternative splicing (AS). We showed that the dysregulated expression of eukaryotic translation initiation factor eIF4E elevated selective splice-factor production, thereby impacting multiple spliceosome complexes, including factors mutated in AML such as SF3B1 and U2AF1. These changes generated a splicing landscape that predominantly supported altered splice-site selection for ~800 transcripts in cell lines and ~4,600 transcripts in specimens from high-eIF4E AML patients otherwise harboring no known SF mutations. Nuclear RNA immunoprecipitations, export assays, polysome analyses, and mutational studies together revealed that eIF4E primarily increased SF production via its nuclear RNA export activity. By contrast, eIF4E dysregulation did not induce known SF mutations or alter spliceosome number. eIF4E interacted with the spliceosome and some pre-mRNAs, suggesting its direct involvement in specific splicing events. eIF4E induced simultaneous effects on numerous SF proteins, resulting in a much larger range of splicing alterations than in the case of mutation or dysregulation of individual SFs and providing a novel paradigm for splicing control and dysregulation.

Dynamic states of eIF6 and SDS variants modulate interactions with uL14 of the 60S ribosomal subunit.

Assembly of ribosomal subunits into active ribosomal complexes is integral to protein synthesis. Release of eIF6 from the 60S ribosomal subunit primes 60S to associate with the 40S subunit and engage in translation. The dynamics of eIF6 interaction with the uL14 (RPL23) interface of 60S and its perturbation by somatic mutations acquired in Shwachman-Diamond Syndrome (SDS) is yet to be clearly understood. Here, by using a modified strategy to obtain high yields of recombinant human eIF6 we have uncovered the critical interface entailing eight key residues in the C-tail of uL14 that is essential for physical interactions between 60S and eIF6. Disruption of the complementary binding interface by conformational changes in eIF6 disease variants provide a mechanism for weakened interactions of variants with the 60S. Hydrogen-deuterium exchange mass spectrometry (HDX-MS) analyses uncovered dynamic configurational rearrangements in eIF6 induced by binding to uL14 and exposed an allosteric interface regulated by the C-tail of eIF6. Disrupting key residues in the eIF6-60S binding interface markedly limits proliferation of cancer cells, which highlights the significance of therapeutically targeting this interface. Establishing these key interfaces thus provide a therapeutic framework for targeting eIF6 in cancers and SDS.

A noncanonical function of EIF4E limits ALDH1B1 activity and increases susceptibility to ferroptosis.

Ferroptosis is a type of lipid peroxidation-dependent cell death that is emerging as a therapeutic target for cancer. However, the mechanisms of ferroptosis during the generation and detoxification of lipid peroxidation products remain rather poorly defined. Here, we report an unexpected role for the eukaryotic translation initiation factor EIF4E as a determinant of ferroptotic sensitivity by controlling lipid peroxidation. A drug screening identified 4EGI-1 and 4E1RCat (previously known as EIF4E-EIF4G1 interaction inhibitors) as powerful inhibitors of ferroptosis. Genetic and functional studies showed that EIF4E (but not EIF4G1) promotes ferroptosis in a translation-independent manner. Using mass spectrometry and subsequent protein-protein interaction analysis, we identified EIF4E as an endogenous repressor of ALDH1B1 in mitochondria. ALDH1B1 belongs to the family of aldehyde dehydrogenases and may metabolize the aldehyde substrate 4-hydroxynonenal (4HNE) at high concentrations. Supraphysiological levels of 4HNE triggered ferroptosis, while low concentrations of 4HNE increased the cell susceptibility to classical ferroptosis inducers by activating the NOX1 pathway. Accordingly, EIF4E-dependent ALDH1B1 inhibition enhanced the anticancer activity of ferroptosis inducers in vitro and in vivo. Our results support a key function of EIF4E in orchestrating lipid peroxidation to ignite ferroptosis.

High expression of serine and arginine-rich splicing factor 9 (SRSF9) is associated with hepatocellular carcinoma progression and a poor prognosis.

BACKGROUND: Serine and arginine-rich splicing factor 9 (SRSF9) has been linked to the occurrence and progression of various cancers; however, its effects and mechanism of action hepatocellular carcinoma (HCC) have not been reported. In this study, we used a bioinformatics approach and in vitro assays to evaluate the expression of SRSF9 in HCC, its prognostic value, and its underlying regulatory mechanisms, including analyses of related pathways and the role of methylation. METHODS: Transcriptomic and DNA methylation data for 357 HCC cases and 50 paratumor tissues in The Cancer Genome Atlas database were obtained. Additionally, protein expression data for cell lines and tissue samples were obtained from the Human Protein Atlas. The CMap databased was used to predict candidate drugs targeting SRSF9. Various cell lines were used for in vitro validation. RESULTS: SRSF9 expression was significantly elevated in HCC and was negatively regulated by its methylation site cg06116271. The low expression of SRSF9 and hypermethylation of cg06116271 were both associated with a longer overall survival time. A correlation analysis revealed ten genes that were co-expressed with SRSF9; levels of CDK4, RAN, DENR, RNF34, and ANAPC5 were positively correlated and levels of RBP4, APOC1, MASP2, HP, and HPX were negatively correlated with SRSF9 expression. The knockdown of SRSF9 in vitro inhibited the proliferation and migration of HCC cells and significantly reduced the expression of proteins in the Wnt signaling pathway (DVL2 and ß-catenin) and cell cycle pathway (Cyclin D and Cyclin E). A CMap analysis identified two drugs, camptothecin and apigenin, able to target and inhibit the expression of SRSF9. CONCLUSIONS: This study expands our understanding of the molecular biological functions of SRSF9 and cg06116271 and provides candidate diagnostic and therapeutic targets for HCC.

Inhibition of eIF6 Activity Reduces Hepatocellular Carcinoma Growth: An In Vivo and In Vitro Study.

Nonalcoholic fatty liver disease (NAFLD) is characterized by the accumulation of lipids in the liver. Given the high prevalence of NAFLD, its evolution to nonalcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) is of global concern. Therapies for managing NASH-driven HCC can benefit from targeting factors that play a continuous role in NAFLD evolution to HCC. Recent work has shown that postprandial liver translation exacerbates lipid accumulation through the activity of a translation factor, eukaryotic initiation factor 6 (eIF6). Here, we test the effect of eIF6 inhibition on the progression of HCC. Mice heterozygous for eIF6 express half the level of eIF6 compared to wt mice and are resistant to the formation of HCC nodules upon exposure to a high fat/high sugar diet combined with liver damage. Histology showed that nodules in eIF6 het mice were smaller with reduced proliferation compared to wt nodules. By using an in vitro model of human HCC, we confirm that eIF6 depletion reduces the growth of HCC spheroids. We also tested three pharmacological inhibitors of eIF6 activity-eIFsixty-1, eIFsixty-4, and eIFsixty-6-and all three reduced eIF6 binding to 60S ribosomes and limited the growth of HCC spheroids. Thus, inhibition of eIF6 activity is feasible and limits HCC formation.

Inhibitors of eIF4G1-eIF1 uncover its regulatory role of ER/UPR stress-response genes independent of eIF2α-phosphorylation.

During translation initiation, eIF4G1 dynamically interacts with eIF4E and eIF1. While the role of eIF4E-eIF4G1 is well established, the regulatory functions of eIF4G1-eIF1 are poorly understood. Here, we report the identification of the eIF4G1-eIF1 inhibitors i14G1-10 and i14G1-12. i14G1s directly bind eIF4G1 and inhibit translation in vitro and in the cell, and their effects on translation are dependent on eIF4G1 levels. Translatome analyses revealed that i14G1s mimic eIF1 and eIF4G1 perturbations on the stringency of start codon selection and the opposing roles of eIF1-eIF4G1 in scanning-dependent and scanning-independent short 5' untranslated region (UTR) translation. Remarkably, i14G1s activate ER/unfolded protein response (UPR) stress-response genes via enhanced ribosome loading, elevated 5'UTR translation at near-cognate AUGs, and unexpected concomitant up-regulation of coding-region translation. These effects are, at least in part, independent of eIF2α-phosphorylation. Interestingly, eIF4G1-eIF1 interaction itself is negatively regulated by ER stress and mTOR inhibition. Thus, i14G1s uncover an unknown mechanism of ER/UPR translational stress response and are valuable research tools and potential drugs against diseases exhibiting dysregulated translation.

The search for genetic dark matter and lessons learned from the journey.

In this review, I describe our scientific journey to unearth the impact of RNA metabolism in cancer using the eukaryotic translation initiation factor eIF4E as an exemplar. This model allowed us to discover new structural, biochemical, and molecular features of RNA processing, and to reveal their substantial impact on cell physiology. This led us to develop proof-of-principle strategies to target these pathways in cancer patients leading to clinical benefit. I discuss the important role that the unexpected plays in research and the necessity of embracing the data even when it clashes with dogma. I also touch on the importance of equity, diversity, and inclusion to the success of the scientific enterprise.

DENR controls JAK2 translation to induce PD-L1 expression for tumor immune evasion.

RNA-binding proteins (RBPs) can recognize thousands of RNAs that help to maintain cell homeostasis, and RBP dysfunction is frequently observed in various cancers. However, whether specific RBPs are involved in tumor immune evasion by regulating programmed death ligand-1 (PD-L1) is unclear. Here, we perform targeted RBP CRISPR/Cas9 screening and identify density regulated re-initiation and release factor (DENR) as a PD-L1 regulator. DENR-depleted cancer cells exhibit reduced PD-L1 expression in vitro and in vivo. DENR depletion significantly suppresses tumor growth and enhances the tumor-killing activity of CD8+ T cells. Mechanistically, DENR antagonizes the translational repression of three consecutive upstream open reading frames (uORFs) upstream of Janus kinase 2 (Jak2); thus, DENR deficiency impairs JAK2 translation and the IFNγ-JAK-STAT signaling pathway, resulting in reduced PD-L1 expression in tumors. Overall, we discover an RBP DENR that could regulate PD-L1 expression for tumor immune evasion, and highlight the potential of DENR as a therapeutic target for immunotherapy.

Aberrant protein synthesis and cancer development: The role of canonical eukaryotic initiation, elongation and termination factors in tumorigenesis.

In tumourigenesis, oncogenes or dysregulated tumour suppressor genes alter the canonical translation machinery leading to a reprogramming of the translatome that, in turn, promotes the translation of selected mRNAs encoding proteins involved in proliferation and metastasis. It is therefore unsurprising that abnormal expression levels and activities of eukaryotic initiation factors (eIFs), elongation factors (eEFs) or termination factors (eRFs) are associated with poor outcome for patients with a wide range of cancers. In this review we discuss how RNA binding proteins (RBPs) within the canonical translation factor machinery are dysregulated in cancers and how targeting such proteins is leading to new therapeutic avenues.

AGO1 regulates pericentromeric regions in mouse embryonic stem cells.

Argonaute proteins (AGOs), which play an essential role in cytosolic post-transcriptional gene silencing, have been also reported to function in nuclear processes like transcriptional activation or repression, alternative splicing and, chromatin organization. As most of these studies have been conducted in human cancer cell lines, the relevance of AGOs nuclear functions in the context of mouse early embryonic development remains uninvestigated. Here, we examined a possible role of the AGO1 protein on the distribution of constitutive heterochromatin in mouse embryonic stem cells (mESCs). We observed a specific redistribution of the repressive histone mark H3K9me3 and the heterochromatin protein HP1α, away from pericentromeric regions upon Ago1 depletion. Furthermore, we demonstrated that major satellite transcripts are strongly up-regulated in Ago1_KO mESCs and that their levels are partially restored upon AGO1 rescue. We also observed a similar redistribution of H3K9me3 and HP1α in Drosha_KO mESCs, suggesting a role for microRNAs (miRNAs) in the regulation of heterochromatin distribution in mESCs. Finally, we showed that specific miRNAs with complementarity to major satellites can partially regulate the expression of these transcripts.

Cyclin B/CDK1 and Cyclin A/CDK2 phosphorylate DENR to promote mitotic protein translation and faithful cell division.

DENR and MCTS1 have been identified as oncogenes in several different tumor entities. The heterodimeric DENR·MCTS1 protein complex promotes translation of mRNAs containing upstream Open Reading Frames (uORFs). We show here that DENR is phosphorylated on Serine 73 by Cyclin B/CDK1 and Cyclin A/CDK2 at the onset of mitosis, and then dephosphorylated as cells exit mitosis. Phosphorylation of Ser73 promotes mitotic stability of DENR protein and prevents its cleavage at Asp26. This leads to enhanced translation of mRNAs involved in mitosis. Indeed, we find that roughly 40% of all mRNAs with elevated translation in mitosis are DENR targets. In the absence of DENR or of Ser73 phosphorylation, cells display elevated levels of aberrant mitoses and cell death. This provides a mechanism how the cell cycle regulates translation of a subset of mitotically relevant mRNAs during mitosis.

Seeking the truth behind the myth: Argonaute tales from "nuclearland".

Argonaute proteins have been traditionally characterized as a highly evolutionary conserved family engaged in post-transcriptional gene silencing pathways. The Argonaute family is mainly grouped into the AGO and PIWI clades. The canonical role of Argonaute proteins relies on their ability to bind small-RNAs that recognize complementary sequences on target mRNAs to induce either mRNA degradation or translational repression. However, there is an increasing amount of evidence supporting that Argonaute proteins also exert multiple nuclear functions that subsequently regulate gene expression. In this line, genome-wide studies showed that members from the AGO clade regulate transcription, 3D chromatin organization, and splicing of active loci located within euchromatin. Here, we discuss recent work based on high-throughput technologies that have significantly contributed to shed light on the multivariate nuclear functions of AGO proteins in different model organisms. We also analyze data supporting that AGO proteins are able to execute these nuclear functions independently from small RNA pathways. Finally, we integrate these mechanistic insights with recent reports highlighting the clinical importance of AGO in breast and prostate cancer development.

HAX1 maintains the glioma progression in hypoxia through promoting mitochondrial fission.

HCLS1-associated protein X-1 (HAX1), an anti-apoptotic molecular, overexpresses in glioma. However, the role of HAX1 in glioma cell surviving in hypoxic environment remains unclear. Western blotting, qRT-PCR, Transwell assay, TUNEL assay, wounding healing assay, clone formation, tumour xenograft model and immunohistochemical staining were used to investigate the role of HAX1 in glioma. HAX1 regulated by HIF-1α was increased in glioma cells cultured in hypoxia. Silencing of HAX1 could cause an increased apoptosis of glioma cells cultured in hypoxia. Silencing of HAX1 also decreased the proliferation, migration and invasion of glioma cells cultured in hypoxia. Increased mitochondrial fission could prevent glioma cells from the damage induced by HAX1 knockdown in hypoxia. Furthermore, HAX1 was found to regulate glioma cells through phosphorylated AKT/Drp signal pathway. In conclusion, our study suggested that HAX1 promoted survival of glioma cells in hypoxic environment via AKT/Drp signal pathway. Our study also provided a potential therapeutic target for glioma.

RSK Inhibition Induces Apoptosis by Downregulating Protein Synthesis in a Variety of Acute Myeloid Leukemia Cell Lines.

Fms-like tyrosine kinase 3 (FLT3) and isocitrate dehydrogenase 1/2 (IDH1/2) mutations drive malignancy in acute myeloid leukemia (AML), which accounts for approximately 40% of AML cases. Treatment with FLT3 or IDH1/2 inhibitors is used for such patients; however, it is not considered for most patients with AML who lack mutations on the respective genes. In this study, p90 ribosomal S6 kinase (RSK) was found to serve as a new therapeutic target in various AMLs with or without FLT3 mutations. BI-D1870, a potent inhibitor of RSK, significantly suppressed the proliferation of AML cell lines, among which three encoded wild-type FLT3 and three contained FLT3 driver mutations, compared with chronic myeloid leukemia K562 cells or other adherent cancer cells. BI-D1870 inhibited protein synthesis by dephosphorylating the p70 S6 kinase and eukaryotic initiation factor 4E-binding protein 1 in all AML cells except KG-1a cells. Meanwhile, the expression of microtubule-associated protein light chain 3B-I and -II increased in KG-1a cells treated with BI-D1870. BI-D1870 induced caspase-dependent apoptosis in all AML cells, including KG-1a cells. We next investigated the synergistic effect of BI-D1870 with cytarabine, a traditional anticancer drug used in AML. Synergistic effects of BI-D1870 and cytarabine were not observed in any of the cell lines. The findings suggested that BI-D1870 alone exerts an adequate antiproliferative effect on AML with or without FLT3 mutations and serves as a novel AML therapeutic agent.

Translation Initiation Machinery as a Tumor Selective Target for Radiosensitization.

Towards improving the efficacy of radiotherapy, one approach is to target the molecules and processes mediating cellular radioresponse. Along these lines, translational control of gene expression has been established as a fundamental component of cellular radioresponse, which suggests that the molecules participating in this process (i.e., the translational machinery) can serve as determinants of radiosensitivity. Moreover, the proteins comprising the translational machinery are often overexpressed in tumor cells suggesting the potential for tumor specific radiosensitization. Studies to date have shown that inhibiting proteins involved in translation initiation, the rate-limiting step in translation, specifically the three members of the eIF4F cap binding complex eIF4E, eIF4G, and eIF4A as well as the cap binding regulatory kinases mTOR and Mnk1/2, results in the radiosensitization of tumor cells. Because ribosomes are required for translation initiation, inhibiting ribosome biogenesis also appears to be a strategy for radiosensitization. In general, the radiosensitization induced by targeting the translation initiation machinery involves inhibition of DNA repair, which appears to be the consequence of a reduced expression of proteins critical to radioresponse. The availability of clinically relevant inhibitors of this component of the translational machinery suggests opportunities to extend this approach to radiosensitization to patient care.

eIF5B regulates the expression of PD-L1 in prostate cancer cells by interacting with Wig1.

BACKGROUND: Eukaryotic translation initiation factors (eIFs) are the key factors to synthesize translation initiation complexes during the synthesis of eukaryotic proteins. Besides, eIFs are especially important in regulating the immune function of tumor cells. However, the effect mechanism of eIFs in prostate cancer remains to be studied, which is precisely the purpose of this study. METHODS: In this study, three groups of prostate cancer cells were investigated. One group had its eIF5B gene knocked down; another group had its Programmed death 1 (PD-L1) overexpressed; the final group had its Wild-type p53-induced gene 1 (Wig1) overexpressed. Genetic alterations of the cancer cells were performed by plasmid transfection. The expression of PD-L1 mRNA was detected by quantitative real-time PCR (qRT-PCR), and the expressions of PD-L1 and eIF5B proteins were observed by western blot assays. Cell Counting Kit-8 (CCK-8), flow cytometry, Transwell and Transwell martrigel were used to investigated cell proliferation, apoptosis, migration and invasion, respectively. The effect of peripheral blood mononuclear cells (PBMCs) on tumor cells was observed, and the interaction between eIF5B and Wig1 was revealed by co-immunoprecipitation (CoIP) assay. Finally, the effects of interference with eIF5B expression on the growth, morphology, and immunity of the tumor, as well as PD-L1 expression in the tumor, were verified by tumor xenograft assays in vivo. RESULTS: Compared with normal prostate epithelial cells, prostate cancer cells revealed higher expressions of eIF5B and PD-L1 interference with eIF-5B expression can inhibit the proliferation, migration, invasion and PD-L1 expression of prostate cancer cells. Meanwhile, the cancer cell group with interference with eIF5B expression also demonstrated greater, apoptosis and higher vulnerability to PBMCs. CoIP assays showed that Wig1 could bind to eIF5B in prostate cancer cells, and its overexpression can inhibit the proliferation, migration, invasion and PD-L1 expression of cancer cells while promoting apoptosis. Moreover, interference with eIF5B expression can inhibit tumor growth, destroy tumor morphology, and suppress the proliferation of tumor cells. CONCLUSION: eIF5B can promote the expression of PD-L1 by interacting with Wig1. Besides, interference with eIF5B expression can inhibit the proliferation, migration, invasion and immunosuppressive response of prostate cancer cells. This study proposes a new target, eIF5B, for immunotherapy of prostate cancer.

eIF4A, a target of siRNA derived from rice stripe virus, negatively regulates antiviral autophagy by interacting with ATG5 in Nicotiana benthamiana.

Autophagy is induced by viral infection and has antiviral functions in plants, but the underlying mechanism is poorly understood. We previously identified a viral small interfering RNA (vsiRNA) derived from rice stripe virus (RSV) RNA4 that contributes to the leaf-twisting and stunting symptoms caused by this virus by targeting the host eukaryotic translation initiation factor 4A (eIF4A) mRNA for silencing. In addition, autophagy plays antiviral roles by degrading RSV p3 protein, a suppressor of RNA silencing. Here, we demonstrate that eIF4A acts as a negative regulator of autophagy in Nicotiana benthamiana. Silencing of NbeIF4A activated autophagy and inhibited RSV infection by facilitating autophagic degradation of p3. Further analysis showed that NbeIF4A interacts with NbATG5 and interferes with its interaction with ATG12. Overexpression of NbeIF4A suppressed NbATG5-activated autophagy. Moreover, expression of vsiRNA-4A, which targets NbeIF4A mRNA for cleavage, induced autophagy by silencing NbeIF4A. Finally, we demonstrate that eIF4A from rice, the natural host of RSV, also interacts with OsATG5 and suppresses OsATG5-activated autophagy, pointing to the conserved function of eIF4A as a negative regulator of antiviral autophagy. Taken together, these results reveal that eIF4A negatively regulates antiviral autophagy by interacting with ATG5 and that its mRNA is recognized by a virus-derived siRNA, resulting in its silencing, which induces autophagy against viral infection.