Activation of the mechanistic/mammalian target of rapamycin (mTOR) kinase in models of acute and chronic pain is strongly implicated in mediating enhanced translation and hyperalgesia. However, the molecular mechanisms by which mTOR regulates nociception remain unclear. Here we show that deletion of the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1), a major mTOR downstream effector, which represses eIF4E activity and cap-dependent translation, leads to mechanical, but not thermal pain hypersensitivity. Mice lacking 4E-BP1 exhibit enhanced spinal cord expression of neuroligin 1, a cell-adhesion postsynaptic protein regulating excitatory synapse function, and show increased excitatory synaptic input into spinal neurons, and a lowered threshold for induction of synaptic potentiation. Pharmacological inhibition of eIF4E or genetic reduction of neuroligin 1 levels normalizes the increased excitatory synaptic activity and reverses mechanical hypersensitivity. Thus, translational control by 4E-BP1 downstream of mTOR effects the expression of neuroligin 1 and excitatory synaptic transmission in the spinal cord, and thereby contributes to enhanced mechanical nociception.

The integrated stress response (ISR) is activated in response to diverse stress stimuli to maintain homeostasis in neurons. Central to this process is the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2α). Here, we report a critical role for ISR in regulating the mammalian circadian clock. The eIF2α kinase GCN2 rhythmically phosphorylates eIF2α in the suprachiasmatic circadian clock. Increased eIF2α phosphorylation shortens the circadian period in both fibroblasts and mice, whereas reduced eIF2α phosphorylation lengthens the circadian period and impairs circadian rhythmicity in animals. Mechanistically, phosphorylation of eIF2α promotes mRNA translation of Atf4. ATF4 binding motifs are identified in multiple clock genes, including Per2, Per3, Cry1, Cry2, and Clock. ATF4 binds to the TTGCAGCA motif in the Per2 promoter and activates its transcription. Together, these results demonstrate a significant role for ISR in circadian physiology and provide a potential link between dysregulated ISR and circadian dysfunction in brain diseases.

MicroRNAs (miRNAs) have emerged as critical regulators of cellular metabolism. To characterise miRNAs crucial to the maintenance of hepatic lipid homeostasis, we examined the overlap between the miRNA signature associated with inhibition of peroxisome proliferator activated receptor-α (PPAR-α) signaling, a pathway regulating fatty acid metabolism, and the miRNA profile associated with 25-hydroxycholesterol treatment, an oxysterol regulator of sterol regulatory element binding protein (SREBP) and liver X receptor (LXR) signaling. Using this strategy, we identified microRNA-7 (miR-7) as a PPAR-α regulated miRNA, which activates SREBP signaling and promotes hepatocellular lipid accumulation. This is mediated, in part, by suppression of the negative regulator of SREBP signaling: ERLIN2. miR-7 also regulates genes associated with PPAR signaling and sterol metabolism, including liver X receptor β (LXR-β), a transcriptional regulator of sterol synthesis, efflux, and excretion. Collectively, our findings highlight miR-7 as a novel mediator of cross-talk between PPAR, SREBP, and LXR signaling pathways in the liver.

MicroRNAs (miRNAs) are part of a complex regulatory network that modulates cellular lipid metabolism. Here, we identify miR-124 as a regulator of triglyceride (TG) metabolism. This study advances our knowledge of the role of miR-124 in human hepatoma cells. Transcriptional profiling of Huh7.5 cells overexpressing miR-124 reveals enrichment for host factors involved in fatty acid oxidation among repressed miRNA targets. In addition, miR-124 down-regulates arylacetamide deacetylase (AADAC) and adipose triglyceride lipase, lipases proposed to mediate breakdown of hepatic TG stores for lipoprotein assembly and mitochondrial β-oxidation. Consistent with the inhibition of TG and fatty acid catabolism, miR-124 expression promotes cellular TG accumulation. Interestingly, miR-124 inhibits the production of hepatitis C virus, a virus that hijacks lipid pathways during its life cycle. Antiviral activity of miR-124 is consistent with repression of AADAC, a pro-viral host factor. Overall, our data highlight miR-124 as a novel regulator of TG metabolism in human hepatoma cells.

The protozoan parasite Leishmania donovani (L. donovani) causes visceral leishmaniasis, a chronic infection which is fatal when untreated. Herein, we investigated whether in addition to altering transcription, L. donovani modulates host mRNA translation to establish a successful infection. Polysome-profiling revealed that one third of protein-coding mRNAs expressed in primary mouse macrophages are differentially translated upon infection with L. donovani promastigotes or amastigotes. Gene ontology analysis identified key biological processes enriched for translationally regulated mRNAs and were predicted to be either activated (e.g. chromatin remodeling and RNA metabolism) or inhibited (e.g. intracellular trafficking and antigen presentation) upon infection. Mechanistic in silico and biochemical analyses showed selective activation mTOR- and eIF4A-dependent mRNA translation, including transcripts encoding central regulators of mRNA turnover and inflammation (i.e. PABPC1, EIF2AK2, and TGF-β). L. donovani survival within macrophages was favored under mTOR inhibition but was dampened by pharmacological blockade of eIF4A. Overall, this study uncovers a vast yet selective reprogramming of the host cell translational landscape early during L. donovani infection, and suggests that some of these changes are involved in host defense mechanisms while others are part of parasite-driven survival strategies. Further in vitro and in vivo investigation will shed light on the contribution of mTOR- and eIF4A-dependent translational programs to the outcome of visceral leishmaniasis.

The biological impact of ionizing radiation (IR) on humans depends not only on the physical properties and absorbed dose of radiation but also on the unique susceptibility of the exposed individual. A critical target of IR is DNA, and the DNA damage response is a safeguard mechanism for maintaining genomic integrity in response to the induced cellular stress. Unrepaired DNA lesions lead to various mutations, contributing to adverse health effects. Cellular sensitivity to IR is highly correlated with the ability of cells to repair DNA lesions, in particular coding sequences of genes that affect that process and of others that contribute to preserving genomic integrity. However, accurate profiling of the molecular events underlying individual sensitivity requires techniques with sensitive readouts. Here we summarize recent studies that have used whole-genome analysis and identified genes that impact individual radiosensitivity. Whereas microarray and RNA-seq provide a snapshot of the transcriptome, RNA interference (RNAi) and CRISPR-Cas9 techniques are powerful tools that enable modulation of gene expression and characterizing the function of specific genes involved in radiosensitivity or radioresistance. Notably, CRISPR-Cas9 has altered the landscape of genome-editing technology with its increased readiness, precision, and sensitivity. Identifying critical regulators of cellular radiosensitivity would help tailor regimens that enhance the efficacy of therapeutic treatments and fast-track prediction of clinical outcomes. It would also contribute to occupational protection based on average individual sensitivity, as well as the formulation of countermeasures to the harmful effects of radiation.

We established a split nanoluciferase complementation assay to rapidly screen for inhibitors that interfere with binding of the receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein with its target receptor, angiotensin-converting enzyme 2 (ACE2). After a screen of 1,200 US Food and Drug Administration (FDA)-approved compounds, we identified bifonazole, an imidazole-based antifungal agent, as a competitive inhibitor of RBD-ACE2 binding. Mechanistically, bifonazole binds ACE2 around residue K353, which prevents association with the RBD, affecting entry and replication of spike-pseudotyped viruses as well as native SARS-CoV-2 and its variants of concern (VOCs). Intranasal administration of bifonazole reduces lethality in K18-hACE2 mice challenged with vesicular stomatitis virus (VSV)-spike by 40%, with a similar benefit after live SARS-CoV-2 challenge. Our screen identified an antiviral agent that is effective against SARS-CoV-2 and VOCs such as Omicron that employ the same receptor to infect cells and therefore has high potential to be repurposed to control, treat, or prevent coronavirus disease 2019 (COVID-19).

Macrophages undergo swift changes in mRNA abundance upon pathogen invasion. Herein we describe early remodelling of the macrophage transcriptome during infection by amastigotes or promastigotes of Leishmania donovani. Approximately 10-16% of host mRNAs were differentially modulated in L. donovani-infected macrophages when compared to uninfected controls. This response was partially stage-specific as a third of changes in mRNA abundance were either exclusively driven by one of the parasite forms or significantly different between them. Gene ontology analyses identified categories associated with immune functions (e.g. antigen presentation and leukocyte activation) among significantly downregulated mRNAs during amastigote infection while cytoprotective-related categories (e.g. DNA repair and apoptosis inhibition) were enriched in upregulated transcripts. Interestingly a combination of upregulated (e.g. cellular response to IFNβ) and repressed (e.g. leukocyte activation, chemotaxis) immune-related transcripts were overrepresented in the promastigote-infected dataset. In addition, Ingenuity Pathway Analysis (IPA) associated specific mRNA subsets with a number of upstream transcriptional regulators predicted to be modulated in macrophages infected with L. donovani amastigotes (e.g. STAT1 inhibition) or promastigotes (e.g. NRF2, IRF3, and IRF7 activation). Overall, our results indicate that early parasite stage-driven transcriptional remodelling in macrophages contributes to orchestrate both protective and deleterious host cell responses during L. donovani infection.

Transgenes deliver therapeutic payloads to improve oncolytic virus immunotherapy. Transgenes encoded within oncolytic viruses are designed to be highly transcribed, but protein synthesis is often negatively affected by viral infection, compromising the amount of therapeutic protein expressed. Studying the oncolytic herpes simplex virus-1 (HSV1), we found standard transgene mRNAs to be suboptimally translated in infected cells.

MicroRNA-122 positively modulates hepatitis C virus (HCV) through direct interactions with viral RNA. Three microRNA-122 recognition elements (MREs) have been previously identified: two in the 5'UTR and one in the 3'UTR. Herein, we report the relative affinity of microRNA-122 to these sites using viral RNA-coated magnetic beads, with mutagenesis and probes to disrupt interactions of microRNA-122 at specific sites. We demonstrate cooperativity in binding between the closely spaced MREs within the 5'UTR in vitro. We also identified a well conserved fourth site in the coding region and showed that it is the highest affinity MRE site. Site-directed mutagenesis of the MREs in HCV subgenomic replicons expressed in Huh-7.5 cells demonstrated competing roles of the stimulatory MREs in the 5'UTR with the inhibitory role of an MRE in the open reading frame (ORF). These data have important implications in elucidating the mechanism of interaction between microRNA-122 and HCV RNA.

To identify therapeutic opportunities for oncolytic viral therapy, we conducted genome-wide RNAi screens to search for host factors that modulate rhabdoviral oncolysis. Our screens uncovered the endoplasmic reticulum (ER) stress response pathways as important modulators of rhabdovirus-mediated cytotoxicity. Further investigation revealed an unconventional mechanism whereby ER stress response inhibition preconditioned cancer cells, which sensitized them to caspase-2-dependent apoptosis induced by a subsequent rhabdovirus infection. Importantly, this mechanism was tumor cell specific, selectively increasing potency of the oncolytic virus by up to 10,000-fold. In vivo studies using a small molecule inhibitor of IRE1α showed dramatically improved oncolytic efficacy in resistant tumor models. Our study demonstrates proof of concept for using functional genomics to improve biotherapeutic agents for cancer.

We report that combining a DNA analog (2'F-ANA) with rigid RNA analogs [2'F-RNA and/or locked nucleic acid (LNA)] in siRNA duplexes can produce gene silencing agents with enhanced potency. The favored conformations of these two analogs are different, and combining them in a 1-1 pattern led to reduced affinity, whereas alternating short continuous regions of individual modifications increased affinity relative to an RNA:RNA duplex. Thus, the binding affinity at key regions of the siRNA duplex could be tuned by changing the pattern of incorporation of DNA-like and RNA-like nucleotides. These heavily or fully modified duplexes are active against a range of mRNA targets. Effective patterns of modification were chosen based on screens using two sequences targeting firefly luciferase. We then applied the most effective duplex designs to the knockdown of the eIF4E binding proteins 4E-BP1 and 4E-BP2. We identified modified duplexes with potency comparable to native siRNA. Modified duplexes showed dramatically enhanced stability to serum nucleases, and were characterized by circular dichroism and thermal denaturation studies. Chemical modification significantly reduced the immunostimulatory properties of these siRNAs in human peripheral blood mononuclear cells.

Rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, is an aggressive cancer with a poor prognosis. Despite current management, the 5-year survival rate for patients with metastatic RMS is ∼30%; underscoring the need to develop better treatment strategies. We have recently reported that pannexin 1 (PANX1) levels are downregulated in RMS and that restoring its expression inhibits RMS progression. Here, we have surveyed and characterized the molecular changes induced by PANX1 re-expression in RMS. We cataloged transcriptomic changes in this context by RNA sequencing. At the protein level, we unveiled PANX1 interactors using BioID, complemented by co-immunoprecipitation coupled to high-performance liquid chromatography/electrospray ionization tandem mass spectrometry performed in PANX1-enriched fractions. Using these data, we generated searchable public databases for the PANX1 interactome and changes to the RMS transcriptome occurring when PANX1 expression is restored. STRING network analyses revealed a PANX1 interactome involving plasma membrane and cytoskeleton-associated proteins including the previously undescribed interactor AHNAK. Indeed, AHNAK knockdown abrogated the PANX1-mediated reduction in RMS cell viability and migration. Using these unbiased approaches, we bring insight to the mechanisms by which PANX1 inhibits RMS progression, identifying the cell migration protein AHNAK as a key modifier of PANX1-mediated changes in RMS malignant properties.

Type I interferons (IFNs) are critical cytokines in the host defense against invading pathogens. Sustained production of IFNs, however, is detrimental to the host, as it provokes autoimmune diseases. Thus, the expression of IFNs is tightly controlled. We report that the mRNA 5' cap-binding protein 4EHP plays a key role in regulating type I IFN concomitant with controlling virus replication, both in vitro and in vivo. Mechanistically, 4EHP suppresses IFN-β production by effecting the miR-34a-induced translational silencing of Ifnb1 mRNA. miR-34a is upregulated by both RNA virus infection and IFN-β induction, prompting a negative feedback regulatory mechanism that represses IFN-β expression via 4EHP. These findings demonstrate the direct involvement of 4EHP in virus-induced host response, underscoring a critical translational silencing mechanism mediated by 4EHP and miR-34a to impede sustained IFN production. This study highlights an intrinsic regulatory function for miRNA and the translation machinery in maintaining host homeostasis.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

The coronavirus disease 2019 (COVID-19) pandemic requires the continued development of safe, long-lasting, and efficacious vaccines for preventive responses to major outbreaks around the world, and especially in isolated and developing countries. To combat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), we characterize a temperature-stable vaccine candidate (TOH-Vac1) that uses a replication-competent, attenuated vaccinia virus as a vector to express a membrane-tethered spike receptor binding domain (RBD) antigen. We evaluate the effects of dose escalation and administration routes on vaccine safety, efficacy, and immunogenicity in animal models. Our vaccine induces high levels of SARS-CoV-2 neutralizing antibodies and favorable T cell responses, while maintaining an optimal safety profile in mice and cynomolgus macaques. We demonstrate robust immune responses and protective immunity against SARS-CoV-2 variants after only a single dose. Together, these findings support further development of our novel and versatile vaccine platform as an alternative or complementary approach to current vaccines.

Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.

LARP1 is a key repressor of TOP mRNA translation. It binds the m7Gppp cap moiety and the adjacent 5'TOP motif of TOP mRNAs, thus impeding the assembly of the eIF4F complex on these transcripts. mTORC1 controls TOP mRNA translation via LARP1, but the details of the mechanism are unclear. Herein we elucidate the mechanism by which mTORC1 controls LARP1's translation repression activity. We demonstrate that mTORC1 phosphorylates LARP1 in vitro and in vivo, activities that are efficiently inhibited by rapamycin and torin1. We uncover 26 rapamycin-sensitive phospho-serine and -threonine residues on LARP1 that are distributed in 7 clusters. Our data show that phosphorylation of a cluster of residues located proximally to the m7Gppp cap-binding DM15 region is particularly sensitive to rapamycin and regulates both the RNA-binding and the translation inhibitory activities of LARP1. Our results unravel a new model of translation control in which the La module (LaMod) and DM15 region of LARP1, both of which can directly interact with TOP mRNA, are differentially regulated: the LaMod remains constitutively bound to PABP (irrespective of the activation status of mTORC1), while the C-terminal DM15 'pendular hook' engages the TOP mRNA 5'-end to repress translation, but only in conditions of mTORC1 inhibition.

Viruses evade the innate immune response by suppressing the production or activity of cytokines such as type I interferons (IFNs). Here we report the discovery of a mechanism by which the SARS-CoV-2 virus coopts an intrinsic cellular machinery to suppress the production of the key immunostimulatory cytokine IFN-β. We reveal that the SARS-CoV-2 encoded nonstructural protein 2 (NSP2) directly interacts with the cellular GIGYF2 protein. This interaction enhances the binding of GIGYF2 to the mRNA cap-binding protein 4EHP, thereby repressing the translation of the Ifnb1 mRNA. Depletion of GIGYF2 or 4EHP significantly enhances IFN-β production, which inhibits SARS-CoV-2 replication. Our findings reveal a target for rescuing the antiviral innate immune response to SARS-CoV-2 and other RNA viruses.

Myxoma virus (MyxV) is a rabbit-specific poxvirus. However, its ability to selectively target tumor cells has established it as a safe and effective anticancer therapy. To strengthen its preclinical efficacy, transgenes that can prolong cancer cell infection and enhance anti-tumor effector functions are currently being investigated. We engineered MyxV armed with CD47, to turn on a 'do not eat me' signal within infected cells with actively replicating viruses, and with IFN-γ to further activate host immune anticancer responses. Tumor suppressive activities were significantly enhanced by the dual-armed MyxV_CD47/IFN-γ compared to parental MyxV or single-armed MyxV_CD47 or MyxV_IFN-γ. In addition, significant increases in IFN-γ+ CD8+T-cells and CD4+ T-cells populations within tumor-infiltrating lymphocytes (TIL) were observed after MyxV_CD47/IFN-γ treatment. Notably, all groups treated with MyxV showed a marked reduction in Foxp3+ CD4+ regulatory T-cells (Tregs) within TIL. We also show that MyxV infection induces PD-L1 up-regulation in cancer cells, and combinational treatment of MyxV with anti-mouse PD-L1 antibodies (αPD-L1) further controlled tumor burden and increased survival in the syngeneic melanoma model B16F10. Our data demonstrate that a CD47 and IFNγ dual-armed MyxV is an effective oncolytic viral immunotherapeutic. These findings strongly support further preclinical investigations to develop next-generation MyxV-based immunotherapy approaches.