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Downloaded October, 2015) 83 bioRxiv preprint doi: https://doi.org/10.1101/522607; this version posted January 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Induction of an alternative 5’ leader enhances translation of Inpp5e and resistance to oncolytic virus infection Huy-Dung Hoang1,Δ, Tyson E. Graber1,2,Δ, Jian-Jun Jia1, Nasana Vaidya1, Victoria Gilchrist1, Wencheng Li3, Christos G. Gkogkas4, Maritza Jaramillo5, Seyed Mehdi Jafarnejad6, and Tommy Alain1* 1 Children's Hospital of Eastern Ontario Research Institute, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, Ontario, Canada 2 Department of Biochemistry and Goodman Cancer Center, McGill University, Montreal, Quebec, Canada 3 Department of Biochemistry and Molecular Biology, Rutgers New Jersey Medical School, Newark, NJ, USA 4 Centre for Discovery Brain Sciences, University of Edinburgh, Edinburgh, UK. 5 INRS Institut Armand-Frappier Research Centre, Laval, Quebec, Canada 6 Centre for Cancer Research and Cell Biology, The Queen’s University of Belfast, Belfast, UK *correspondence: Tommy Alain Children’s Hospital of Eastern Ontario Research Institute 401 Smyth Road Ottawa, Ontario, Canada Δ These authors contributed equally to this work. Short Title: Oncolytic viruses trigger an innate RNA translation response ABSTRACT variant that lacks repressive upstream open reading frames (uORFs) within its 5’ leader and is consequently Residual cell-intrinsic innate immunity in cancer cells efficiently translated. Furthermore, we show that INPP5E hampers infection with oncolytic viruses. mRNA contributes to antiviral immunity by altering virus translation is an important component of innate attachment. These findings uncover a role for immunity, yet the targeted cellular mRNAs remain ill- translational control through alternative 5’ leader defined. We characterized the translatome of resistant expression and assign ciliary proteins such as INPP5E to murine “4T1” breast cancer cells infected with three of the cellular antiviral response. the most clinically advanced oncolytic viruses: Herpes Simplex virus 1, Reovirus and Vaccinia virus. Common among all three infections were translationally de- KEYWORDS (3-10) repressed mRNAs involved in ciliary homeostasis Oncolytic virus, ribosome profiling, translation, uORF, including Inpp5e, encoding an inositol 5-phosphatase that INPP5E, BBS9, RNA variant, isoform switch, alternative modifies lipid second messenger signalling. splicing, breast cancer. Translationally repressed in the uninfected condition, viral infection induced expression of an Inpp5e mRNA 1 bioRxiv preprint doi: https://doi.org/10.1101/522607; this version posted January 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. INTRODUCTION alleviate viral resistance, an undesirable feature of tumour Mammalian cells possess a sophisticated cell- cells in the context of oncolytic virus therapy, which intrinsic innate antiviral program that is activated upon represents a promising class of cancer therapeutics that infection. Transcriptional induction of type I interferon relies on natural or engineered cancer cell tropism and expression (IFN-α and β) and downstream interferon mobilization of adaptive, anti-tumour immunity 18,19. stimulated genes (ISGs) is a major, well-characterized arm of the innate immune response to infection 1,2. In this work, we queried which specific Another essential but less characterized feature of this substrates of translation contribute to the viral resistance response is the mRNA translation arm of innate immunity of 4T1 breast cancer cells with each of three leading – a reprogramming of protein synthesis to permit oncolytic viruses: Herpes Simplex Virus 1-1716 from expression of cellular antiviral proteins while Sorrento Therapeutics (HSV1), Reovirus Type 3 Dearing concurrently thwarting production of viral proteins 3,4. - “Reolysin” from Oncoytics Biotech (Reovirus), and Vaccinia Virus “JX-594” from Sillajen (formerly Translation initiation can be modulated by Jennerex Biotherapeutics) (VACV). Comparing viral several eukaryotic initiation factors (eIF) and RNA versus mock infected, we identified translationally binding proteins (RBP) 5,6. In addition to the m7G cap upregulated host mRNAs common to all three infections. structure which helps recruit eIFs, other cis-acting We show that these mRNAs, whose 5’ leader are enriched sequence elements that lie within 5´ leaders, such as 5´ in uORFs, are translationally repressed in mock condition terminal oligopyrimidine (TOP) motifs, upstream open and become de-repressed upon infection. Interestingly, reading frames (uORF), internal ribosome entry sites this subset includes mRNAs encoding proteins associated (IRES), RBP binding sites or localized secondary with primary cilium homeostasis. We characterized the structure can govern the translational efficiency (TE) of important ciliopathy gene Inpp5e, encoding an inositol 5- mRNAs 6,7. During infection, signalling cascades that phosphatase, and describe a virus-induced RNA variant feed into mRNA translation such as switch that releases its uORF-mediated translation PI3K/mTORC1/(S6K or 4E-BP) and ERK/MNK/eIF4E repression. This response limits viral propagation as cells were shown to enhance translation of antiviral mRNAs deficient in INPP5E exhibit increased cell surface including IRF7 and ISG158–10. Conversely, translation attachment of virions and subsequent infection efficiency. initiation can be transiently suppressed following Together, these findings highlight the dynamic landscape infection, by preventing efficient ribosome assembly of alternative 5’ leader usage during viral infection and through activation of eIF2α kinases and subsequent identify INPP5E as a translationally induced antiviral phosphorylation of the alpha-subunit of eIF2 (P-eIF2α) 3. effector that limits oncolytic virus efficacy. Paradoxically, some cellular mRNAs which have uORFs (e.g. ATF4 11) and/or IRES (e.g. cIAP1/BIRC2 12) in their RESULTS 5´ leaders display enhanced TE in conditions of high Distinct transcriptional and translational host responses phospho-eIF2α. Alternative mRNA transcription, splicing and polyadenylation can also indirectly modify to oncolytic viruses. translational output by altering 5´ leaders and 3’ To assess for translationally regulated innate immune genes, we used HSV1-1716 (HSV1), Reolysin untranslated regions (UTR), thus changing the composition of sequence elements that affect TE 13–15. (Reovirus), and JX-594 (VACV) individually to infect 4T1 cells, a murine mammary carcinoma model that is refractory to viral oncolysis and closely resembles stage Viruses use a plethora of methods to maximize 20,21 TE of their own mRNAs, from evolving 5´ leaders that IV human breast cancer . Each of these viruses has a are better substrates for translation due to the presence of different rate of infection that eventually results in the shutdown of host cell translation 22,23. We therefore IRES, to deploying proteins that shutdown global cellular translation (host-shutoff)4. Surveying which cellular and selected an effective dose that is cytopathic for 50% viral mRNAs are translated in this highly dynamic (ED50) of 4T1 cells at 48 hours post-infection (Fig. S1A). At 18 hours post-infection, polysome profiles (Fig. 1A) environment has been the subject of several recent studies 35 16,17. However, these investigations did not specifically and S-methionine labelling (Fig. 1B) showed robust address how translation of mRNAs encoding putative viral protein synthesis while that of the host cells was only slightly affected. The majority of cells were infected pro- or antiviral effectors might modulate infection. Furthermore, identification of host mRNAs under at this dose and timepoint, confirmed by co-expression of translation control during infection could provide a virion-derived GFP transgene in the case of HSV1 and targetable strategies to improve antiviral therapies or VACV (Fig. S1B). 2 bioRxiv preprint doi: https://doi.org/10.1101/522607; this version posted January 17, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Figure 1. Generating the transcriptome and translatome profiles of breast cancer cells undergoing viral infection. (A) Polysome profiling of virus- vs. mock-infected 4T1 cells at 18 hours post-infection, or (B) metabolic labelling with 35S-methionine followed by SDS-PAGE to resolve labelled, nascent peptides, showed efficient viral translation and no global changes in cellular translation. (C) Schematic illustration of the ribosome profiling strategy used in this study. (D) Differential expression of sequenced genes (total number shown in top right of plots) at both the transcriptional and translational genome levels. The average RNA expression or translation efficiency from two biological replicates is shown. Genes that were up- or downregulated more than 1.5-fold were considered to be differentially expressed. (E) Correlation analysis of RNA (transcriptome) and RPF (translatome) normalized abundance between viruses (all relative to mock-infected). Pearson’s correlation coefficients are presented. We profiled both the transcriptome and translatome mRNA species was quantitated 25. In contrast
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