bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 Norovirus replication in human intestinal epithelial cells is restricted by the 2 interferon-induced JAK/STAT signalling pathway and RNA Polymerase II 3 mediated transcriptional responses. 4 5 Myra Hosmilloa#, Yasmin Chaudhrya, Komal Nayakb , Frederic Sorgeloosa, Bon- 6 Kyoung Kooc,d, Alessandra Merendac*, Reidun Lillestole, Lydia Drumrighte, 7 Matthias Zilbauerb and Ian Goodfellowa# 8 9 a Division of Virology, Department of Pathology, University of Cambridge, 10 Cambridge, UK 11 b Department of Paediatrics, University of Cambridge, Cambridge, UK 12 c Wellcome Trust-Medical Research Council Stem Cell Institute, University of 13 Cambridge, Cambridge, UK 14 d Institute of Molecular Biotechnology of the Austrian Academy of Sciences 15 (IMBA), Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria. 16 e Department of Medicine, Addenbrooke’s Hospital, University of Cambridge, 17 Cambridge, UK 18 19 Running title: Restriction of HuNoV infection in intestinal mucosa 20 21 #Address correspondence to Ian Goodfellow, [email protected] and Myra 22 Hosmillo, [email protected] 23 *Present address: bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 24 Alessandra Merenda, Oncode Institute and Department of Cell Biology, Centre 25 for Molecular Medicine, University Medical Centre Utrecht, 3584 CX Utrecht, the 26 Netherlands 27 28 Abstract: 217 words 29 Text: 7,105 words 30 bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 31 Abstract 32 Human noroviruses (HuNoV) are a leading cause of viral gastroenteritis 33 worldwide and a significant cause of morbidity and mortality in all age groups. 34 The recent finding that HuNoV can be propagated in B cells and mucosa derived 35 intestinal epithelial organoids (IEOs), has transformed our capability to dissect 36 the life cycle of noroviruses. Using RNA-Seq of HuNoV infected intestinal 37 epithelial cells (IECs), we have found that replication of HuNoV in IECs results in 38 interferon-induced transcriptional responses and that HuNoV replication in IECs 39 is sensitive to IFN. This contrasts with previous studies that suggest that the 40 innate immune response may play no role in the restriction of HuNoV replication 41 in immortalised cells. We demonstrate that the inhibition of JAK1/JAK2 42 enhances HuNoV replication in IECs. Surprisingly, targeted inhibition of cellular 43 RNA polymerase II-mediated transcription was not detrimental to HuNoV 44 replication, but enhanced replication to a greater degree compared to blocking 45 of JAK signalling directly. Furthermore, we demonstrate for the first time that 46 IECs generated from genetically modified intestinal organoids, engineered to 47 be deficient in the interferon response, are more permissive to HuNoV 48 infection. Together our work identifies the IFN-induced transcriptional 49 responses restrict HuNoV replication in IECs and demonstrates that the 50 inhibition of these responses by modifications to the culture conditions can 51 greatly enhance the robustness of the norovirus culture system. 52 53 bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 54 Importance 55 Noroviruses are a major cause of gastroenteritis worldwide yet the challenges 56 associated with their growth culture has greatly hampered the development of 57 therapeutic approaches and has limited our understanding of cellular pathways 58 that control infection. Here we show that human intestinal epithelial cells, the 59 first point of entry of human noroviruses into the host, limit virus replication by 60 the induction of the innate responses. Furthermore we show that modulating 61 the ability of intestinal epithelial cells to induce transcriptional responses to 62 HuNoV infection can significantly enhance human norovirus replication in 63 culture. Collectively our findings provide new insights into the biological 64 pathways that control norovirus infection but also identify mechanisms to 65 enhance the robustness of norovirus culture. 66 67 bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 68 Introduction 69 The induction of the host innate response plays an essential role in the 70 suppression of pathogen infection. The synthesis of interferons (IFN) and the 71 subsequent signalling cascades that leads to the induction of IFN-stimulated 72 genes (ISGs), determine the outcome of viral infection (1, 2). An understanding of 73 the mechanisms underlying the interplay between pathogens and innate immune 74 responses is vital to understanding viral pathogenesis and can greatly aid the 75 identification of potential therapeutic and/or preventive strategies. 76 77 Human noroviruses (HuNoV) are widely recognised as the leading cause of viral 78 gastroenteritis worldwide (3). Noroviruses are classified into at least seven 79 genogroups based on the sequence of the major capsid protein VP1 and regions 80 within ORF1 (3–5). HuNoVs belong to one of three norovirus genogroups (GI, 81 GII, or GIV), which are further divided into >25 genetic clusters or genotypes (6– 82 8). Epidemiological studies reveal that over 75% of confirmed human norovirus 83 infections are associated with HuNoV GII (9, 10). Whilst norovirus gastroenteritis 84 typically results in an acute and self-limiting disease, the socioeconomic impact in 85 both developed and developing countries is estimated to be more than $60.3 86 billion per annum (11). HuNoV infection is particularly severe and prolonged in 87 immunocompromised patients, including young children, elderly, or patients 88 receiving treatment for cancer. In these cases infections can last from months to 89 years (12, 13). 90 bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 91 Our understanding of the molecular mechanisms that control HuNoV infection 92 has been limited by the lack of robust culture systems that facilitate the detailed 93 analysis of the viral life cycle. As a result, murine norovirus (MNV) and other 94 members of the Caliciviridae family of positive sense RNA viruses, such as feline 95 calicivirus (FCV) and porcine sapovirus (PSaV), are often used as surrogate 96 models (14–17). MNV, FCV and PSaV can all be efficiently cultured in 97 immortalised cells and are amenable to reverse genetics (16–20). These model 98 systems have been critical to understanding many aspects of the life cycle of 99 members of the Caliciviridae (15). 100 101 Recent efforts have led to the establishment of two HuNoV culture systems 102 based on immortalised B cells (21, 22) and intestinal epithelial cells (IECs) 103 generated from biopsy-derived human intestinal epithelial organoids (IEOs) (23). 104 Whilst authentic replication of HuNoV can be observed in both the B-cell and 105 IEC-based culture systems, repeated long-term passage of HuNoV and the 106 generation of high titre viral stocks is not possible, suggesting that replication is 107 restricted in some manner. In the current study we sought to better understand 108 the cellular response to HuNoV infection and to identify pathways that restrict 109 HuNoV replication in organoid-derived IECs. Using RNA-Seq we observed that 110 HuNoV infection of IECs results in an interferon-mediated antiviral transcriptional 111 response. We show for the first time that HuNoV replication in IECs is sensitive to 112 both Type I and III interferon and that HuNoV replication is restricted by virus- 113 induced innate response. Pharmacological inhibition of the interferon response 114 or genetic modification of organoids to prevent the activation of the interferon bioRxiv preprint doi: https://doi.org/10.1101/731802; this version posted August 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 115 response significantly improved HuNoV replication in IECs. Furthermore, we 116 show that ongoing HuNoV replication is enhanced by the inhibition of RNA Pol II 117 mediated transcription. Overall this work provides new insights into the cellular 118 responses to HuNoV infection of the gut epithelium and identifies modifications to 119 the HuNoV culture system that significantly enhances its utility. 120 121 122 Materials and Methods. 123 Stool samples. Stool specimens were anonymized with written consent from 124 patients at Addenbrooke’s Hospital, Cambridge, who tested positive of HuNoV 125 infection.