key: cord-0745815-qfpgu02l authors: Wing, Peter A.C.; Prange-Barczynska, Maria; Cross, Amy; Crotta, Stefania; Orbegozo Rubio, Claudia; Cheng, Xiaotong; Harris, James M.; Zhuang, Xiaodong; Johnson, Rachel L.; Ryan, Kathryn A.; Hall, Yper; Carroll, Miles W.; Issa, Fadi; Balfe, Peter; Wack, Andreas; Bishop, Tammie; Salguero, Francisco J.; McKeating, Jane A. title: Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model date: 2022-03-15 journal: bioRxiv DOI: 10.1101/2022.03.15.484379 sha: 2136515cf391468ac7f7c01ba374410fd7107e4f doc_id: 745815 cord_uid: qfpgu02l Understanding the host pathways that define susceptibility to SARS-CoV-2 infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in Syrian hamsters. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced the levels of infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities. We have reported that hypoxic activation of HIF-1 inhibits SARS-CoV-2 entry and replication in 81 primary and immortalised lung epithelial cells (Wing et al., 2021a) . HIF-1 downregulates the 82 expression of two key entry factors ACE2 and TMPRSS2, thereby limiting SARS-CoV-2 83 internalisation, whilst also restricting the establishment of viral replication complexes. These data 84 show an essential role for hypoxia/HIF-1 in multiple aspects of the SARS-CoV-2 life cycle and it is 85 timely to address the role of HIFs in an immune competent animal model of COVID-19 disease. and clinical studies (Provenzano et al., 2016) . In the control group, animals were treated with vehicle 105 24h prior to infection which continued throughout the study in the same manner as treated animals 106 Since FG-4592 reduced the level of infectious virus in the lung we were interested to assess whether 166 treatment impacts the viral transcriptome. Mapping the viral reads across the 30kb SARS-CoV-2 167 genome demonstrated an increasing read depth from ORF1ab to the 3'UTR consistent with the 168 transcription of sub-genomic (sg) RNAs (Supplementary Fig.4A ). In addition to the gRNA, the viral 169 transcriptome includes 9 canonical sub-genomic (sg) RNAs that encode the structural proteins, which 170 are essential for the genesis of nascent virus particles. Quantifying the junction spanning reads 171 between the common 5' leader sequence and the start of each sgRNA (as previously described (Kim 172 et al., 2020)), enabled us to infer their approximate abundance. FG-4592 reduced the abundance of 173 most sgRNAs with a greater variability in the treated groups and a significant reduction in the 174 nucleocapsid (N) transcript, the most abundant of the viral RNAs (Fig.3A) . We extended these 175 observations to study the effect of HIF-signalling in SARS-CoV-2 transcription in the lung Calu-3 176 epithelial cell line (Sampaio et al., 2021) and showed a significant reduction in S, E, M, ORF6, 177 ORF7A, ORF7B, ORF8 and N junction spanning reads (Fig.3B) . The relative abundance of SARS-178 CoV-2 transcripts were similar in Calu-3 and infected hamster lung tissue. Analysing samples from 179 the infected Calu-3 cells by northern blotting confirmed that FG-4592 treatment reduced viral 180 transcripts (Fig.3C) . Furthermore, FG-4592 treatment inhibited N protein expression in infected Calu-181 3 cells (Fig.3D) , providing an explanation for the antiviral activity of HIFs. 182 183 Finally, we assessed SARS-CoV-2 sequence variation to determine whether treatment associated 184 with genetic changes. Viral sequences were conserved across the genome in the vehicle or treated 185 lung tissues (Supplementary Fig.4B ) and no changes in the consensus sequence were seen in the 186 treated animals or Calu-3 cells, with 100% conservation of the nucleotide sequence across the 187 genome (Supplementary Table 2 ). Together these data show that FG-4592 treatment had no effect 188 on the sequence of SARS-CoV-2 in the lung but reduced sgRNAs. 189 190 Spatial analysis of SARS-CoV-2 RNA and nucleocapsid expression in the respiratory tract. 191 As FG-4592 reduced the clinical signs and levels of infectious virus in the upper and lower respiratory 192 tract we explored the impact of treatment on virus-associated pathology. Sequential sections from the 193 nasal cavity and lung tissue were stained with haematoxylin and eosin (H&E) and with RNA-scope in 194 situ hybridization (ISH) probes targeting the Spike gene to assess the tissue distribution of CoV-2 RNA. We noted extensive inflammatory cell exudate in the nasal cavity and mild to moderate 196 necrosis in both the olfactory and respiratory epithelia ( Supplementary Fig.5A-C) . We assessed 197 these pathological changes using a semi-quantitative scoring system and showed a reduction in the 198 nasal histopathological score in the post-infection treatment group (Fig.4B, Supplementary Table 3) . 199 Viral RNA primarily localised to the epithelia and exudate in the nasal cavity and FG-4592 reduced 200 epithelial staining, the major site of virus replication (Fig.4B) . A similar observation was noted for viral 201 RNA signals in the exudate (Fig.4B) ; however, these results may be compromised by the daily 202 collection of nasal washes. 203 In agreement with previous studies (Dowall et al., 2021; Gruber et al., 2020; Nouailles et al., 2021) , 205 SARS-CoV-2 infected lung tissue showed pulmonary lesions consisting of broncho-interstitial 206 pneumonia extending into the alveoli and multifocal areas of consolidation, consistent with 207 inflammatory cell infiltration and oedema (Fig.4A, Supplementary Fig.5) . Digital image analysis 208 showed that FG-4592 treatment did not alter the severity of lung histopathology (Fig.4C ). Viral RNA 209 was detected in the bronchiolar epithelia, bronchiolar inflammatory exudates, as well as in the lung 210 parenchyma of the control vehicle animals (Fig.4A) and FG-4592 treatment had no detectable effect 211 on the viral RNA signals in the parenchyma or airways (Fig.4C) . 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