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1 Characterization and functional interrogation of SARS-CoV-2 RNA interactome 2 3 Athéna Labeau1, Alain Lefevre-Utile2, Lucie Bonnet-Madin1, Luc Fery-Simonian1 4 Vassili Soumelis2, Vincent Lotteau3, Pierre-Olivier Vidalain3, Ali Amara1#, Laurent 5 Meertens1#° 6 7 8 1 INSERM U944 CNRS 7212, Université de Paris, Institut de Recherche Saint-Louis, 9 Hôpital Saint-Louis, 75010 Paris, France 10 11 2 INSERM U976, Université de Paris, Institut de Recherche Saint-Louis, Hôpital 12 Saint-Louis, 75010 Paris, France 13 14 3 Centre International de Recherche en Infectiologie (CIRI), Inserm U1111, Université 15 Claude Bernard Lyon 1, CNRS UMR5308, ENS de Lyon, 69007 Lyon, France 16 17 18 # Corresponding author: [email protected], [email protected] 19 20 ° Lead contact
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21 SUMMARY
22 Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative
23 agent of COVID-19 pandemic, which has caused a devastating global health crisis.
24 The emergence of highly transmissible novel viral strains that escape neutralizing
25 responses emphasizes the urgent need to deepen our understanding of SARS-CoV-2
26 biology and to develop additional therapeutic strategies. Using a comprehensive
27 identification of RNA binding proteins (RBP) by mass spectrometry (ChIRP-M/S)
28 approach, we identified 142 high-confidence cellular factors that bind the SARS-CoV-
29 2 viral genome during infection. By systematically knocking down their expression in a
30 human lung epithelial cell line, we found that the majority of the RBPs identified in our
31 study are proviral factors that regulate SARS-CoV-2 genome replication. We showed
32 that some of these proteins represented drug targets of interest for inhibiting SARS-
33 CoV-2 infection. In conclusion, this study provides a comprehensive view of the SARS-
34 CoV-2 RNA interactome during infection and highlights candidates for host-centered
35 antiviral therapies.
36
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37 Introduction
38
39 SARS-CoV-2, which belongs to the Coronaviridae family, has been identified as
40 the causative agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19)
41 (Zhu et al., 2020). As of March 7, 2021, SARS-CoV-2 has spread Worldwide, with
42 about 116 million confirmed cases and above 2.5 million deaths according to the WHO.
43 Vaccination appears as one of the most expected health interventions with over 200
44 vaccine candidates under development as of December, 2020 (Mellet and Pepper,
45 2021) with those that received an emergency approval targeting the viral spike protein
46 (Poland et al., 2020). However, the emergence of SARS-CoV-2 variants with mutations
47 in the spike protein that reduces susceptibility to neutralizing antibodies (Choi et al.,
48 2020; Kemp et al., 2021; Liu et al., 2021; McCarthy et al., 2021; Thomson et al., 2021),
49 raises the concern of future variants escaping from natural or vaccine-induced
50 neutralizing immunity. Furthermore, a recent study conducted in Manaus, Brazil,
51 suggested a potential antibody wane at 8 months post-exposure (Sabino et al., 2021).
52 Thus, there is an urgent need to improve our understanding of SARS-CoV-2 replication
53 mechanisms to develop additional therapeutic strategies.
54 The genome of SARS-CoV-2 is composed of a single stranded positive RNA with
55 a length of approximatively 30 kb (Zhou et al., 2020). In the cytoplasm of infected cells
56 the viral RNA is translated by the host machinery in two overlapping replicase
57 polyproteins that undergo subsequent viral-protease-mediated processing to generate
58 16 non-structural proteins (NSP) (de Wilde et al., 2018). These NSPs assemble and
59 recruit poorly-characterized host cell factors to form the replication and transcription
60 complex (RTC), which localizes in a virus-induced network composed of double-
61 membrane vesicles (DMV)(Cortese et al., 2020; Eymieux et al., 2021). Within the RTC,
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62 the RNA-dependent-RNA polymerase (RdRp) complex uses the genomic RNA as a
63 template to generate negative strand intermediates that serve for the synthesis of both
64 progeny genomic RNA and subgenomic viral RNA (sgvRNA) (Snijder et al., 2016).
65 SgvRNAs are then translated in 4 structural (S, E, M and N) and seven accessory
66 proteins (ORF3a-9b) (Kim et al., 2020). Viral genomic RNA associates with the
67 nucleocapsid (N) to form the ribonucleocapsid that drives, with the other structural
68 proteins, the assembly of new viral particles, which bud in the lumen of the
69 endoplasmic reticulum-Golgi intermediate compartment (ERGIC).
70 Like all viruses, SARS-CoV-2 heavily relies on cellular proteins to accomplish its
71 infectious life cycle. This host dependency represents a potential “Achilles’ heel” that
72 could be exploited to develop new host-centered approaches to treat SARS-CoV-2
73 infection. In this context, several strategies were used to decipher the molecular
74 interactions occurring between the virus and host cell during infection. Affinity
75 purification combined with mass spectrometry allowed the identification SARS-CoV-2
76 virus-host protein-protein interactions (PPI) (Gordon et al., 2020a, 2020b; Li et al.,
77 2020) while CRISPR-Cas9 phenotypic screens have provided valuable information
78 about the host genes and cellular pathways important for the SARS-CoV-2 life cycle
79 (Daniloski et al., 2020; Hoffmann et al., 2021; Schneider et al., 2020; Wang et al., 2021;
80 Wei et al., 2020). These studies have led to the identification of potential cellular targets
81 for drug repurposing, such as the PI3K complex, the sigma-1 and -2 receptors as well
82 as the cholesterol homeostasis (Daniloski et al., 2020; Gordon et al., 2020a; Hoffmann
83 et al., 2021; Wang et al., 2021).
84 In this study, using a comprehensive identification of RNA-binding protein by
85 mass spectrometry (ChIRP-M/S) approach, we provide a global map of the host
86 proteins that associate with the SARS-CoV-2 genome during infection. By combining
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87 these proteomics data with gene silencing experiments, we identified a set of new
88 SARS-CoV-2 host dependency factors that impact infection and could be targetable
89 for antiviral therapies.
90
91 Results and Discussion
92
93 Characterization of the SARS-CoV-2 RNA interactome in human cells
94 To characterize the host factors that interact with SARS-CoV-2 genome during
95 infection, we performed a comprehensive identification of RNA-binding proteins by
96 mass spectrometry approach (ChIRP-M/S) (Figure 1A). Originally developed to study
97 endogenous ribonucleoprotein (RNP) complexes, this technique has been recently
98 adapted to investigate viral RNA-host protein interactions (Ooi et al., 2019). Briefly, we
99 challenged 293T cells stably expressing the human ACE2 receptor (293T-ACE2) with
100 a primary clinical SARS-CoV-2 220/95 strain (EPI_ISL_469284) isolated by our
101 laboratory in March 2020 (Onodi et al., 2021). Forty-eight hours later, SARS-CoV-2
102 infected and uninfected cells were treated with formaldehyde to crosslink RNA with
103 RBPs in order to preserve RNP complexes. To specifically capture SARS-CoV-2 RNA,
104 we designed 129 biotinylated antisense probes (Table S1) complementary of the full-
105 length viral RNA, and subsequently used streptavidin beads to pulldown the RNPs.
106 Co-immunoprecipitated proteins were separated by SDS-PAGE, visualized by silver-
107 staining (Figure 1B), and subjected to mass spectrometry (MS) analysis (Table S2).
108 As expected, the SARS-CoV-2 N protein, which is the main viral RBP, was the most
109 enriched protein in our study (Figure 1B, C). The structural M and S proteins, the
110 replicase polyprotein pp1ab and the accessory protein ORF9b were also significantly
111 and reproducibly enriched in our experiments (Figure 1C, 1D). Of the 16 NSPs
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112 released from pp1ab, 9 were enriched in our ChIRP-M/S (Figure 1D) and are part of
113 the coronavirus RTC (V’kovski et al., 2019), where NSP2-NSP16 play essential
114 functions involved in RNA replication (V’kovski et al., 2021). NSP1 is known to interact
115 with 40S subunit of the ribosome to inhibit cellular translation, and to bind the viral 5’
116 untranslated region (UTR) to promote vRNA translation (Schubert et al., 2020). The
117 structural protein M and S associate with the viral RNA during virus assembly and
118 budding. Finally, ORF9b has be shown to localize at the mitochondria membrane and
119 antagonize the RIG-I-MAVS antiviral signaling through its association with TOM70
120 (Gordon et al., 2020b; Jiang et al., 2020). Together, these data provide strong
121 evidences that our ChIRP-M/S approach reproductively and specifically pulldown viral
122 RNP complexes involved in different steps of SARS-CoV-2 life cycle, from
123 replication/transcription to particle assembly.
124
125 A view of the SARS-CoV-2 RNA-host protein interactome
126 Using SAINTexpress scoring algorithms, we identified 142 high-confident human
127 proteins that reproductively associated with the SARS-CoV-2 RNA (SAINT score
128 ≥0.79; Figure 1E, Table S3). Among them, 89 % (127 out of 142) were annotated by
129 the ontology term “RNA binding” (GO: 0003723), suggesting a direct interaction with
130 the viral genome. Next, we compared our RNA interactome with recently published
131 data sets obtained by RNA-centric (RAP-M/S and ChIRP-M/S) approaches (Flynn et
132 al., 2021; Lee et al., 2020; Schmidt et al., 2020). We found that 24% to 38% (Schmidt
133 et al.: 35/144; Flynn et al.: 77/228 and Lee et al.: 74/193) of the SARS-CoV-2 RNA
134 host factors identified in these studies were also enriched in our ChIRP-M/S
135 interactome (Table S4). Interestingly, we highlighted 58 common hits which defined a
136 reproducible set of cellular SARS-CoV-2 RNA RBP (Figure S1). These include multiple
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137 RBPs involved in nucleic acid metabolism including splicing, mRNA stability and
138 transcriptional regulation such as several heterogenous nuclear ribonucleoproteins
139 (hnRNPs) (Geuens et al., 2016), the serine/arginine-rich splicing factor (SRSF)
140 (Shepard and Hertel, 2009), poly(A) binding protein (PABP) (Mangus et al., 2003) and
141 the Insulin-like growth factor 2 mRNA binding protein (IGF2BP) (Bell et al., 2013). We
142 also found several components of the small 40S ribosome subunit (RPS3, RPS3A,
143 RPS16, RPS19 and RPS20), components of the translation initiation complex
144 (EIF4G1, EIF3F and EIF4H) and translation elongation factor (EEF2). Surprisingly, we
145 did not identify EIF4E, the CAP binding protein, suggesting that SARS-CoV-2 might
146 use an alternate CAP-dependent EIF4E-independent translation mechanism (de la
147 Parra et al., 2018). Chaperone proteins (HSP90AB1, CCT2 and CCT8) were also
148 significantly enriched and are likely involved in viral protein folding. Characteristic
149 RBPs involved in ribonucleoprotein granules such as paraspeckles (PS) (RBM14,
150 NONO, MATR3, SFPQ) and stress granules (SG) (G3BP1, G3BP2, TIA1, CAPRIN1,
151 ATXN2L) (Nakagawa et al., 2018; Yang et al., 2020) were among the highest-scoring
152 candidates. Many PS-interacting proteins were also highly represented in our ChIRP-
153 M/S data set (DAZAP1, ESWR1, FAM98A, FUS, RBMX, UBAP2L, as well as
154 hnRNPA1, A1L2, H1, H3, K, R, UL1) (Naganuma et al., 2012).
155 Functional analyses of the RBPs revealed more than 150 statistically enriched
156 GO terms, which cluster into 23 functional groups (Figure 2A), and are also found in
157 the three other SARS-CoV-2 RNA interactome studies (Figure 2B). Among them, we
158 found host molecules that participate in RNA metabolic process, translation, RNA
159 stabilization, ribonucleoprotein complex assembly, viral process and innate immune
160 response (Figure 2A, 2C). Based on its predominant nuclear localization and its role in
161 mRNA maturation, the significant enrichment of RNA splicing GO term (p=3.55x10-43)
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162 was unexpected. Pre-mRNA splicing is critical for mRNA nucleo-cytoplasmic export,
163 thereby contributing to the regulation of gene expression (Reed and Hurt, 2002). It is
164 tempting to speculate that SARS-CoV-2 RNA may sequester host RBP involved in
165 splicing in order to hamper cellular mRNA nuclear export, thus favoring the cytoplasmic
166 translation of viral RNAs. This hypothesis is consistent with recent findings showing
167 that SARS-CoV-2 NPS16 disrupts mRNA splicing to antagonize innate immunity at a
168 post-transcriptional level (Banerjee et al., 2020).
169
170 Overlapping of the ChIRP interactome with known coronavirus-host interactions
171 identifies host factors involved in central carbon metabolism as a new target of
172 SARS-CoV-2
173 To gain insights on the interactions between host factors and viral proteins we
174 compared our SARS-CoV-2 interactome with a reference coronavirus interactome,
175 which corresponds to a meta-analysis of the viral-host protein-protein interactions of
176 13 coronaviruses collected built upon 112 publications (Perrin-Cocon et al., 2020). Of
177 the 142 high-confidence hits identified by our ChIRP approach, 80 overlapped with the
178 1,140 host proteins (56%) of the reference coronavirus interactome. As illustrated in
179 Figure 3A, these host proteins captured by our ChIRP assay are known interactors of
180 one or several coronaviruses viral proteins. Interestingly, a majority of these proteins
181 (79%) were previously identified as N protein-binding partners. Shared interactors with
182 S, NSP1, NSP2, NSP9, NSP12, NSP13, NSP14, NSP15, ORF6 and ORF9b were also
183 identified (Figure 3A). In our interaction network, the ATP-dependent RNA helicase
184 DDX1 was the most connected factor interacting with both N and NSP14 from both
185 TGEV and PEDV, NSP14 from SARS-CoV-1 and NSP2 from MHV. Altogether, this
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186 supports a key role of DDX1 in SARS-CoV-2 replication cycle, as previously reported
187 for other coronaviruses (Wu et al., 2014; Xu et al., 2010).
188 We then analyzed the subset of cellular proteins that did not overlap with the
189 reference coronavirus interactome, which essentially corresponds to novel SARS-
190 CoV-2 and coronavirus interactors. First, we compared the fold enrichment in the
191 ChIRP experiments for the 62 new and 80 known interactors and observed a similar
192 distribution, indicating that the two subsets were qualitatively equivalent and
193 correspond to robust interactions (Figure 3B). Next, we investigated distribution of
194 biological processes covered by the subset of new interactors, and found a distribution
195 of the functional annotations similar to what we observed with the full data set.
196 However, eight proteins (ALDOA, CBR1, FASN, GPI, LDHB, MTHFD1, NTPCR and
197 PGK1) belonged to metabolic pathways according to KEGG database. Interestingly,
198 five proteins (GPI, ALDOA, PGK1, LDHB, FASN) were involved in glycolysis,
199 lactagenesis and lipogenesis which are tightly connected pathways (Figure 3C). These
200 data are consistent with recent a study showing that glycolysis and lactate pathways
201 sustained SARS-CoV-2 replication (Codo et al., 2020). Furthermore, fatty acid
202 synthesis is necessary for the formation of lipid droplets (LD) which are required for
203 SARS-CoV-2 viral replication (Dias et al., 2020). Together, these data suggest that
204 SARS-CoV-2 targets the central carbon metabolism to accomplish its life cycle.
205
206 Functional interrogation of the SARS-CoV-2 RBP identified in our ChIRP-M/S
207 approach
208 To further pinpoint the function of the identified RBPs in SARS-CoV-2 infection,
209 we silenced their expression by RNA interference (RNAi) and determined
210 consequences on virus infection. We designed a library of siRNA pool targeting 138
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211 RBPs identified in our study and performed a loss-of-function screen in the human lung
212 epithelial cell line A549 stably expressing ACE2. SiRNA targeting the cathepsin L
213 protease (CTSL) and the vacuolar ATPase subunit ATP6V1B2, two host molecules
214 important for SARS-CoV-2 viral entry (Daniloski et al., 2020), were used as positive
215 controls, whilst a non-targeting siRNA pool (siNT) was used as negative control. SiRNA
216 transfected cells were challenged with SARS-CoV-2 for 24 hours and viral infection
217 was assessed either by quantifying the intracellular accumulation of the N protein by
218 flow cytometry (viral replication) or the viral RNA released in the supernatant of infected
219 cells by RT-qPCR (release of viral particles) (Figure 4A). All readouts were normalized
220 to the siNT-transfected cells. Transfected cells were also monitored for cell viability,
221 and 7% (9/138) of the siRNA affecting cell viability were removed from the analysis.
222 As expected, transfection of siRNA targeting CTSL and ATP6V1B2, resulted in a
223 strong decrease of infection in both assays (Figure 4B-C). RT-qPCR experiments
224 revealed that 45 siRNAs reduced by at least 50% viral particles production whereas 4
225 siRNAs increased it by 200 % (Figure 4B). Flow cytometry studies showed that 21
226 siRNAs decreased the percent of infected cells by more than 50 %, while 12 enhanced
227 it by 150% (Figure 4C). By intersecting the data from both assays, we identified 25
228 RBPs that impacted viral infection. Interestingly, most of them (19/25) are host
229 dependency factors (HDF) and few (6/25) act as host restriction factors (HRF) (Figure
230 4D). This contrasts with the data recently published by Flynn et al. showing that the
231 majority of SARS-CoV-2 RBP factors identified in their study has an antiviral function
232 (Flynn et al., 2021). We observed a high correlation coefficient (r=0.742) between our
233 RT-qPCR and flow cytometry data set indicating that most of the identified RBPs are
234 required for viral replication (Figure 4D), and only very few act at a late step
235 (assembly/particle egress) of the SARS-CoV-2 life cycle (Figure 4D, green circles).
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236 Interestingly, several of HDF such as ILF3, HNRNPU, HNRNPK, FAM120A and QKI
237 were predicted to bind the 5’- or 3’-UTR of SARS-CoV-2 RNA (Sun et al., 2021).
238 Others, including SFPQ, DDX5, EWSR1, HNRNPK, ADAR, PA2G4 were previously
239 reported to promote replication of other RNA viruses (Garcia-Moreno et al., 2019;
240 Gélinas et al., 2011; Landeras-Bueno et al., 2011; Li et al., 2013; Oakland et al., 2013;
241 Thompson et al., 2020; Zhou et al., 2019a), supporting the role of these RBPs in SARS-
242 CoV-2 replication. Interestingly, ILF3 was shown to participate to antiviral type I
243 interferon response by promoting the expression of IFNB1 and interferon-stimulated
244 gene (ISG) during host translational shutoff (Watson et al., 2020). QKI was shown to
245 repress host interferon response by downregulating MAVS expression at a post-
246 transcriptional level (Liao et al., 2020). This suggests that SARS-CoV2 has evolved
247 several mechanisms to dampen the type I interferon response by targeting specific
248 RBPs.
249 Next, we looked at the DrugBank, ChEMBL, DGIdb (Drug-gene interaction
250 database) and PanDrugs database for drugs that target our host RBP. We identified
251 camptothecin (XRCC5), cerulenin (FASN), dabigatran (HNRNPC), geldanamycin
252 (HSP90AB1), phenethyl isothiocyanate (HNRNPK), sunitinib malate (EWSR1) and
253 artemisinin (an antimalarial interacting with multiple RBPs) as potential SARS-CoV-2
254 antiviral candidates. To test whether these compounds impact infection, A549-ACE2
255 cells were incubated with the compounds at a concentration of 10 and 1 µM and then
256 challenged with SARS-CoV-2. We used equivalent dilution of DMSO as negative
257 control and similar concentration of remdesivir, an inhibitor SARS-CoV-2 replication
258 (Wang et al., 2020) as positive control. We monitored the release of progeny viruses
259 in the supernatant as well as cell viability 24 hours post-infection. Of all the compounds
260 tested, we observed a significant inhibitory effect for phenethyl isothiocyanate (40 %
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261 inhibition) and sunitinib malate (80 % inhibition) at a concentration of 1 µM without
262 toxicity (Figure S2A). Sunitinib malate is a promising therapeutic for the treatment of
263 patient suffering from sarcoma such as DSRCT (Desmoplastic Small Round Cell
264 Tumor) or EMC which expressed an EWSR1-WT1 or EWSR1-NR4A3 fusion,
265 respectively (Mello et al., 2021; Stacchiotti et al., 2014). Sunitinib malate inhibited the
266 release of SARS-CoV-2 particles in infected cells in dose-response manner with an
267 IC50 of 0.49 µM (Figure 4E). Sunitinib malate inhibits also the N protein production in
268 infected cells, consistent with a role of EWSR1 in genome replication (Figure S3B).
269 Similar results were obtained in 293T-ACE2 cells (Figure S3C).
270 In conclusion, our data provides a landscape of functional interactions that the
271 SARS-CoV-2 RNA genome establishes with the host cell during infection. We provide
272 several evidences that SARS-CoV-2 exploit several cellular RBP to facilitate viral
273 replication and highlight host molecules that could be targeted for antiviral intervention.
274
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275 ACKNOWLEDGMENTS
276 This works was supported by French Government's Investissement d'Avenir
277 program, Laboratoire d'Excellence "Integrative Biology of Emerging Infectious
278 Diseases" (grant n°ANR-10-LABX-62-IBEID), the Fondation pour la Recherche
279 Medicale (grant FRM - EQU202003010193 and FDM201806006187), the Agence
280 Nationale de la Recherche (ANR) (ANR-20-COVI-000 project IDISCOVR and ANR-
281 20-CO11-0004 project FISHBP) and University of Paris (Plan de Soutien Covid-19:
282 RACPL20FIR01-COVID-SOUL). Laurent Meertens and Ali Amara dedicate this work
283 to the memory of Professor Jean-Louis Virelizier (Unité d Immunologie Virale, Institut
284 Pasteur, Paris) and Professor Renaud Mahieux (Ecole Normale Supérieure, Lyon,
285 France), who left us during the SARS-CoV-2 epidemic. The authors thank Alessia
286 Zamborlini for critical reading of the manuscript.
287 288 AUTHOR CONTRIBUTION
289 Conceptualization, L.M. and A.A.; L.M. supervised the project. L.M. performed the
290 ChIRP experiments and analyzed the data with the help of A.L and P.O; A.L-U.; P.O.
291 and V.S. performed the bioinformatic analysis; L.B-M and L.M produced the SARS-
292 CoV-2 virus stocks used in this study. L.F-S participated in the ChIRP experiments.
293 P.O.V and V.L. analyzed the viral protein-RBP protein interaction; A.L and L.M.
294 performed the siRNA screen; A.L. screened the compounds; L.M., P.O.V., A.L. and
295 A.A. wrote the manuscript with the inputs from all authors; Funding Acquisition, A.A
296 and L.M.
297 298 DECLARATION OF INTEREST
299 The authors declare no competing interest.
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300 Figure legends
301 Figure 1. Purification of the SARS-CoV-2 RNA interactome. (A) Schematic
302 illustrating the ChIRP-M/S approach used to identify host factors bound to SARS-CoV-
303 2 RNA. (B) Proteins enriched by ChIRP from uninfected and SARS-CoV-2-infected
304 cells were resolved by SDS-PAGE and visualized by silver staining. (C) Quantification
305 of the peptides enrichment in SARS-CoV-2 infected cells over uninfected cells (n=5
306 independent replicates). Viral proteins are marked by a red circle. (D) (top) Schematic
307 representing unique peptides distribution along the enriched viral proteins. (bottom)
308 Table showing the number of unique peptides for each viral protein. Data from one
309 representative replicate were shown. (E) Fold change (SAINTexpress FC-A score) of
310 the 140 high-confident host proteins identified in our SARS-CoV-2 ChIRP-M/S.
311 (Proteins Histone H3.2 and H2B type 1-H were not represented).
312
313 Figure 2. Biological analysis of the SARS-CoV-2 RNA interactome (A) GO
314 enrichment analysis of SARS-CoV-2 RNA interactome proteins. Circles represent
315 enriched function for an annotated ontology term and circle size correspond to the
316 number of enriched proteins within that term. (B) Heatmap of the top-ranked GO
317 biological processes enriched across all SARS-CoV-2 RNA interactomes (C) Selected
318 biological process networks of SARS-CoV-2 interactome proteins.
319
320 Figure 3. Overlapping of our ChIRP interactome with known coronavirus-host
321 interactions identifies the central carbon metabolism as SARS-CoV-2 target. (A)
322 Map showing interactions between the cellular proteins of our ChIRP dataset and viral
323 proteins of SARS-CoV-2 or other coronaviruses. This analysis was performed using
324 the reference coronavirus interactome previously published (Perrin-Cocon et al.,
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325 2020). Round circles correspond to host proteins. Squares correspond to viral proteins.
326 (B) Fold change scores of cellular proteins that are either known coronavirus targets
327 or new interactors identified for the first time by the ChIRP study. Fold change scores
328 (FC-A) were calculated by Saint-Express from the average mean of spectral counts.
329 No significant differences were observed between the two distributions (p-value>0.05;
330 Mann Whitney test). (C) Simplified metabolic pathways showing functional connections
331 between five enzymes identified in our ChIRP analysis and targeted by SARS-CoV-2.
332
333 Figure 4. Functional interrogation of the SARS-CoV-2 RNA interactome and
334 compounds screening. (A) Schematic illustrating the loss-of-function screen
335 procedure. (B and C) A549-ACE2 cells were transfected with an arrayed siRNA library
336 and challenged with SARS-CoV-2 (MOI 0.05) for 24h hours. (B) Yield of viral particles
337 released in the supernatant of infected cells was quantified by RT-qPCR and
338 normalized to the siNT-transfected cells. (C) Viral replication was assessed by flow
339 cytometry using anti-N protein mAb, and normalized to the siNT-transfected cells. Data
340 shown are means of two independent experiments. Adjusted p-values were calculated
341 by one-way ANOVA with Benjamini and Hochberg correction. Host dependency factors
342 are marked in blue and host restriction factors are marked in red. Positive controls
343 (CTSL and ATP6V1B2) are highlighted in yellow. (D) Intersection of the data obtained
344 from N protein quantification by flow cytometry and virus release in supernatant of
345 infected cells by RT-qPCR. Data shown are means of two independent experiments.
346 Host dependency factors are marked in blue and host restriction factors are marked in
347 red. (E) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in continuous
348 presence of increased concentrations of remdesivir or sunitinib malate. Virus released
349 in supernatant was quantified 24 hpi by RT-qPCR (red lane). Cell viability was
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350 assessed in parallel (black lane). Data shown are mean +/- SD of three independent
351 experiments in duplicate.
352
353 Tables titles
354
355 Table S1. Sequences of the 129 biotinylated ChIRP probes. Related to figure 1.
356
357 Table S2. Datasets of the mass spectrometry analyses. Related to figure 1.
358
359 Table S3. SAINTexpress analyses of the ChIRP-M/S dataset. Related to figure 1.
360
361 Table S4. List of RBP identified in ChIRP-M/S that overlap with other SARS-CoV-2
362 interactome. Related to figure 1.
363
364 Supplementary Figure Legends
365
366 Figure S1. Intersection of SARS-CoV-2 interactomes identified a set of core
367 SARS-CoV-2 RNA RBP. Related to Figure 1. Venn Diagram comparing the
368 significantly enriched RBP from SARS-CoV-2 RNA interactomes.
369
370 Figure S2. Screening of compounds with antiviral activity targeting SARS-CoV-
371 2 host RBP. Related to Figure 4. (A) A549-ACE2 were infected with SARS-CoV-2
372 (MOI 0.05) in continuous presence of compounds (10 and 1 µM). Virus released in
373 supernatant was quantified 24 hpi by RT-qPCR (top panel). Cell viability was assessed
374 in parallel (bottom panel). Data shown are mean +/- SD of three independent
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375 experiments in duplicate. Significance was calculated using two-way ANOVA statistical
376 test with Dunnett’s multiple comparisons test. (ns not significant, ** p< 0.01; *** p<
377 0.001; **** p<0.0001). (B) A549-ACE2 were infected with SARS-CoV-2 (MOI 0.05) in
378 continuous presence of increased concentrations of remdesivir or sunitinib malate.
379 Percentage of infected cells was quantified 24 hpi by flow cytometry using anti-N mAb
380 (blue lane). Cell viability was assessed in parallel (black lane). Data shown are mean
381 +/- SD of three independent experiments in duplicate. (C) 293T-ACE2 were infected
382 with SARS-CoV-2 (MOI 0.05) in continuous presence of increased concentrations of
383 remdesivir or sunitinib malate. Virus released in supernatant was quantified 24 hpi by
384 RT-qPCR (red lane). Cell viability was assessed in parallel (black lane). Data shown
385 are mean +/- SD of three independent experiments in duplicate.
386
387
388 EXPERIMENTAL METHODS
389
390 Cell lines
391 Lenti-X 293T cells (human embryonic kidney cells), A549 cells (Human alveolar basal
392 epithelial carcinoma), and Vero E6 cells (African green monkey kidney cells) were
393 maintained in Dulbecco Modified Eagle Medium (DMEM; Invitrogen Life Technologies)
394 supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1%
395 penicillin/streptomycin (P/S) and 1% GlutaMAX (Life Technologies). All cell lines were
396 periodically tested negative for mycoplasma contamination prior to use in experiments.
397
398 Virus preparation and titration
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399 SARS-CoV-2 strain 220_95 (EPI_ISL_469284) was isolated from nasopharyngeal
400 swab specimens collected at Service de Virologie (Hospital Saint Louis, Paris) (Onodi
401 et al., 2021). Virus was propagated on Vero E6 in DMEM-2% (DMEM supplemented
402 with 2% FBS, 1% P/S, 1% GlutaMAX, and 25 mM Hepes). Viruses were passaged
403 three times before being used for experiments. For the last passage, viruses were
404 purified through a 20% sucrose cushion by ultracentrifugation at 80,000xg for 2 hours
405 at 4°C. Pellets were resuspended in TNE1X pH7.4 (Tris 50 mM, NaCl 100mM, EDTA
406 0.5 mM), aliquoted and stored at -80°C.
407 Viruses titer was ascertained by plaque assays in Vero E6 cells and expressed as PFU
408 per ml. Cells were incubated for one hour with 10-fold serial dilution of viral stocks. The
409 inoculum was then replaced with Avicel 2.4% mixed at equal volume with DMEM
410 supplemented with 4% FBS, 2% Glutamax, 50mM MgCl2, 0.225 % of NaHCO3, and
411 incubated 3 days at 37°C. Then, Vero E6 cells were washed twice with phosphate-
412 buffered saline (PBS) and stained with an aqueous solution containing 1%
413 formaldehyde, 0,5% crystal violet, and 0,89% sodium chloride, for 15 min at room
414 temperature. Vero E6 cells were then washed with water to visualize lysis plaques.
415
416 Generation of cell lines stably expressing ACE2
417 The human ACE2 coding DNA sequence (CDS) was amplified by RT-PCR from pLenti-
418 ACE2 plasmid (a gift from Olivier Schwartz, Virus and Immunity, Institut Pasteur) using
419 the ACE2 forward and reverse primers. Amplification product was cloned into pLVX-
420 IRES-ZsGreen1 vector using XhoI/NotI restriction enzymes. The ACE2 protein-
421 expressing lentiviral vectors pseudotyped with vesicular stomatitis virus glycoprotein
422 G (VSV-G) were generated by transfecting lenti-X 293T (5x106) cells with pLVX-ACE2-
423 ZsGreen, psPAX2, and pVSV-G (4:3:1 ratio) using calcium phosphate transfection
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424 (Promega) following the manufacturer’s instructions. Supernatants were harvested 48
425 hours after transfection, cleared by centrifugation, and filtered. Lentiviral vectors were
426 purified as describe above (see ‘virus preparation and titration’ section). 293T and
427 A549 cells (2x105) were seeded in 6 well plate and transduced by spinofection at
428 1,000xg for 2 hours at 33°C. Cell populations were sorted based on GFP expression
429 with a BD FACSAria II (Becton Dickinson) with FACSDiva 6.1.2 software (Becton
430 Dickinson).
431
432 Flow cytometry analysis of ACE2 cell surface expression
433 Cells were detached with 2.5 mM EDTA in PBS and incubated with anti-human ACE2
434 mAb (5 mg/ml) in 100 ml of PBS with 0.02% NaN3 and 5% FBS for 1 hour at 4°C.
435 Following the primary staining, cells were washed and stained with incubated with the
436 Alexa 647-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) for 30 min at
437 4°C. Acquisition was performed on an Attune NxT Flow Cytometer (Thermo Fisher
438 Scientific) and analysis was done by using FlowJo software (Tree Star)
439
440 Comprehensive identification of RNA binding proteins by mass spectrometry
441 ChIRP-M/S was performed as previously described by Ooi and colleagues (Ooi et al.,
442 2019). SARS-CoV-2 probes were designed using the online tool
443 (https://www.biosearchtech.com/stellaris-designer), with a repeat masking setting of 5
444 and even coverage of the whole viral genome (1 oligos every 200 bp). Oligos were
445 synthetized with 3’ biotin-TEG modification at Eurofins Genomics. The 129 oligos were
446 resuspended at 100 µM and mixed at equal volume to prepare the probes pool.
447
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448 293T cells stably expressing ACE2 were seeded at 9x106 cells per T150 flask (3 flasks
449 per condition) and inoculated with SARS-CoV-2 at a multiplicity of infection (MOI) of
450 0.001 or mock-treated. The media was removed 48 hours later and the cells were
451 rinsed once with PBS, trypsinized and pooled for each condition. Cells were
452 centrifugated at 1,500 rpm for 5 min and pellets were washed twice with PBS. The
453 cells were resuspended with 35 ml of PBS containing 3% formaldehyde and incubated
454 under agitation for 30 min at room temperature (RT). Of note, all the buffers used
455 throughout the ChIRP experiments were freshly prepared. Cross-linking was
456 terminated by adding glycine to a final concentration of 0.125 mM for 5 min at RT.
457 Cross-linked cells were centrifugated at 1,000xg for 5 min, washed once with PBS,
458 weighted, then snap frozen and kept at -80°C until used. Cells were resuspended in 1
459 ml of lysis buffer (50 mM Tris-HCl, pH7.0, 10 mM EDTA, 1% SDS supplemented with
460 SUPERase-IN and protease inhibitor) per 100 mg of cell pellet. The lysates were
461 sonicated using a Diagenode Bioruptor. At this stage, 10 µl of lysates were kept as
462 sample inputs. Lysates were precleared by adding 30 µl of washed MyOne C1 beads
463 per ml of lysate and rocked for 30 min at 37 °C. Beads were removed from the lysates
464 using a magnetic stand and 2 ml of ChIRP hybridization buffer (750 mM NaCl, 1%
465 SDS, 50 mM Tris-HCl pH 7.0, 1 mM EDTA, 15% formamide supplemented with 1 mM
466 PMSF, protease inhibitor and SUPERase-in) and 1.5 µl of probes pool were added for
467 every ml of lysate. After mixing, hybridization took place for 16 hours at 37 °C under
468 rotation. Then, 100 µl of washed MyOne C1 beads per microliter of probes pool were
469 added to each sample and incubated for 45 min at 37°C. Magnetic beads were
470 collected on a magnetic stand and washed 5 times with 1 ml of washing buffer (2x
471 SSC, 0.5% SDS, add 1 mM PMSF) for 5 min at 37°C. To eluate the enriched
472 ribonucleoprotein complexes, the beads were incubated with 300 µl of biotin elution
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473 buffer (12.5 mM D-biotin, 7.5mM Hepes pH 7.5, 75 mM NaCl, 1.5 mM EDTA, 0.15 %
474 SDS, 0.075% sarkosyl and 0.02% Na-Doexycholate) for 20 min at RT under rotation
475 and then 15 min at 65 °C with shaking. Eluate was collected in a fresh tube and the
476 beads subject to elution again. The two eluents were pooled (600 µl) and 150 µl of
477 trichloroacetic acid (25 % of the total eluate volume) was added, mixed vigorously and
478 let overnight at 4°C for precipitation. The next day, the samples were centrifugated at
479 20,000 g for 45 min at 4°C. Pellets were washed once with ice-cold acetone and spun
480 at 16,000 rpm for 5 min at 4°C. The acetone supernatant was removed, the tubes
481 centrifuged briefly to carefully remove the residual acetone and the pellets were left to
482 air-dry on the bench top. The pellets were solubilized in 30 µl of Laemmli 2X buffer with
483 20 mM DTT and boiled at 95 °C for 30 min with a periodic mixing (every 5 min) to
484 reverse the cross-linking.
485 One-tenth of the protein samples and their corresponding inputs (pre-diluted 1/10)
486 were size-separated on a bolt 4-12% Bis-Tris plus (Thermo Fisher Scientific) and
487 stained with the silver stain kit (Thermo Fisher Scientific) as per manufacturer’s
488 instructions. The remaining samples were size-separated on a bolt 10 % Bis-Tris plus
489 and stained with the Imperial protein stain (Thermo Fisher Scientific) according to
490 manufacturer’s instructions. For each ChIRP experiments one gel slice from the SARS-
491 CoV-2 and mock-infected samples were cut from the SDS-page. Gel slices from 5
492 independent replicates were sent for mass spectrometry analysis at Taplin Biological
493 Mass Spectrometry Facility (Harvard Medical School) as previously described
494 (Hafirassou et al., 2017).
495
496 High-confidence RNA binding protein scoring
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497 ChIRP-M/S data were analyzed using the online server (https://reprint-apms.org/) to
498 run SAINTexpress (Teo et al., 2014). Total peptides counts from each of the five
499 replicates, including both mock- and SARS-CoV-2-infected cells, were used as
500 quantitative values to run the analysis. To determine the final list of high-confidence
501 hits we applied stringent filtering criteria with MIST scoring > 0.7, and a SAINTexpress
502 score above 0.78. Data shown in figure 1E correspond to the fold change scores (FC-
503 A) calculated by SAINTexpress from the average mean of spectral counts.
504
505 Identification of known and new interactors of coronaviruses in the ChIRP
506 dataset
507 A recently published meta-analysis by Perrin-Cocon et al. was used as a reference for
508 known interactions between coronavirus and host proteins (Perrin-Cocon et al., 2020).
509 In this report, the literature was manually curated to assemble a dataset of 1,311
510 coronavirus-host interactions encompassing 1,140 distinct cellular proteins. Host
511 proteins in the ChIRP dataset overlapping with those present in this reference
512 interactome were considered as previously reported or « known » interactors of
513 coronaviruses. They were displayed together with the interacting-coronavirus proteins
514 as an interaction network using Cytoscape (Shannon et al., 2003). Cellular proteins in
515 the ChIRP dataset that were not reported in Perrin-Cocon et al. were considered as
516 « new » interactors of SARS-CoV-2 and coronaviruses in general. This list of new
517 interactors was analyzed with the Functional Annotation Tool of the online knowledge
518 base DAVID Bioinformatics Resources 6.8, NIAID/NIH (Dennis et al., 2003) to
519 identify statistical enrichments in KEGG pathway annotations (Kanehisa and Goto,
520 2000).
521
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522 siRNA screen assay
523 An arrayed ON-TARGETplus SMARTpool siRNA library (Horizon Discovery) targeting
524 138 of the 142 RNA binding proteins significantly enriched in our ChRIP-M/S was
525 purchased from Horizon Discovery. A549 cells stably expressing ACE2 were seeded
526 in 24-well plate format (viral infection) or a 96-well plate (viability) and then were
527 reverse transfected in duplicate with 30 nM of siRNA using the Lipofectamine
528 RNAiMAX reagent (Invitrogen) according to manufacturer’s instruction. 48 hours post-
529 transfection, cells seeded in a 24-well format were challenged with SARS-CoV-2 at a
530 MOI of 0.05 in DMEM containing 5 % FBS. After three hours at 37°C, virus inoculum
531 was removed and replace with fresh medium. 24 hours post-infection, cells and 200
532 µL of supernatants were collected for N protein staining and viral RNA quantification
533 as described in ‘Viral infection quantification assays’. Throughout the screening assay,
534 siRNA-transfected cells seeded in 96-well format were incubated for 72 hours at 37 °C
535 and used to assess cell viability.
536
537 Drug treatment and SARS-CoV-2 infection
538 Cells grown in 24-well plates were treated with the indicated compound concentrations
539 for two hours prior infection and compounds were maintained throughout the course
540 of infection. Cells treated with equivalent concentration of DMSO were used as control.
541 The cells were challenged with SARS-CoV-2 at a MOI of 0.05 for 3 hours, then washed
542 once with PBS and incubated with fresh media. 24 hours post-infection, cells and 200
543 µL of supernatants were collected for N protein staining and viral RNA quantification,
544 as described in ‘Viral infection quantification assays’. In parallel cells seeded in 96-well
545 format were incubated 24 hours with the drugs to assess cell viability
546
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547 Viral infection quantification assays
548 For infection quantification by flow cytometry analysis, 24 hours post-infection cells
549 were trypsinized and fixed with 2% (v/v) paraformaldehyde (PFA) diluted in PBS for 15
550 min at room temperature. Cells were incubated for 45 min at 4°C with 1 μg/ml of the
551 anti-N mAb diluted in permeabilization flow cytometry buffer (PBS supplemented with
552 5% FBS, 0.5% (w/v) saponin, 0.1% Sodium azide). After washing, cells were incubated
553 with 1 μg/ml of Alexa Fluor 647-conjugated goat anti-mouse IgG diluted in
554 permeabilization flow cytometry buffer for 30 min at 4°C. Acquisition was performed on
555 an Attune NxT Flow Cytometer (Thermo Fisher Scientific) and analysis was done by
556 using FlowJo software (Tree Star). To assess infectious viral particles release during
557 infection by RT-qPCR, viruses were first inactivated by incubating the supernatants v/v
558 with 1% Triton X-100 (Sigma) in PBS for 30 min under agitation at RT. Yields of viral
559 RNA were quantified by real-time qPCR by using SARS-CoV-2 specific primers
560 targeting the E gene with the Luna® Universal One-Step RT-qPCR Kit (New England
561 Biolabs) in a LightCycler 480 thermocycler (Roche) according to the manufacturer’s
562 protocol. The number of viral genomes is expressed as PFU equivalent/ml and was
563 calculated by performing a standard curve with a similarly treated supernatant from a
564 viral stock with a known titer as described by Gordon et al (Gordon et al., 2020b).
565
566 Cell viability assay
567 Cell viability and proliferation were assessed using the CellTiter-Glo 2.0 Assay
568 (Promega). Briefly, cells were plated in 96-well plates (5x103). At specific times, 100 µl
569 of CellTiter-Glo reagent mixed v/v with PBS were added to each well. After 10 min
570 incubation, 90 μl from each well were transferred to an opaque 96-well plate (Cellstar,
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571 Greiner bio-one) and luminescence was measured on a TriStar2 LB 942 (Berthold)
572 with 0.1 second integration time.
573
574 QUANTIFICATION AND STATISTICAL ANALYSIS
575 Statistical analyses
576 Graphical representation and statistical analyses were performed using Prism7
577 software (GraphPad Software). Unless otherwise stated, results are shown as means
578 +/- standard deviation (SD) from at least 2 independent experiments in duplicates.
579 Differences were tested for statistical significance using One-way or Two-way Anova
580 with multiple comparisons post-test.
581
582 Protein functional enrichment was performed in Cytoscape (Version. 3.8.2). Gene
583 Ontology (GO) Biological process were enriched with the ClueGO (Bindea et al., 2009)
584 extension (Version. 2.5.7) using a two-sided hypergeometric test with Bonferroni
585 correction. Adjusted p-values < 0.5 were considered as significant. P-values and
586 percentage of represented genes per GO terms were computed to select the most
587 enriched functional families. This was illustrated with ggplot2 package (Version. 3.3.2)
588 in R Studio interface (Version. 1.2.1335). Most representative terms were represented
589 in networks using the Cytoscape’s Genemania extension (Version. 3.5.2). Comparison
590 of functional enrichment between our datasets and previous studies was performed in
591 Metascape (Zhou et al., 2019b). GO Biological process with p-value < 0.01, enrichment
592 score > 1.5 and overlap = 4 were selected.
593
594 References
595 Banerjee, A.K., Blanco, M.R., Bruce, E.A., Honson, D.D., Chen, L.M., Chow, A., Bhat, P., 596 Ollikainen, N., Quinodoz, S.A., Loney, C., et al. (2020). SARS-CoV-2 Disrupts Splicing,
25 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
597 Translation, and Protein Trafficking to Suppress Host Defenses. Cell 183, 1325- 598 1339.e21. 599 Bell, J.L., Wächter, K., Mühleck, B., Pazaitis, N., Köhn, M., Lederer, M., and Hüttelmaier, S. 600 (2013). Insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs): post- 601 transcriptional drivers of cancer progression? Cell Mol Life Sci 70, 2657–2675. 602 Bindea, G., Mlecnik, B., Hackl, H., Charoentong, P., Tosolini, M., Kirilovsky, A., Fridman, 603 W.-H., Pagès, F., Trajanoski, Z., and Galon, J. (2009). ClueGO: a Cytoscape plug-in to 604 decipher functionally grouped gene ontology and pathway annotation networks. 605 Bioinformatics 25, 1091–1093. 606 Choi, B., Choudhary, M.C., Regan, J., Sparks, J.A., Padera, R.F., Qiu, X., Solomon, I.H., Kuo, 607 H.-H., Boucau, J., Bowman, K., et al. (2020). Persistence and Evolution of SARS-CoV- 608 2 in an Immunocompromised Host. N Engl J Med 383, 2291–2293. 609 Codo, A.C., Davanzo, G.G., Monteiro, L. de B., de Souza, G.F., Muraro, S.P., Virgilio-da- 610 Silva, J.V., Prodonoff, J.S., Carregari, V.C., de Biagi Junior, C.A.O., Crunfli, F., et al. 611 (2020). Elevated Glucose Levels Favor SARS-CoV-2 Infection and Monocyte Response 612 through a HIF-1α/Glycolysis-Dependent Axis. Cell Metabolism 32, 437-446.e5. 613 Cortese, M., Lee, J.-Y., Cerikan, B., Neufeldt, C.J., Oorschot, V.M.J., Köhrer, S., Hennies, J., 614 Schieber, N.L., Ronchi, P., Mizzon, G., et al. (2020). Integrative Imaging Reveals SARS- 615 CoV-2-Induced Reshaping of Subcellular Morphologies. Cell Host & Microbe 28, 853- 616 866.e5. 617 Daniloski, Z., Jordan, T.X., Wessels, H.-H., Hoagland, D.A., Kasela, S., Legut, M., Maniatis, 618 S., Mimitou, E.P., Lu, L., Geller, E., et al. (2020). Identification of Required Host Factors 619 for SARS-CoV-2 Infection in Human Cells. Cell 0. 620 Dennis, G., Sherman, B.T., Hosack, D.A., Yang, J., Gao, W., Lane, H.C., and Lempicki, R.A. 621 (2003). DAVID: Database for Annotation, Visualization, and Integrated Discovery. 622 Genome Biol 4, P3. 623 Dias, S.S.G., Soares, V.C., Ferreira, A.C., Sacramento, C.Q., Fintelman-Rodrigues, N., 624 Temerozo, J.R., Teixeira, L., Silva, M.A.N. da, Barreto, E., Mattos, M., et al. (2020). 625 Lipid droplets fuel SARS-CoV-2 replication and production of inflammatory mediators. 626 PLOS Pathogens 16, e1009127. 627 Eymieux, S., Rouillé, Y., Terrier, O., Seron, K., Blanchard, E., Rosa-Calatrava, M., Dubuisson, 628 J., Belouzard, S., and Roingeard, P. (2021). Ultrastructural modifications induced by 629 SARS-CoV-2 in Vero cells: a kinetic analysis of viral factory formation, viral particle 630 morphogenesis and virion release. Cell. Mol. Life Sci. 631 Flynn, R.A., Belk, J.A., Qi, Y., Yasumoto, Y., Wei, J., Alfajaro, M.M., Shi, Q., Mumbach, 632 M.R., Limaye, A., DeWeirdt, P.C., et al. (2021). Discovery and functional interrogation 633 of SARS-CoV-2 RNA-host protein interactions. Cell S009286742100297X. 634 Garcia-Moreno, M., Noerenberg, M., Ni, S., Järvelin, A.I., González-Almela, E., Lenz, C.E., 635 Bach-Pages, M., Cox, V., Avolio, R., Davis, T., et al. (2019). System-wide Profiling of 636 RNA-Binding Proteins Uncovers Key Regulators of Virus Infection. Molecular Cell 74, 637 196-211.e11. 638 Gélinas, J.-F., Clerzius, G., Shaw, E., and Gatignol, A. (2011). Enhancement of Replication of 639 RNA Viruses by ADAR1 via RNA Editing and Inhibition of RNA-Activated Protein 640 Kinase▿. J Virol 85, 8460–8466. 641 Geuens, T., Bouhy, D., and Timmerman, V. (2016). The hnRNP family: insights into their role 642 in health and disease. Hum Genet 135, 851–867. 643 Gordon, D.E., Jang, G.M., Bouhaddou, M., Xu, J., Obernier, K., White, K.M., O’Meara, M.J., 644 Rezelj, V.V., Guo, J.Z., Swaney, D.L., et al. (2020a). A SARS-CoV-2 protein interaction 645 map reveals targets for drug repurposing. Nature 583, 459–468. 646 Gordon, D.E., Hiatt, J., Bouhaddou, M., Rezelj, V.V., Ulferts, S., Braberg, H., Jureka, A.S.,
26 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
647 Obernier, K., Guo, J.Z., Batra, J., et al. (2020b). Comparative host-coronavirus protein 648 interaction networks reveal pan-viral disease mechanisms. Science. 649 Hafirassou, M.L., Meertens, L., Umaña-Diaz, C., Labeau, A., Dejarnac, O., Bonnet-Madin, L., 650 Kümmerer, B.M., Delaugerre, C., Roingeard, P., Vidalain, P.-O., et al. (2017). A Global 651 Interactome Map of the Dengue Virus NS1 Identifies Virus Restriction and Dependency 652 Host Factors. Cell Reports 21, 3900–3913. 653 Hoffmann, H.-H., Sánchez-Rivera, F.J., Schneider, W.M., Luna, J.M., Soto-Feliciano, Y.M., 654 Ashbrook, A.W., Le Pen, J., Leal, A.A., Ricardo-Lax, I., Michailidis, E., et al. (2021). 655 Functional interrogation of a SARS-CoV-2 host protein interactome identifies unique and 656 shared coronavirus host factors. Cell Host & Microbe 29, 267-280.e5. 657 Jiang, H., Zhang, H., Meng, Q., Xie, J., Li, Y., Chen, H., Zheng, Y., Wang, X., Qi, H., Zhang, 658 J., et al. (2020). SARS-CoV-2 Orf9b suppresses type I interferon responses by targeting 659 TOM70. Cellular & Molecular Immunology 17, 998–1000. 660 Kanehisa, M., and Goto, S. (2000). KEGG: kyoto encyclopedia of genes and genomes. Nucleic 661 Acids Res 28, 27–30. 662 Kemp, S.A., Collier, D.A., Datir, R.P., Ferreira, I.A.T.M., Gayed, S., Jahun, A., Hosmillo, M., 663 Rees-Spear, C., Mlcochova, P., Lumb, I.U., et al. (2021). SARS-CoV-2 evolution during 664 treatment of chronic infection. Nature. 665 Kim, D., Lee, J.-Y., Yang, J.-S., Kim, J.W., Kim, V.N., and Chang, H. (2020). The Architecture 666 of SARS-CoV-2 Transcriptome. Cell 181, 914-921.e10. 667 Landeras-Bueno, S., Jorba, N., Pérez-Cidoncha, M., and Ortín, J. (2011). The Splicing Factor 668 Proline-Glutamine Rich (SFPQ/PSF) Is Involved in Influenza Virus Transcription. PLOS 669 Pathogens 7, e1002397. 670 Lee, S., Lee, Y., Choi, Y., Son, A., Park, Y., Lee, K.-M., Kim, J., Kim, J.-S., and Kim, V.N. 671 (2020). The SARS-CoV-2 RNA interactome. BioRxiv 2020.11.02.364497. 672 Li, C., Ge, L., Li, P., Wang, Y., Sun, M., Huang, L., Ishag, H., Di, D., Shen, Z., Fan, W., et al. 673 (2013). The DEAD-box RNA helicase DDX5 acts as a positive regulator of Japanese 674 encephalitis virus replication by binding to viral 3′ UTR. Antiviral Research 100, 487– 675 499. 676 Li, J., Guo, M., Tian, X., Wang, X., Yang, X., Wu, P., Liu, C., Xiao, Z., Qu, Y., Yin, Y., et al. 677 (2020). Virus-Host Interactome and Proteomic Survey Reveal Potential Virulence 678 Factors Influencing SARS-CoV-2 Pathogenesis. Med S2666634020300155. 679 Liao, K.-C., Chuo, V., Fagg, W.S., Bradrick, S.S., Pompon, J., and Garcia-Blanco, M.A. (2020). 680 The RNA binding protein Quaking represses host interferon response by downregulating 681 MAVS. RNA Biol 17, 366–380. 682 Liu, Z., VanBlargan, L.A., Bloyet, L.-M., Rothlauf, P.W., Chen, R.E., Stumpf, S., Zhao, H., 683 Errico, J.M., Theel, E.S., Liebeskind, M.J., et al. (2021). Identification of SARS-CoV-2 684 spike mutations that attenuate monoclonal and serum antibody neutralization. Cell Host 685 & Microbe 0. 686 Mangus, D.A., Evans, M.C., and Jacobson, A. (2003). Poly(A)-binding proteins: 687 multifunctional scaffolds for the post-transcriptional control of gene expression. Genome 688 Biol 4, 223. 689 McCarthy, K.R., Rennick, L.J., Nambulli, S., Robinson-McCarthy, L.R., Bain, W.G., Haidar, 690 G., and Duprex, W.P. (2021). Recurrent deletions in the SARS-CoV-2 spike glycoprotein 691 drive antibody escape. Science. 692 Mellet, J., and Pepper, M.S. (2021). A COVID-19 Vaccine: Big Strides Come with Big 693 Challenges. Vaccines 9, 39. 694 Mello, C.A., Campos, F.A.B., Santos, T.G., Silva, M.L.G., Torrezan, G.T., Costa, F.D., 695 Formiga, M.N., Nicolau, U., Nascimento, A.G., Silva, C., et al. (2021). Desmoplastic 696 Small Round Cell Tumor: A Review of Main Molecular Abnormalities and Emerging
27 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
697 Therapy. Cancers 13, 498. 698 Naganuma, T., Nakagawa, S., Tanigawa, A., Sasaki, Y.F., Goshima, N., and Hirose, T. (2012). 699 Alternative 3′-end processing of long noncoding RNA initiates construction of nuclear 700 paraspeckles. The EMBO Journal 31, 4020–4034. 701 Nakagawa, S., Yamazaki, T., and Hirose, T. (2018). Molecular dissection of nuclear 702 paraspeckles: towards understanding the emerging world of the RNP milieu. Open Biol 703 8. 704 Oakland, T.E., Haselton, K.J., and Randall, G. (2013). EWSR1 Binds the Hepatitis C Virus cis- 705 Acting Replication Element and Is Required for Efficient Viral Replication. Journal of 706 Virology 87, 6625–6634. 707 Onodi, F., Bonnet-Madin, L., Meertens, L., Karpf, L., Poirot, J., Zhang, S.-Y., Picard, C., Puel, 708 A., Jouanguy, E., Zhang, Q., et al. (2021). SARS-CoV-2 induces human plasmacytoid 709 predendritic cell diversification via UNC93B and IRAK4. Journal of Experimental 710 Medicine 218. 711 Ooi, Y.S., Majzoub, K., Flynn, R.A., Mata, M.A., Diep, J., Li, J.K., van Buuren, N., Rumachik, 712 N., Johnson, A.G., Puschnik, A.S., et al. (2019). An RNA-centric dissection of host 713 complexes controlling flavivirus infection. Nat Microbiol 4, 2369–2382. 714 de la Parra, C., Ernlund, A., Alard, A., Ruggles, K., Ueberheide, B., and Schneider, R.J. (2018). 715 A widespread alternate form of cap-dependent mRNA translation initiation. Nat Commun 716 9. 717 Perrin-Cocon, L., Diaz, O., Jacquemin, C., Barthel, V., Ogire, E., Ramière, C., André, P., 718 Lotteau, V., and Vidalain, P.-O. (2020). The current landscape of coronavirus-host 719 protein-protein interactions. J Transl Med 18, 319. 720 Poland, G.A., Ovsyannikova, I.G., and Kennedy, R.B. (2020). SARS-CoV-2 immunity: review 721 and applications to phase 3 vaccine candidates. The Lancet 396, 1595–1606. 722 Reed, R., and Hurt, E. (2002). A Conserved mRNA Export Machinery Coupled to pre-mRNA 723 Splicing. Cell 108, 523–531. 724 Sabino, E.C., Buss, L.F., Carvalho, M.P.S., Prete, C.A., Crispim, M.A.E., Fraiji, N.A., Pereira, 725 R.H.M., Parag, K.V., Peixoto, P. da S., Kraemer, M.U.G., et al. (2021). Resurgence of 726 COVID-19 in Manaus, Brazil, despite high seroprevalence. The Lancet 0. 727 Schmidt, N., Lareau, C.A., Keshishian, H., Ganskih, S., Schneider, C., Hennig, T., Melanson, 728 R., Werner, S., Wei, Y., Zimmer, M., et al. (2020). The SARS-CoV-2 RNA–protein 729 interactome in infected human cells. Nat Microbiol. 730 Schneider, W.M., Luna, J.M., Hoffmann, H.-H., Sánchez-Rivera, F.J., Leal, A.A., Ashbrook, 731 A.W., Le Pen, J., Ricardo-Lax, I., Michailidis, E., Peace, A., et al. (2020). Genome-Scale 732 Identification of SARS-CoV-2 and Pan-coronavirus Host Factor Networks. Cell 733 S0092867420316767. 734 Schubert, K., Karousis, E.D., Jomaa, A., Scaiola, A., Echeverria, B., Gurzeler, L.-A., 735 Leibundgut, M., Thiel, V., Mühlemann, O., and Ban, N. (2020). SARS-CoV-2 Nsp1 736 binds the ribosomal mRNA channel to inhibit translation. Nature Structural & Molecular 737 Biology 27, 959–966. 738 Shannon, P., Markiel, A., Ozier, O., Baliga, N.S., Wang, J.T., Ramage, D., Amin, N., 739 Schwikowski, B., and Ideker, T. (2003). Cytoscape: a software environment for 740 integrated models of biomolecular interaction networks. Genome Res 13, 2498–2504. 741 Shepard, P.J., and Hertel, K.J. (2009). The SR protein family. Genome Biol 10, 242. 742 Snijder, E.J., Decroly, E., and Ziebuhr, J. (2016). Chapter Three - The Nonstructural Proteins 743 Directing Coronavirus RNA Synthesis and Processing. In Advances in Virus Research, 744 J. Ziebuhr, ed. (Academic Press), pp. 59–126. 745 Stacchiotti, S., Pantaleo, M.A., Astolfi, A., Dagrada, G.P., Negri, T., Dei Tos, A.P., Indio, V., 746 Morosi, C., Gronchi, A., Colombo, C., et al. (2014). Activity of sunitinib in extraskeletal
28 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
747 myxoid chondrosarcoma. European Journal of Cancer 50, 1657–1664. 748 Sun, L., Li, P., Ju, X., Rao, J., Huang, W., Ren, L., Zhang, S., Xiong, T., Xu, K., Zhou, X., et 749 al. (2021). In vivo structural characterization of the SARS-CoV-2 RNA genome identifies 750 host proteins vulnerable to repurposed drugs. Cell. 751 Teo, G., Liu, G., Zhang, J., Nesvizhskii, A.I., Gingras, A.-C., and Choi, H. (2014). 752 SAINTexpress: improvements and additional features in Significance Analysis of 753 INTeractome software. J Proteomics 100, 37–43. 754 Thompson, M.G., Dittmar, M., Mallory, M.J., Bhat, P., Ferretti, M.B., Fontoura, B.M., Cherry, 755 S., and Lynch, K.W. (2020). Viral-induced alternative splicing of host genes promotes 756 influenza replication. ELife 9, e55500. 757 Thomson, E.C., Rosen, L.E., Shepherd, J.G., Spreafico, R., Filipe, A. da S., Wojcechowskyj, 758 J.A., Davis, C., Piccoli, L., Pascall, D.J., Dillen, J., et al. (2021). Circulating SARS-CoV- 759 2 spike N439K variants maintain fitness while evading antibody-mediated immunity. Cell 760 0. 761 V’kovski, P., Gerber, M., Kelly, J., Pfaender, S., Ebert, N., Braga Lagache, S., Simillion, C., 762 Portmann, J., Stalder, H., Gaschen, V., et al. (2019). Determination of host proteins 763 composing the microenvironment of coronavirus replicase complexes by proximity- 764 labeling. ELife 8, e42037. 765 V’kovski, P., Kratzel, A., Steiner, S., Stalder, H., and Thiel, V. (2021). Coronavirus biology 766 and replication: implications for SARS-CoV-2. Nature Reviews Microbiology 19, 155– 767 170. 768 Wang, M., Cao, R., Zhang, L., Yang, X., Liu, J., Xu, M., Shi, Z., Hu, Z., Zhong, W., and Xiao, 769 G. (2020). Remdesivir and chloroquine effectively inhibit the recently emerged novel 770 coronavirus (2019-nCoV) in vitro. Cell Research 30, 269–271. 771 Wang, R., Simoneau, C.R., Kulsuptrakul, J., Bouhaddou, M., Travisano, K.A., Hayashi, J.M., 772 Carlson-Stevermer, J., Zengel, J.R., Richards, C.M., Fozouni, P., et al. (2021). Genetic 773 Screens Identify Host Factors for SARS-CoV-2 and Common Cold Coronaviruses. Cell 774 184, 106-119.e14. 775 Watson, S.F., Bellora, N., and Macias, S. (2020). ILF3 contributes to the establishment of the 776 antiviral type I interferon program. Nucleic Acids Research 48, 116–129. 777 Wei, J., Alfajaro, M.M., DeWeirdt, P.C., Hanna, R.E., Lu-Culligan, W.J., Cai, W.L., Strine, 778 M.S., Zhang, S.-M., Graziano, V.R., Schmitz, C.O., et al. (2020). Genome-wide CRISPR 779 screens reveal host factors critical for SARS-CoV-2 infection. Cell S0092867420313921. 780 de Wilde, A.H., Snijder, E.J., Kikkert, M., and van Hemert, M.J. (2018). Host Factors in 781 Coronavirus Replication. Curr Top Microbiol Immunol 419, 1–42. 782 Wu, C.-H., Chen, P.-J., and Yeh, S.-H. (2014). Nucleocapsid phosphorylation and RNA 783 helicase DDX1 recruitment enables coronavirus transition from discontinuous to 784 continuous transcription. Cell Host Microbe 16, 462–472. 785 Xu, L., Khadijah, S., Fang, S., Wang, L., Tay, F.P.L., and Liu, D.X. (2010). The cellular RNA 786 helicase DDX1 interacts with coronavirus nonstructural protein 14 and enhances viral 787 replication. J Virol 84, 8571–8583. 788 Yang, P., Mathieu, C., Kolaitis, R.-M., Zhang, P., Messing, J., Yurtsever, U., Yang, Z., Wu, J., 789 Li, Y., Pan, Q., et al. (2020). G3BP1 Is a Tunable Switch that Triggers Phase Separation 790 to Assemble Stress Granules. Cell 181, 325-345.e28. 791 Zhou, B., Wu, F., Han, J., Qi, F., Ni, T., and Qian, F. (2019a). Exploitation of nuclear protein 792 SFPQ by the encephalomyocarditis virus to facilitate its replication. Biochemical and 793 Biophysical Research Communications 510, 65–71. 794 Zhou, P., Yang, X.-L., Wang, X.-G., Hu, B., Zhang, L., Zhang, W., Si, H.-R., Zhu, Y., Li, B., 795 Huang, C.-L., et al. (2020). A pneumonia outbreak associated with a new coronavirus of 796 probable bat origin. Nature 579, 270–273.
29 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
797 Zhou, Y., Zhou, B., Pache, L., Chang, M., Khodabakhshi, A.H., Tanaseichuk, O., Benner, C., 798 and Chanda, S.K. (2019b). Metascape provides a biologist-oriented resource for the 799 analysis of systems-level datasets. Nat Commun 10, 1523. 800 Zhu, N., Zhang, D., Wang, W., Li, X., Yang, B., Song, J., Zhao, X., Huang, B., Shi, W., Lu, R., 801 et al. (2020). A Novel Coronavirus from Patients with Pneumonia in China, 2019. N Engl 802 J Med 382, 727–733. 803
30 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
A In vivo crosslinking B Streptavidin bead Input (0.1%) ChIRP
Sonication Hybridization Nucleus MW Pull Down UninfectedInfected UninfectedInfected (kDa) Viral RNA 185 115 293T-ACE2 cells 129 Biotinylated Biotin 80 Infected with SARS-CoV-2 tiling oligos elution 65
N 50 Viral RBP RNA binding protein (RBP) complex Identification by LC-MS/MS 30 Host Factor RBP 25
Proteins complexed to RBP 15 10
C D
8 N Unique peptide 6 NSP1 NSP3 NSP5 NSP8 NSP14 S M N 4 pp1ab 2/Mock) - M NSP2 NSP9 NSP13 NSP16 3a 6 9b ORF9b
CoV 2 S - Number of unique peptide/ viral protein 0 Structural Non-structural proteins Auxiliary proteins proteins Peptides enrichment Peptides
log2 (SARS -2 NSP1 NSP2 NSP3 NSP5 NSP8 NSP9 NSP13 NSP14 NSP16 S M N ORF3a ORF6 ORF9b
2 1 11 2 1 2 1 1 1 31 5 49 1 1 4 -4
E
HNRNPL HNRNPA0 HSPA4 RRBP1 G3BP1 HDLBP FLNB CAPRIN1 MATR3 IGF2BP1 EWSR1 DDX3Y HNRNPM HNRNPH1 RALY EIF3F IGF2BP3 UBA1 ATXN2L STRAP PABPC1 SFPQ PARK7 XRCC5 NONO HNRNPA2B1 NUDT21 RPS16 SYNCRIP SND1 SAFB SARNP ILF3 USO1 POLDIP3 TARDBP PTBP1 PABPC4 KPNA2 PURA CCAR2 PCBP1 ADAR PDLIM1 HNRNPH3 XRCC6 LARP4B EIF4H DDX5 ALDOA ST13 FASN PRKDC ELAVL1 HNRNPD RPS20 RTCB MAP4 TRIM28 ZFR FUBP1 HNRNPF PA2G4 USP10 DHX9 UBAP2L DAZAP1 STIP1 RBMX FLNA FAM98A DDX6 DDX1 KPNB1 HSP90AB1 RPL12 HNRNPA1L2 LDHB CBR1 NUFIP2 SRSF1 PARP1 HNRNPUL2 PPIA HNRNPR HNRNPC RPS3A CRKL FUS KHSRP C14ORF166 MRPL22 UPF1 PRRC2A HNRNPUL1 CCT8 FAM120A ZC3HAV1 EIF4G1 NTPCR MOV10 HNRNPDL TAGLN2 BUB3 HNRNPH2 HNRNPCL2 EEF2 SRSF3 FUBP3 PGK1 PCBP2 SERBP1 IGF2BP2 RPS19 SRSF7 YBX1 HNRNPA3 MTHFD1 CCT2 LARP1B G3BP2 HNRNPK RANGAP1 RPS10P5 RBM14 DDX17 TIA1 RACK1 GPI QKI SNRPD2 YBX3 HNRNPA1 ZNF326 RBM4 NCL ILF2 HNRNPU RAN RPS3 0 2 4 6 0 1 2 3 4 5 0 1 2 3 4 5 0 1 2 3 4 5 Fold Change FC-A score Fold Change FC-A score Fold Change FC-A score Fold Change FC-A score (SARS-CoV-2/Mock) (SARS-CoV-2/Mock) (SARS-CoV-2/Mock) (SARS-CoV-2/Mock)
Figure 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
A B 50 RNA metabolic process RNA Splicing
40
RNA catabolic process log10)
- 30 Translation
RNA stabilization Viral process Value (
- 20
Adj. P RNA localization 10 Ribonucleoprotein complex assembly
Stress granule assembly 20 40 60 Percentage of selected genes
C
RNA trafficking Translation process Regulation of RNA stability
Immune response Virus biology Biology of stress granule
Figure 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
A SARS-CoV-2 PEDV IBV
SARS-CoV TGEV MHV NSP15 NSP1 HCoV-OC43
HNRNPD HNRNPF PPIA HNRNPA1
SYNCRIP HDLBP USP10 S PABPC1 RBM14 FUS IGF2BP1 LARP4B
SERBP1 ATXN2L XRCC6
XRCC5 NSP12 UBAP2L EIF3F FLNA YBX1
NCL RPS19 G3BP1
NSP9 KPNA2 G3BP2 EIF4H PABPC4 HNRNPU ORF6 NSP2 MOV10 FAM98A KPNB1 UPF1 DHX9
HNRNPA0 HNRNPA3 HNRNPA2B1 HNRNPC HNRNPDL HNRNPH1 CCT8 STIP1 RRBP1 DDX1 N
ORF9B HNRNPH3 HNRNPK HNRNPL HNRNPM HNRNPR SFPQ NUFIP2 EIF4G1 DDX3Y
CRKL RANGAP1 NSP13 DDX5 DDX6 DDX17 SRSF1 SRSF3 SRSF7 PCBP2 RPS20 NSP14
RPS3 RPS3A RPS16 RPL12 RTCB RBMX
NONO CAPRIN1 YBX3 RTRAF ELAVL1 SND1
ILF2 ILF3 RACK1 IGF2BP3 PARP1 PTBP1
B C
Glucose-6P ns GPI 5 Fructose-6P 4 Fructose-1,6P
3 ALDOA FC
vs Control) Control) vs Glyceraldehyde 3P 2 Fold Change ChIRP ( 1 PGK1
0 3P-Glycerate YES NO known coronavirus interactors LDHB FASN Lactate Pyruvate Fatty acids
Figure 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.03.23.436611; this version posted March 23, 2021. 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-NC-ND 4.0 International license.
A SARS-CoV-2 inoculation siRNA transfection of 293T- and A549-ACE2 cells Level of infection by Flow Cytometry
Supernatant Virus quantification for viral titers (SARS-CoV-2 RT-qPCR)
Cell Viability
-48h 0h 30h
B C Viral titer (RT-qPCR) Replication (Flow cytometry) 10 16 EEF2 CTSL ATP6V1B2
14 ILF3 TRIM28 HNRNPK HNRNPU 8
12 CTSL EWSR1 DDX1 ADAR 10 DDX5 6 log10) log10) FAM120A - - SRSF3 HNRNPC G3BP2 DDX17 ATP6V1B2 8 QKI XRCC5
PA2G4 ( Value Value ( Value - - SFPQ CCT2 4 KPNB1 6 HNRNPK XRCC5 GPI HNRNPA3 HNRNPUL2 Adj. P Adj. Adj. P Adj. PPIA EWSR1 USO1 4 PPIA HNRNPA1L2 G3BP2 ADAR 2 UBA1 ATXN2L EIF4G1 ILF3 2 TRIM28 MAP4 HNRNPA2B1 FAM120A DDX5 HNRNPH3 0 0 -7 -5 -3 -1 1 3 -8 -6 -4 -2 0 2 4 Ratio of viral titers Ratio of infection log2 (siTarget/siNT) log2 (siTarget/siNT) D E
2 Remdesivir 120 (Spearman r=0.742 p< 0.0001) PPIA G3BP1 EIF4G1 TRIM28 100
SARNP 1 PRRC2A 80 KPNB1 60 Viability USO1 Percent 40 Viral RNA log2 (Infection Ratio) HNRNPH3 0
-4 -3 -2 -1 0 1 2 (relative to DMSO) 20 ATXN2L 0 IC50= 0.016 μM HNRNPUL2 HNRNPA2B1 HNRNPH1 -1 0.01 0.1 1 10 IGF2BP3 Concentration (µM) HSPA4 YBX1 XRCC5 CCT2 Sunitinib malate 120 PA2G4 -2 HNRNPR SFPQ 100
ADAR 80 QKI -3 60 Viability EWSR1 DDX1 Percent 40 Viral RNA DDX5 SRSF3 -4 (relative to DMSO) 20
FAM120A 0 HNRNPU IC50= 0.49 μM ILF3 0.01 0.1 1 10 HNRNPK log2 (viral titer Ratio) Concentration (µM) -5 Figure 4 This study Flynn et al. (ChIRP-M/S in 293T-ACE2 cells) (ChIRP-M/S in Huh7.5 cells) Core set of SARS-CoV-2 RNA-binding proteins DAZAP1 DDX17 DDX5 DHX9 EIF4H ELAVL1 EWSR1 FAM120A FAM98A 19 FUBP1 FUBP3 FUS 49 119 G3BP1 G3BP2 HDLBP HNRNPA0 58 HNRNPA1 HNRNPA2B1 HNRNPA3 HNRNPC 16 32 HNRNPD HNRNPDL HNRNPH1 HNRNPH3 HNRNPK HNRNPL 83 HNRNPM HNRNPR IGF2BP2 IGF2BP3 ILF2 ILF3 KHSRP MAP4 MATR3 MOV10 NONO PA2G4 PABPC1 Lee et al. PABPC4 (RAP-M/S in VeroE6 cells) PCBP2 POLDIP3 PTBP1 PURA QKI RALY RBM14 RBMX SFPQ SND1 SRSF1 SRSF3 SRSF7 SYNCRIP TARDBP YBX1 YBX3 ZC3HAV1
Figure S1 A
ns 140 120 ns ns ns 10 μM ns 120 ns ns 10 μM ns ns ns ns 100 ns ns ns * 1 μM 1 μM ** 100 *** **** 80 **** 80 ** **** 60 **** 60 **** Virus titer Virus **** **** 40 **** viability Cell 40 **** (relative to DMSO) **** (relative to DMSO) 20 **** 20
**** **** 0 **** 0
malate malate Cerulenin Cerulenin RemdesivirArtemisinin Dabigatran RemdesivirArtemisinin Dabigatran Camptothecin Geldanamycinisothiocyanate Camptothecin Geldanamycinisothiocyanate Sunitinib Sunitinib
Phenethyl Phenethyl B
Remdesivir Sunitinib malate 120 120 N protein N protein 100 100 Viability Viability 80 80
60 60 Percent Percent 40 40 (relative to DMSO) (relative to DMSO) 20 20
0 0 IC50= 0.035 μM IC50= 0.38 μM 0.01 0.1 1 10 0.01 0.1 1 10 Concentration (µM) Concentration (µM)
C
Remdesivir Sunitinib malate 120 120 Viability Viability 100 100 Viral RNA Viral RNA 80 80
60 60 Percent Percent 40 40 (relative to DMSO) (relative to DMSO) 20 20
0 0 IC50= 0.035 μM IC50= 0.78 μM 10-2 10-1 100 101 0.01 0.1 1 10 Concentration (µM) Concentration (µM)
Figure S2