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Orthogonal genome-wide screenings in bat cells identify MTHFD1 as a target of broad antiviral therapy

Bats are responsible for the zoonotic transmission of several major viral diseases including the 2003 SARS outbreak and the ongoing COVID-19 pandemic. While bat genomic sequencing studies have revealed characteristic adaptations of the innate immune system, functional genomic studies are urgently needed to provide a foundation for the molecular dissection of the tolerance of viral infections in bats. Here we report the establishment and screening of genome-wide RNAi library and CRISPR library for the model megabat, Pteropus Alecto. We used the complementary RNAi and CRISPR libraries to interrogate Pteropus Alecto cells for infection with two different viruses, mumps virus and Influenza A virus, respectively. Screening results converged on the endocytosis pathway and the protein secretory pathway as required for both viral infections. Additionally, we revealed a general dependence of the C-1-tetrahydrofolate synthase gene, MTHFD1, for viral replication in bat cells as well as in human cells. MTHFD1 inhibitor carolacton potently blocked replication of several RNA viruses including SARS-CoV-2. Our studies provide a resource for systematic inquiry into the genetic underpinnings of bat biology and a potential target for developing broad spectrum antiviral therapy.

viruses, causing the epidemics of the Ebola 1 , SARS 2 , Henipavirus 3 , MERS 4 and COVID-19 5,6 . 20 Bats are widely considered the largest reservoir of numerous virus species but are resilient to the 21 diseases associated with many of these viruses 7,8 . How bats tolerate the infections with these 22 viruses is of intense interest due to the scientific and public health relevance. Genomic 23 sequencing studies have revealed several genetic adaptions underlying this tolerance of viral 24 infections. For example, the DNA damage checkpoint pathway and the NF-kB pathway are 25 known to be positively selected in the bat genomes 9 . In addition, in the type I interferon gene 26 family, the MHC class I genes and natural killer cell receptor family have been reported to be 27 highly expanded in the bat genomes 10 . These studies provided evidence to support the hypothesis 28 that bats have evolved a highly specific genetic configuration, especially in the innate immunity 29 pathways. A major caveat is the lack of systematic examination of the functions of these genes 30 due to shortage of functional genomic tools. 31 CRISPR and RNAi technologies have enabled genome-wide screening of gene functions for a 1 variety of cell types, yielding an unprecedented wealth of information revealing the complexity of 2 biological processes 11,12 . This is especially true in the field of virus-host interactions, where a 3 plethora of host factors have been identified for a variety of viruses [13][14][15] . Both technologies have 4 advantages and disadvantages and provide complementary approaches to achieve comprehensive 5 genetic perturbations [16][17][18] . CRISPR permits complete knockout of a gene, thus affords generation 6 of truly null genotype. A caveat is that the lethality associated with knockout of essential genes 7 prevents functional parsing of these genes in different biological processes. In addition, CRISPR 8 screening is usually used a pool-based format that requires the generation of stable cell lines 9 through long term culturing. Therefore, CRISPR is suited for studying phenotypes that require a 10 longer time to manifest. Based on previous studies, CRISPR screens demonstrated excellent 11 specificity but limited sensitivity 17 . In contrast, siRNA screening relies on temporary knockdown 12 of genes in an individual well format, a methodology suited for examining short-term phenotypic 13 changes. Due to the transient nature of the assay, siRNA screening can tolerate growth defects due 14 to gene knockdown and can elucidate hypomorphic phenotypes induced by siRNAs with varying 15 degrees of knockdown. In general, RNAi screens allow comprehensive identification of genes in 16 a biological process at the cost of more false positives due to pervasive off-target effects 17 . To fully 17 realize the power of CRISPR and RNAi screening as complementary strategies to interrogate gene 18 function in bats, here we developed a pool-based CRISPR library and a plate-based siRNA library 19 for the whole genome of the model megabat Pteropus Alecto. We screened two viruses, influenza 20 A virus and mumps virus, using the CRISPR and RNAi libraries, respectively, in the hope of 21 uncovering common host factors for different viruses. From these two screens, common pathways 22 such as the endocytosis pathway and protein secretory pathway have been identified, 23 demonstrating conserved pathways between bat and other mammals in hosting viral infections. 24 We extensively characterized a common hit from the two screens, MTHFD1, and identified it as a 25 critical factor for viral replication in both bats and humans. We further demonstrate that MTHFD1 26 is a potential target for developing broad-spectrum antiviral drugs. MTHFD1 inhibitor carolacton 27 demonstrated broad antiviral activities against Zika virus, mumps virus, and importantly, SARS-28 CoV-2. In summary, we developed two orthogonal libraries for genome-wide loss-of-function 29 screening in bats and demonstrated their utility in discovery of important host factors for viral 30 infections. 31

Design and validation of genome-wide CRISPR of P. Alecto 1
We designed and synthesized the CRISPR and RNAi libraries based on the genome of P. alecto. 2 For the CRISPR library, we adopted the algorithm developed for the construction of the popular 3 GeCKO library to select single guide RNAs (sgRNAs) 19 . We designed 85,275 sgRNAs targeting 4 21,334 genes annotated in the P. alecto genome with 21,330 genes with 4 sgRNAs for each gene. 5 The other 34 genes have 1-3 sgRNAs due to the constraints of the gene sequences for designing 6 sgRNAs. Selection of sgRNAs was based on the calculations of off-target scores to lower potential 7 off-target effects of the sgRNAs. The sgRNAs designs were validated for 21 sgRNAs targeting 11 8 genes using T7E1 endonuclease cleavage assay or Western blotting. We observed 90.5% of 9 effectiveness in gene editing or gene knockdown, supporting the quality of the designed library 10 (Supplementary Figure 1). 11

CRISPR screening identified influenza A virus host factors 13
We performed a CRISPR screen with influenza A virus in PaKi cells to identify viral host factors 14 (Fig. 1a). PaKi cells stably expressing SgCas9 and sgRNA library were infected with the H1N1 15 PR8 strain of influenza A virus, in duplicate. We used a high multiplicity-of-infection (MOI) so 16 that naïve PaKi cells all died before the 9th day post infection. We then collected the surviving 17 cells and compared sgRNA abundance with mock-infected cells. The two replicates demonstrated 18 a high correlation, indicating high reproducibility (Supplementary Fig. 2). We calculated the 19 results using the RIGER algorithm and identified 21 host factors required for the viral infection or 20 pathogenesis 20 (Fig. 1b). Three of the top four hits belong to the V-type proton ATPase subunits 21 (ATP6V1B2, ATP6V1F and ATP6V0D1), which are involved in the acidification of endosome 22 and have previously been previous identified as key host factors for influenza virus entry in several 23 screens performed in human cells (Fig. 1b) 21 . Among the other hits were two genes, COPZ1 and 24 SEC23B, involved in the protein secretory pathway, which are also known to be required for the 25 replication of many viruses including influenza virus and flavivirus (Fig. 1b) [21][22][23] . The enrichment 26 of previously known host factors in the identified hits in the CRISPR screen validated our library 27 and the screening methodology. 28 29

RNAi screen results converge with CRISPR screening on key pathways 30
We synthesized the sgRNA library in an array-based oligonucleotide library and cloned them into 1 the lentiGuide-Puro lentivirus expression vector 24 . A custom bat siRNA library was designed to 2 target 18,328 genes using 40,016 siRNA duplexes. We performed RNAi screening in PaKi cells 3 for identification of host factors for the infection of mumps virus, a paramyxovirus (Fig. 1c). The 4 screen was performed in 384-well format with a mumps virus strain expressing EGFP from an 5 additional open reading frame 25 . PaKi cells were infected 48 hours after transfection of the siRNA 6 library. High-content imaging analysis was utilized to quantitate the infection rate as well as the 7 total cell number each well, indicating cytotoxicity of the siRNA. Excluding siRNAs with 8 significant cytotoxicity, we selected 45 host dependency factors whose knockdown resulted in 9 significantly reduced viral infection and 45 host restriction factors whose knockdown promoted 10 viral infection based on the Z score of infection rate (Fig. 1d). We selected a number of genes for 11 individual validation with more replicates and obtained a high validation rate (11/12 of host 12 dependency factors and 7/12 of the host restriction factors validated with at least one siRNA) 13 ( Supplementary Fig. 3), supporting the reproducibility of our screening methodology. 14 15 Although there is only one gene overlap between the host factors identified in both CRISPR 16 and RNAi screens, we observed that the endocytosis pathway and protein secretory pathway were 17 enriched in both screens, albeit represented by different genes (Fig. 1b and 1d). For the 18 endocytosis pathway, two genes encoding components of clathrin complex, CLTA and CLTC, 19 were among the top three hits (Fig. 1d). Four genes involved in the protein secretory, namely 20 ARF1, ARF4, COPG1 and SEC61B, were identified (Fig. 1d). This is consistent with the idea that 21 both influenza A virus and mumps require the two pathways for viral replication. In addition, 22 protein translation initiation, which was previously reported to be required for influenza A 23 infection 21 , was also represented in the top hits by three genes: EIF3B, EIF3G and EIF4A3. 24 Conversely, RNAi screening revealed genes that restrict mumps infection in PaKi cells. Among 25 the top hits were several known antiviral genes, TRIM39, TRIM43 26 and NAMPT 27 . Overall, we 26 identified many candidate genes that might play a role in viral infection in bats. 27

28
The two pathways identified by both CRISPR screen and siRNA screen, endocytosis and 29 protein secretion, have previously been implicated in human host response to a number of viral 30 pathogens 13, 21,23 To validate the role of these pathways in the bat response to viral infection, we 31 established PaKi stable cell lines expressing shRNAs targeting several hits from the two screens. 1 Stable knockdown of a V-type proton ATPase subunit and SEC23B led to a significant decrease 2 of influenza A virus infection in PaKi cells ( Fig. 1e and 1f). In addition, the small molecule 3 inhibitor of V-type proton ATPase, bafilomycin A (Fig. 1g), potently blocked influenza infection. 4 Taken together, these results demonstrate that bat and human cells share key genes and pathways 5 in hosting viral infection. 6 7 MTHFD1 is a key host factor for a broad spectrum of RNA viruses 8 The gene identified as a host factor from both the CRISPR and RNAi screen was MTHFD1 ( Fig.  9 1c and 1d), which we validated by siRNA knockdown in the context of mumps infection 10 (Supplemental Fig. 3

). We validated its function in influenza A virus replication in PaKi cells 11
with two different sgRNAs targeting MTHFD1 ( Fig. 2a-2b). Similarly, in HEK293T cells with 12 MTHFD1 knocked out by two sgRNAs, influenza replication is also inhibited, suggesting a 13 conserved requirement for this gene by influenza virus in human cells (Fig. 2c). Further virus 14 titration assay demonstrated that MTHFD1 knockdown significantly inhibits viral replication for 15 mumps virus in PaKi cells ( Fig. 2d and 2e). The effect is similar to the knockdown of SEC61B, a 16 well-studied host protein required for viral replication and a top hit in our RNAi screen ( Fig. 2d  17 and 2e) 23 . We additionally tested Melaka virus (PRV3M), a pteropine orthoreovirus that causes 18 acute respiratory disease when transmitted by bats to humans 28 . PRV3M titer was also significantly 19 reduced by MTHFD1 knockdown (Fig. 2e and 2f). Infection of Zika virus, a member of the 20 flavivirus family, was also inhibited by MTHFD1 knockdown using siRNA (Fig. 2g). Alternative 21 knockdown with stable expression of two shRNAs validated the requirement of MTHFD1 for the 22 replication of mumps virus and Zika virus (Fig. 2h, 2i and 2j). Importantly, we also show that this 23 inhibition is completely rescued by overexpression of MTHFD1 from a construct resistant to the 24 shRNA, ruling out potential off-target effects (Fig. 2k). 25 26

MTHFD1's formyl tetrahydrofolate synthetase activity is essential for RNA virus replication 27
MTHFD1 is a trifunctional enzyme involved in the one carbon (C1) metabolism pathway, which 28 is responsible for cellular production of purine, dTMP and methyl groups 29 . MTHFD1 has three 29 enzymatic functions including dehydrogenase and cyclohydrolase activities encoded in the N-30 terminal domain and formyl tetrahydrofolate (Formyl THF) synthetase activity encoded in the C-31 terminal domain ( Fig. 3a and 3b). We suspected that MTHFD1 knockdown would lead to the 1 deficiency of cellular purine levels thereby reducing RNA replication. Indeed, supplementation of 2 purine analogs, either hypoxanthine or inosine in the media almost completely rescued the 3 replication of influenza A and Zika viruses in MTHFD1-knockout or knockdown PaKi cells as 4 shown by both immunofluorescence staining and virus tittering assay (Fig. 3c, 3d and 3e). In 5 contrast, supplementation of methyl-group donor or thymidine had little effect on viral replication, 6 evidence that it is the purine synthesis activity of MTHFD1 that is essential for the viral replication 7 (Fig. 3d). To further pinpoint the enzymatic function of MTHFD1 in viral replication, we 8 performed gene complementation experiments in an shRNA cell line with over 90% knockdown 9 of MTHFD1 (Fig. 2i). The block of Zika virus replication in this cell line was completely rescued 10 by overexpression of an shRNA-resistant MTHFD1 construct or bat homolog ( Fig. 3f and 3g). 11 Mutations of MTHFD1 that are known to disrupt its dehydrogenase and cyclohydrolase activities 12 (D125A and R173C) 30,31 did not affect this rescue (Fig. 3b, 3f and 3g). However, mutations in the 13 Formyl THF synthetase active site (F759A/W788A, or FW/AA) 32 completely abolished this rescue, 14 further supporting that the purine synthesis activity of MTHFD1 is an essential activity for viral 15 replication (Fig. 3b, 3f and 3g). 16

17
We observed a similar dependence on MTHFD1 function for influenza A virus infection in 18 HEK293T cells, a human kidney epithelial cell line (Fig. 3h). Time course experiments 19 demonstrated a viral replication following MTHFD1 knockdown steadily decreased from four 20 hours post-infection with influenza A virus, consistent with a RNA replication defect (Fig. 3i). 21 This defect in replication was completely rescued by supplementing purine analog inosine in the 22 media, supporting a conserved function of MTHFD1 in viral infection in bats and humans (Fig.  23   3h and 3j, Supplementary Fig. 4). 24

MTHFD1 inhibitor carolacton inhibits RNA virus replication 26
Carolacton, a natural product derived from bacteria, has recently been identified as a potent 27 inhibitor of MTHFD1 33 . We tested the effect of this compound on viral replication. Carolacton 28 inhibited Zika virus and mumps virus replication in PaKi cells in a dose dependent fashion (Fig.  29   4a, 4b and 4c). The inhibition was achieved at doses with little cytotoxicity (Fig. 4a). 30 Mechanistically, this inhibition was completely rescued by supplementation of inosine, providing 31 support that carolacton indeed exerts antiviral activities by inhibiting MTHFD1 (Fig. 4b and 4c). 1

We next tested the effect of carolacton on the infection of SARS-CoV-2. The viral infection in 2
Vero E6 cells was effectively inhibited at sub-micromolar concentrations by carolacton (IC 50 =0.14 3 µM) (Fig. 4d). The cytotoxic effect is very moderate in comparison (Fig. 4d), supporting a 4 therapeutic window of this compound in potential clinical applications. 5 6 Discussion 7 We developed the first bat genome-wide RNAi and CRISPR libraries and performed two screens 8 with two different RNA viruses. Although the screening methodologies are quite different, we 9 uncovered similar pathways essential for viral replication, namely the endocytosis pathway and 10 protein secretory pathways. The different genes in these two pathways identified from the screens 11 reflect the complementarity of the methodologies, with RNAi screening useful for observing acute 12 effects on viral infection and CRISPR screening for long-term effects on cell survival. Other than 13 technical differences between screening methods, viral kinetics, sensitivity of the viruses to loss-  Fig. 5a, 5b and 5c). This might be related to the difference in the promoter 26 regions between bats and humans, notably a lack of CCAAT box 34 in the promoter of two bat 27 species (Supplementary Fig. 5d). We show that MTHFD1 knockdown blocks replication of 28 multiple RNA viruses, including influenza virus, mumps virus, Melaka virus and Zika virus. 29 Importantly, host cells have a higher tolerance for MTHFD1 inhibition than viruses, potentially 30 providing a therapeutic window for targeting MTHFD1 with antiviral drugs. Virus infection 31 converts the whole cellular machinery into a factory to massively produce progeny viruses, which 1 explains the vital dependence on MTHFD1. This is analogous to cancer cell's addiction to non-2 oncogenic gene functions such as many metabolic enzymes 35 . Similar to the therapeutic targeting 3 of these non-oncogenic addictions for developing cancer drugs, these viral addictions to metabolic 4 enzymes such as MTHFD1 could open doors to many new opportunities for developing broad-5 spectrum antiviral drugs 36 . These drugs could be readily applied in emerging epidemics, as 6 demonstrated by our evidence here that MTHFD1 inhibitor carolacton can suppress the infection 7 of SARS-CoV-2. We envision the functional genomics tools we provided here will be instrumental 8 to further discoveries by better understanding the bat-virus interaction.   De-multiplexed reads were trimmed by Cutadapt 2 (version 1.18) using 22 bp flanking 23 sequences around the guide sequence. Trimmed reads were mapped to the index of bat sgRNA 24 guide library using Bowtie2 3 . Samtools 4 (version 1.9) was used to process the alignment files to 25 retain perfectly mapped reads only, and a custom-made Python script was used to count the reads 26 mapped to each guide. The reads count of guide for each condition were then normalized using 27 DEseq2. The log2 ratio of counts between conditions were calculated. The log2 fold change of 28 reads count of infected samples to mock infected samples were then used as the input for RIGER 29 algorithm as previously described (RNAi Gene Enrichment Ranking, GENE-E) 1 . The log-fold-30 change metric for ranking sgRNAs and the weighted sum method (weighted sum of first two ranks 31 of sgRNAs for a gene) with 1 × 10 6 permutations were used to convert individual sgRNA to genes. 32 In the output of RIGER analysis, a given gene was assigned a normalized enrichment score (NES) 1 to give the ranked gene list. Hits were defined as those owning p-value smaller than 0.001. imaging (Operetta) as previously described 5 . The siRNA targeted genes were ranked by robust Z 8 scores 6 , and the potential host factors (Z score < -3) or restriction factors (Z score > 6) were 9 identified. 10 11

Validation of sgRNA 12
Single sgRNA was cloned into lentiGuide-puro and packaged in lentivirus, which was 13 transduced into Cas9 stably expressing PaKi cells, followed by puromycin and blasticidin selection. 14 The clonal cells were generated by limiting dilution. The knock out or knock down efficiency was 15 tested by Western blotting. To facilitate the growth of single clone MTHFD1 knock out cells, the 16 cells were cultured with HT medium (DMEM supplemented with 10% FBS, 100µM hypoxanthine 17 and 16µM thymidine).The knock out or knock down efficiency was tested by Western blotting. 18 Validated knockout cells were applied to virus infection assays. For MTHFD1 knock out cells, the 19 HT was removed 8h before virus infection. 20 21

Virus titration 16
Approximately 1x10 4 PaKi cells were plated into each well of 96-well plates. The 17 supernatant containing virus harvested from previously infected cell was subjected to ten-fold 18 serial dilution. Then dilutions were added to the cells and cytopathic effect was observed 96 h after 19 infection. Titres were determined using standard methodology 7 .