key: cord-0869013-vscjyep8
authors: Devignot, Stephanie; Sha, Tim Wai; Burkard, Thomas; Schmerer, Patrick; Hagelkruys, Astrid; Mirazimi, Ali; Elling, Ulrich; Penninger, Josef M.; Weber, Friedemann
title: Low Density Lipoprotein Receptor-Related Protein 1 (LRP1) is a host factor for RNA viruses including SARS-CoV-2
date: 2022-02-18
journal: bioRxiv
DOI: 10.1101/2022.02.17.480904
sha: 61ad074ad65f52a78e4245445f7a6d41dc94f335
doc_id: 869013
cord_uid: vscjyep8

Viruses with an RNA genome are the main causes of zoonotic infections. In order to identify novel pro-viral host cell factors, we screened a haploid insertion-mutagenized mouse embryonic cell library for clones that rendered them resistant to the zoonotic Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales). This screen returned the Low Density Lipoprotein Receptor-Related protein 1 (LRP1, or CD91) as top hit, a 600 kDa plasma membrane protein known to be involved in a wide variety of cell activities. Inactivation of LRP1 expression in human cells reduced RVFV infection at the early stages of infection, including the particle attachment to the cell. In the highly LRP1-positive human HuH-7 cell line, LRP1 was required for the early infection stages also of Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), vesicular stomatitis (VSV; family Rhabdoviridae, order Mononegavirales), Encephalomyocarditis virus (EMCV, family Picornaviridae), and the coronaviruses MERS-CoV, SARS-CoV-1, and SARS-CoV-2. While for RVFV, EMCV, and MERS-CoV the replication cycle could eventually catch up, LRP1 requirement for the late infection stage in HuH-7 cells was observed for SFSV, La Crosse virus (LACV; family Peribunyaviridae, order Bunyavirales), VSV, SARS-CoV-1, and SARS-CoV-2. For SARS-CoV-2, the absence of LRP1 stably reduced viral RNA levels in human lung Calu-3 cells, and both RNA levels and particle production in the hepatic HuH-7 cells. Thus, we identified LRP1 as a host factor that supports various infection cycle stages of a broad spectrum of RNA viruses.

Most pandemics, epidemics, and zoonotic spillover infections are caused by enveloped RNA viruses (1-3). These pathogens contain an RNA genome of positive-sense or negative-sense polarity that is encapsidated by a viral nucleoprotein and surrounded by a lipid bilayer containing transmembrane glycoproteins. Although RNA viruses are phylogenetically very diverse, it is conceivable that all are depending on common basic factors of the cell.

Rift Valley fever virus (RVFV; family Phleboviridae, order Bunyavirales) is an emerging zoonotic negative-strand RNA virus (4) that is listed by the WHO among the pathogens posing the greatest public health risk (2) . Using RVFV as a model, we aimed to identify host cell factors supporting the replication cycle of RNA viruses. An insertion-mutagenized haploid cell library was screened for clones with resistance to the highly cytopathogenic RVFV as an indication that the deleted gene is essential for viral replication. As top ranking hit emerged the Low Density Lipoprotein Receptor-Related protein 1 (LRP1), a large plasma membrane receptor that can bind and internalize more than 40 different ligands.

Indeed, a lack of LRP-1 reduced the ability of RVFV to attach to the cell surface and enter the cytoplasm, whereas RNA levels seemed not to be affected. Subsequent experiments using human HuH-7 knockout cells revealed that LRP1 is also a host cell factor for other, RVFV-related and -unrelated RNA viruses, most prominently human pathogenic coronaviruses SARS-CoV-1 and SARS-CoV-2. Depending on the particular virus, however, LRP1 was important for different infection steps ranging from particle attachment to late-stage viral RNA synthesis. Our study may thus establish LRP-1 as a broad-band host factor for many RNA viruses that acts not exclusively on attachment but in some cases at later stages of the replication cycle. Moreover, we show that SARS-coronaviruses are among the most LRP1dependent viruses.

As RVFV is highly cytolytic, we devised a forward genetic screen that is based on the positive selection of cells deficient in pro-viral genes. A genome-wide library of knockout haploid mouse embryonic stem cells (mESCs), derived from parthenogenetic mouse embryos, was generated by mutagenizing with a retroviral genetrap (Fig. 1A) that disrupts genes in a revertible manner (5) . Altogether 5*10E8 cells were mutagenized by transduction with a genetrap retrovirus at an MOI of 0.02 resulting in approximately 10E7 independent mutations, selected by neomycin and expanded. Infection of the parental, nonmutagenized haploid mESCs with the attenuated RVFV strain MP-12 was productive (Fig. S1A ) and caused cytopathic effect (CPE) (Fig. S1B) . Also after infection of the ~75 million genetrap library cells then identified by an inverted PCR / restriction digest assay on genomic cell DNA, followed by Next Generation Sequencing ( Fig. S3A and B, table S1). The top-ranking gene, found to be genetrap-inserted many times independently (Fig. S4) , was Low Density Lipoprotein Receptor-Related protein 1 (LRP1).

LRP1 is an approximately 600 kDa plasma membrane protein with a 515 kDa extracellular part (6) (7) (8) (9) (10) .

For validation, we employed our genome-wide Haplobank library of more than 100,000 haploid mESC clones that contain a revertible genetrap with individual barcodes ( Fig. 2A) (5) . We took a number of independent Haplobank clones that carried a genetrap insertion in an intron of LRP1 (table 1) . Moreover, Cre-Lox inversion of the genetrap was performed, so each clone exists in a wt and a knockout version (sister clones) (Fig. 2B) . The mutant or wt mESC clones from the Haplobank and their respective reverted sister clones, each labelled either with GFP or mCherry, respectively (see table 1), were then mixed at a 3:7 ratio and subjected to a growth competition assay under RVFV MP-12 infection. Figure   2C shows how LRP1-mutated cell clones exhibit a growth advantage if kept under RVFV MP-12 infection. As controls, we employed Haplobank cell clones mutated in previously identified pro-viral host factors of RVFV, namely prenyltransferase alpha subunit repeat containing 1 (PTAR1; Fig. 2D ) (11) , and the E3 ubiquitin ligase FBXW11 (Fig. 2E) (12, 13) . Interestingly, in our mESC system the inactivation of these published control genes presented a much weaker survival benefit under selection pressure by RVFV than the deletion of LRP1. LRP1 impacts intracellular RNA levels of RVFV, but neither protein synthesis nor particle production To follow up our findings from the mouse ESCs, we performed siRNA knockdown of LRP1 in human A549 cells and tested its effect on RVFV MP-12 replication using RT-qPCR. siRNA-mediated downmodulation of LRP1 mRNA (Fig. S5A ) resulted in an up to 50% reduction of RVFV gene expression at 5 h and 24 h p.i. (Fig. 3A) . Immunoblot analysis however revealed that A549 cells express comparatively little LRP1, whereas in human HuH-7 cells both the 515 kDa alpha chain as well as the 85 kDa beta chain gave strong signals (Fig. S5B) . Thus, we robustly downregulated LRP1 levels in the HuH-7 cells by introducing a CRISPR/Cas9 knockout (table S2 and Fig. S5B ), and studied its phenotype with regard to RVFV replication. Also in the HuH-7 LRP1 knockout cells RT-qPCR analysis showed a suppression of viral gene expression by more than 50% already at 5 p.i. (Fig. 3B) . However, immunoblotting revealed that levels of viral nucleocapsid (N) protein were unchanged between wt and LRP1 knockout cells (Fig. 3C) . Moreover, when supernatants from the cells infected with MOI 0.01 were titrated, we did not observe any differences in virus yields. From these data we concluded that LRP1 is important for maintaining intracellular RNA levels of RVFV in human cells, but that its absence has no consequences for the production of progeny virus particles.

The results obtained so far may indicate that LRP1 is already important at 5 h p.i.. For RVFV, the replication cycle is initiated by attachment of particles to the cell, followed by their endosomal internalisation, fusion of viral and endosomal membranes, and entry of the nucleocapsids into the cytoplasm (4). In order to identify the earliest virus replication step that involves LRP1, we infected HuH-7 wt and LRP1 knockout cells in a synchronized manner, and took cell-associated RNA samples at 0 h (attachment), 2 h (internalisation), 5 h (gene expression after entry into the cytoplasm) and 24 h (late phase of replication). For these analyses we engaged the wt RVFV strain ZH548 which unlike MP- Fig. 3A and B). Thus, is LRP1 is robustly supporting the immediate-early steps of RVFV infection.

The results from the knockout and time course experiments indicate a role of LRP1 as co-factor rather than as a main receptor of RVFV infection. We tested its importance for other RNA viruses, namely the closely related Sandfly fever Sicilian virus (SFSV; family Phleboviridae, order Bunyavirales), the more remotely related La Crosse bunyavirus (LACV; family Peribunyaviridae, order Bunyavirales), and the non-related vesicular stomatitis (VSV; family Rhabdoviridae, order Mononegavirales). Moreover, as these all are negative-strand RNA viruses, we additionally included the non-enveloped, positivestranded Encephalomyocarditis virus (EMCV, family Picornaviridae), and the three human pathogenic members of the positive-strand RNA virus family Coronaviridae, namely MERS-CoV, SARS-CoV-1, and SARS-CoV-2. As shown in figure 5A , for SFSV LRP1 is involved already in particle attachment, as for RVFV. By contrast, for the more remotely related LACV the lack of LRP1 has only a significant impact at 24 h p.i. (Fig. 5B) . The Rhabdovirus VSV, is also affected at 24 h p.i., but additionally at the attachment step (Fig. 5C ). For the picornavirus EMCV, an LRP1-dependency was only apparent at the attachment step of the infection cycle, whereas already at 2 h pi LRP1 was not required any more (Fig.   5D ). For MERS-CoV, the LRP1 knockout impacted particle attachment and the following early steps, but was compensated at 24 h p.i. (Fig. 5E ). The most striking phenotype was however exhibited by the two SARS-coronaviruses, namely SARS-CoV-1 which caused a pandemic in 2002/2003, and the recently emerged SARS-CoV-2. Here, all steps from particle attachment up until the late-stage virus RNA levels at 24 h p.i. were impacted by the lack of LRP1 (Figs. 5F and G). In fact, while for all other tested viruses the typical reduction in viral RNA was around 50% and remained mostly stable or even caught up during the infection cycle, for both SARS-coronaviruses the RNA levels remained low over time, resulting in a 50 to over 95% reduction relative to the LRP1-expressing wt cells at 24 h p.i..

Our comparative time course experiments in HuH-7 cells thus demonstrate that LRP1-dependency is a common trait among RNA viruses. In most cases LPR1 is facilitating virus attachment, and its absence has a rather transient effect on virus infection. For some viruses, however, also the late stage RNA synthesis depends partially oras is the case for the two SARS-coronavirusessubstantially on the presence of an intact LRP1.

For SARS-CoV-2, the causative agent of COVID-19, we further characterized the impact of LRP1 deficiency. To this aim, we employed siRNA knockdown in Calu-3 (Fig. S6A) , a prototypical human lung epithelial cell line used in SARS-CoV-2 research. In these cells, LRP1 was found to be supporting RNA synthesis at 5 h p.i and 24 h p.i, but not at the earlier stages of the replication cycle (Fig. 6A ).

Moreover, analbeit transientimpact on viral N protein synthesis could be observed (Fig. 6B ).

Despite these clear effects on late stages of SARS-CoV-2 infection, however, virus yields were comparable between wt and LRP1-deficient Calu-3 cells at all time points measured (Fig. 6C ).

As observed previously for the human alveolar basal epithelial cell line A549 (see Fig. S5 ), Calu-3 cells express very little LRP-1 (Fig. S6B) . For further investigations, we therefore returned to the HuH-7 cells, which had exhibited both higher LRP1 levels and a more profound impact of LRP1 deficiency on the SARS-CoV-2 replication cycle (see Fig. 5G ). Strikingly, analyses of an infection time course demonstrated that in this system an LRP1 deficiency robustly impacted expression of the viral nucleocapsid protein (N) (Fig. 7A ) as well as virus yields in the supernatants (Fig. 7B) , and the effects were observed throughout the entire replication cycle. In fact, virus yields did not even recover towards the end of the infection cycle, indicating a strong dependency on LRP1. Of note, a markedly reduced SARS-CoV-2 replication was also observed for two other HuH-7 LRP1 knockout cell clones, excluding clonal effects (Fig. 7C ).

Thus, taken together, our forward genetic screen in haploid mESCs enabled us to identify the cellular protein LRP1 as a host factor for RVFV and a series of other RNA viruses, most prominently SARS-CoV-2. Although the strength of the pro-viral effect of LRP1 was cell type-dependent, for most viruses already the attachment phase of the replication cycle was involved. Interestingly, in some cases (e.g. SARS-CoV-2) LRP1 had an impact on the late phase of infection, indicating a role as an auxiliary factor also in RNA synthesis.

LRP1 (or CD91) is a scavenger-type receptor involved in a wide variety of cell activities like migration, proliferation, differentiation, but also regulation of cholesterol homeostasis, inflammation, or clearance of plasma proteins from the blood stream (6) (7) (8) (9) (10) . It is important for the integrity of blood-brain barrier and can bind and internalize more than 40 different ligands (including apoptotic bodies or the Alzheimer disease-associated tau protein). Moreover, LRP1 modulates signalling pathways like e.g. those by JAK/STAT, ERK1/2 or TGF-ß (10, 14) . Our findings indicate that many viruses are aided by LRP1 at their attachment and entry step, as might be expected from a membrane protein involved in constitutive endocytosis. In agreement with these findings, a recent study (published while our manuscript was in preparation) showed that LRP1 acts as a receptor of RVFV by binding to the viral envelope protein and mediating its entry into the cytoplasm (15) .

Interestingly however, some virusesincluding SARS-CoV-2 -also require LRP1 for the later stages of infection. Like all positive-stranded RNA viruses, coronaviruses are known to intensively reorganize internal cell membranes in order to generate compartments that can serve as a safe space for transcription and replication of the genome RNA, and to assemble progeny particles (16) (17) (18) . As LPR1 is mostly cycling between the plasma membrane and the endosomes, it may transport factors critical for the RNA replication of SARS-coronaviruses, e.g. lipids required for the formation of virally-induced intracellular membrane compartments. Curiously, in HuH-7 cells the late-stage requirement for LRP1 was only present for SARS-CoV-1 and SARS-CoV-2 (subgenus Sarbecovirus, genus Betacoronavirus), but not for the related MERS-CoV (subgenus Merbecovirus, genus Betacoronavirus) (19) , while the early-stage requirement for LRP1 was observed for both these betacoronavirus subgenera. It is hence possible that the SARS-coronaviruses engage LRP1 for two different viral activities, attachment and late-stage replication, whereas for MERS-CoV it may be only attachment or, alternatively, the requirement for the later infections stages is not as strict as for the SARS-coronaviruses.

SARS-CoV-2 exhibited the most profound LRP1-dependency in the hepatic HuH-7 cells, impacting both the early attachment step and the subsequent RNA synthesis. HuH-7 cells are of hepatic origin (20) , and SARS-CoV-2 is capable of infecting and damaging organs including the liver (21) (22) (23) . Besides the liver, LRP1 is also detectable in tissues like brain, respiratory system, kidney, sexual organs and connective and soft tissues (24) . Cell types with highest LRP1 levels are hepatocytes, Leydig cells, brain cells like microglia and astrocytes, syncytiotrophoblasts, skeletal myocytes, fibroblasts, peritubular cells, and monocytes/macrophages (25) . Future studies may reveal whether in these and other cell types LRP1 is contributing to the cell tropism of SARS-CoV-2.

In Calu-3 cells, LRP1 was neither required early in infection nor for virus yields, but exclusively for late-stage RNA synthesis. Like Calu-3, alveolar cells and other epithelial cells express much lower levels of LRP1 as hepatocytes, indicating that factors other than LRP1 are mainly supporting SARS-CoV-2 attachment and entry. Thus, apparently, early-stage LRP1 usage by SARS-CoV-2 is cell type-dependent, whereas the late-stage requirement for RNA synthesis is general.

Taken together, we have identified LRP1 as a broadly active pro-viral factor that enables entry of RNA viruses. In some cases, most prominently SARS-CoV-1 and SARS-CoV-2, LRP1 is also facilitating the late stage of infection. It will be interesting to find out how SARS-coronaviruses are engaging LRP1 for their intracellular replication, and by which alternative mechanism MERS-CoV is circumventing LRP1 at the late stage of infection.

A549, BHK, HuH-7, Vero E6, Vero B4, as well as Calu-3 (kindly provided by Marcel Müller, Berlin, Germany) and LRP-1 knockout HuH-7 cells were grown in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal calf serum (FCS), 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin. Medium and supplements were purchased from Thermo Fisher Scientific. The mouse haploid embryonic stem cells AN3-12 were derived and sorted for their haploidy as described (26, 27) .

The AN3-12 cells, the AN3-12 genetrap-mutated library, and the derived genetrap-mutated specific cell lines from the Haplobank (5) 

We used a mouse haploid embryonic stem cell (mESC) barcoded-library (complexity: 9.710E6), 

Experimental details of genomic DNA extraction, restriction digest, ring-ligation and inverse PCR with primers located in the genetrap (see Fig. S3A and B), as well as next generation sequencing of integration sites were described previously (27) . In short, the cell clones that were resistant to infection with RVFV MP-12 were trypsinized, washed in PBS and incubated overnight in gDNA Lysis buffer Purified iPCR products (genetrap-library digested by either MseI or NlaIII) were quantified with a Nanodrop, and mixed 1:1 to be combined into one Next Generation Sequencing flowcell. Raw reads were trimmed to 50 nt and processed as in the NCBI Gene Expression Omnibus entry GSM2227065 (28) . In short, reads were aligned to the genome (mm10) with bowtie (v1.2.2) (29). Insertions of disruptive and undisruptive regions of each gene are summed up (see Fig. S4 ). Binomial test of disruptive insertions against undisruptive regions and against disruptive insertions of a retrovirus input (GSM2227065), respectively, was performed for each gene. Genes were ranked by counts of disruptive insertions (DI) and were selected with a LOFscore <= 1e-20 (loss of function score). If the LOFscore equals 0, we remove genes with less than 10 DIs and without insertions in the background.

Genetrap-mutagenized clones from the Haplobank collection (www.haplobank.at; (5)) were thawed, grown in 10 cm dishes, and split in 6 wells of a 24-well plate. Three of the wells were infected with a MLP-puro-GFP retrovirus, and 3 were infected with a MLP-mCherry-puro-Cre retrovirus (inducing flipping of the genetrap) (5). At 24 h p.i., 1 µg/mL puromycin (Invitrogen) were added and 5 days later cells were split and aliquots were frozen.

For each gene of interest, cells of one GFP-labelled (original clone) and one mCherry-labelled (flipped sister-clone) version were mixed at a ratio of ~30% knockout cells to ~70% wild-type cells, respectively. At 4 h post-seeding, the mixed cells were washed with DMEM and either mock-infected or infected with RVFV MP-12 at MOI 5 for 1 h at 37°C. Medium (ESCM) was then added on top, and cells were further incubated at 37°C. Cells were trypsinized at 2 and 5 days pi, and either fixed in 4% PFA for flow cytometry analysis, or further grown after seeding in a new 24-well plate. The initial ratios between GFP and mCherry-labelled cells were confirmed, followed over-time by flow-cytometry (BD FACS LSR Fortessa, with HTS) and analysed with the FlowJo software. Only conditions with more than 1,000 acquired events were taken into account for final analysis. 

HuH-7 cells with a knockout in genes of interest were generated using the CRISPR-hSpCas9 strategy from the Zhang lab (30, 31) . For the LRP1 gene, sgRNAs (see table S2) were designed using online tools (https://www.addgene.org/crispr/; http://www.e-crisp.org/E-CRISP/designcrispr.html). After cloning of the required plasmids, the lentiviruses expressing either the specific CRISPR-hSpCas9-sgRNAs or the no template control (NTC) CRISPR-hSpCas9 were generated, and then transduced in triplicates into HuH-7 cells. Clonal cell populations were isolated by limiting dilution, following the Addgene protocol (https://www.addgene.org/protocols/limiting-dilution/). Each single colony was further amplified, and screened by western blot. The control cells HuH-7 NTC (clone E5) and the HuH-7 LRP1 knockout cells (clones C5, C8, E3) were then used in further experiments.

Total RNA was extracted from cell lysates using RNeasy (Qiagen) and an aliquot of 100 ng was reverse transcribed with the PrimeScript RT Reagent Kit with gDNA Eraser (Takara) and the included primer mix. An aliquot of 10 ng cDNA was used as template for amplifying sequences of human GAPDH, and LRP1 and VSV with corresponding QuantiTect primers (Qiagen) or specific primers (table S3) , respectively, and the SYBR premix Ex Taq (Tli RnaseH Plus) kit (Takara). RRN18S was amplified in a similar manner, but with 2 ng cDNA as template. RNA levels of EMCV, LACV MERS-CoV, RVFV, SARS-CoV-1, SARS-CoV-2, and SFSV were detected using specific primers and Taqman probes (table   S3) , and the Premix Ex Taq (probe qPCR) kit (Takara). All PCRs were performed in a StepOne plus instrument (Applied Biosystems). The values obtained for each gene were normalized against GAPDH mRNA levels (or RRN18S mRNA levels in the case of the VSV RNA) using the threshold cycle (ΔΔCT) method (32) .

containing protease inhibitors (cOmplete tablets EasyPack, Roche), according to the supplier's protocol.

Samples for immunodetection of LRP1 were mixed with 4 sample buffer (143 mM Tris-HCl pH 6.8 

Supernatants of infected cells were collected and cleared by centrifugation at 400 g for 5 min. Serial dilutions were made to infect subconfluent Vero E6 monolayers, and infected cells were then incubated in medium containing 1.5 % Avicel for 3 days. Cells were washed twice in PBS and stained for 10 min with a cristal violet solution (0.75% crystal violet, 3.75% folmaldehyde, 20% ethanol, 1% methanol).

Cells were then washed, and plaques were counted. Titers were determined as Plaque Forming Units (PFU) per ml.

Statistical analyses performed are described in the figure legends. (see table 1 ). Approximatively 30% of knockout cells were mixed with 70% of their wild-type sister clone, and infected with RVFV MP-12 at MOI 5. Ratio between both sister clones was followed by flow cytometry. n=2, except if event count was below 1,000, in which case the whole data set was removed (n=1) 

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Figure 5. Virus RNA levels in LRP1 knockout cells over the course of infection. (A) Sandfly Sicilian virus (SFSV), (B) LaCrosse virus (LACV), (C) Vesicular stomatitis virus (VSV), (D) encephalomyocarditis virus (EMCV), (E) Middle east respiratory syndrome virus (MERS-CoV), (F)

NTC (no template control) cells were infected with the various viruses at MOI 1, except for VSV that was used at MOI 0.1, washed 3 times and further incubated in medium. Samples were collected after the 3 washes postinfection (attachment step), or at 2 h, 5 h or 24 h post-infection. Two-step RT-qPCR was done to detect viral RNA, as well as the GAPDH and 18S rRNA reference genes. The RNA levels in the infected NTC cells was set-up to 100%

We are indebted to Christian Drosten and Marcel Müller for kindly providing the SARS- CoV-2 virus   and Calu-3 

The authors declare that there are no conflicts of interest.

All data are available in the main text or the supplementary materials.