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LRRC15 inhibits SARS-CoV-2 cellular entry in trans [1]

['Jaewon Song', 'Department Of Molecular Microbiology', 'Immunology', 'Division Of Biology', 'Medicine', 'Brown University', 'Providence', 'Rhode Island', 'United States Of America', 'Ryan D. Chow']

Date: 2022-10

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is mediated by the entry receptor angiotensin-converting enzyme 2 (ACE2). Although attachment factors and coreceptors facilitating entry are extensively studied, cellular entry factors inhibiting viral entry are largely unknown. Using a surfaceome CRISPR activation screen, we identified human LRRC15 as an inhibitory attachment factor for SARS-CoV-2 entry. LRRC15 directly binds to the receptor-binding domain (RBD) of spike protein with a moderate affinity and inhibits spike-mediated entry. Analysis of human lung single-cell RNA sequencing dataset reveals that expression of LRRC15 is primarily detected in fibroblasts and particularly enriched in pathological fibroblasts in COVID-19 patients. ACE2 and LRRC15 are not coexpressed in the same cell types in the lung. Strikingly, expression of LRRC15 in ACE2-negative cells blocks spike-mediated viral entry in ACE2+ cell in trans, suggesting a protective role of LRRC15 in a physiological context. Therefore, LRRC15 represents an inhibitory attachment factor for SARS-CoV-2 that regulates viral entry in trans.

Funding: This study was supported by NIH grants R00 AI141683 (S.L.), 2P20 GM109035-07 (S.L.), K08 AI128043 (C.B.W), R01 AI148467 (C.B.W.), T32 GM007205 (R.D.C.), F30 CA250249 (R.D.C.) and P20 GM119943 (O.D.L.); the Smith Family Awards Program for Excellence in Biomedical Research (S.L.); a Burroughs Wellcome Fund Career Award for Medical Scientists (C.B.W.); the Ludwig Family Foundation (C.B.W.), the Mathers Charitable Foundation (C.B.W.); an Emergent Ventures fast grant (C.B.W). DoD PRMRP IIAR (W81XWH-21-1-0019) (S.C.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we employed a screening method using the CRISPR activation (CRISPRa) technique. We generated a focused CRISPRa library, named surfaceome, that covers all approximately 6,000 known/predicted surface proteins on the cellular plasma membrane. The surfaceome screening with the SARS-CoV-2 spike protein revealed that human LRRC15 (leucin-rich repeat-containing 15) is a novel inhibitory attachment factor for SARS-CoV-2.

Thus far, several cellular factors have been identified to facilitate cellular entry of SARS-CoV-2. However, it is unclear whether there are any host factors that inhibit viral entry. Previous studies indicate that cleavage of spike protein by cellular proteases such as transmembrane protease serine 2 (TMPRSS2), cathepsins, and furin facilitates the entry of SARS-CoV-2 [ 9 , 11 , 17 , 18 ]. Several cellular surface proteins or glycans facilitate viral entry by acting as an attachment factor, which includes neuropilin-1 [ 19 , 20 ], heparan sulfate [ 21 ], and C-type lectins [ 22 ]. Alternative entry factors have been proposed such as AXL [ 23 ] and CD147 [ 24 ]. However, it remains to be elucidated whether any cellular entry factors regulate viral entry in a different manner.

The interaction between the RBD of spike and ACE2 determines several key features of SARS-CoV-2 infection. The high affinity interface between the RBD and ACE2 is associated with higher infectivity of SARS-CoV-2 compared to SARS-CoV-1 [ 13 ], and a single point mutation at the RBD can alter host range and enable mouse infection [ 14 – 16 ]. Spike protein is the primary target antigen for COVID vaccines, and the majority of existing therapeutic antibodies function by blocking RBD and ACE2 interactions, indicating the importance of RBD and its binding to the cellular receptor for controlling SARS-CoV-2.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the causative agent of Coronavirus Disease 2019 (COVID-19), representing a global health threat [ 1 , 2 ]. SARS-CoV-2 belongs to the β-coronavirus family along with Severe Acute Respiratory Syndrome Coronavirus (hereafter SARS-CoV-1) and Middle East Respiratory Syndrome Coronavirus (MERS-CoV) [ 3 , 4 ]. Like SARS-CoV-1, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as a receptor [ 5 , 6 ]. The viral structural protein spike (S), anchored on the surface of the viral envelope as homotrimers, binds to ACE2 and mediates virus entry [ 7 ]. The ectodomain of spike protein consists of the S1 and S2 subunits. The S1 subunit is comprised of the N-terminal domain (NTD) and the receptor-binding domain (RBD) [ 8 ]. The RBD of spike protein directly binds to ACE2, which induces a conformational change that facilitates virus fusion either with endosomal membrane or with the plasma membrane [ 6 , 9 , 10 ]. This fusion event releases the SARS-CoV-2 genome into the cytoplasm [ 11 , 12 ].

Results

A surfaceome CRISPR activation screen identified cellular receptors for spike protein of SARS-CoV-2 To identify host factors that regulate SARS-CoV-2 entry, we performed the surfaceome CRISPRa screen and investigated which cellular proteins regulate spike binding to cells. We specifically selected approximately 6,000 genes encoding plasma membrane proteins that contain either single or multiple transmembrane domains or are associated with the plasma membrane. We designed a CRISPRa library consisting of 4 activating single guide RNAs (sgRNAs) per gene and 1,000 nontargeting control sgRNAs (S1A Fig). The screen was performed in a human melanoma cell line, A375, as this cell line does not express endogenous ACE2 and does not interact with SARS-CoV-2 spike protein without ectopic expression of ACE2 [21]. A375 cells containing catalytically “dead” Cas9 (dCas9) were transduced with the sgRNA library and selected to produce a pool of cells with induced expression of individual surface proteins. We measured the binding of Fc-tagged S1 subunit of SARS-CoV-2 spike to the cells by flow cytometry. Cells exhibiting high fluorescent signal intensity were sorted and subjected to genomic DNA extraction and sgRNA sequencing (Fig 1A and S1 Data). Two biologically independent screen results indicated 2 only distinct hits, ACE2 and LRRC15 (Fig 1B). Other reported spike attachment factors were not significantly enriched in our screen [23,25,26] (S1B Fig). This discrepancy might be from relatively weak spike-binding affinities of previously identified attachment factors [23,26,27] or dependent on cell types expressing different cofactors. LRRC15 is a leucin-rich repeat domain-containing protein, which is an orphan cancer-associated protein [28,29]. There is no reported role of LRRC15 in SARS-CoV-2. An immunoglobulin G (IgG) isotype control and anti-CD45 staining identified IgG receptor genes (FCGR2C, FCGR3B) and CD45-encoding gene, PTPRC, as the top hit, respectively, confirming that the surfaceome CRISPR screening efficiently identifies cellular receptors for targeted proteins (Figs 1B and S1C). PPT PowerPoint slide

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TIFF original image Download: Fig 1. A surfaceome-focused CRISPRa screen identified cellular receptors binding with SARS-CoV-2 spike protein. (A) Schematic of a focused CRISPRa screen for surface proteins interacting with SARS-CoV-2 spike S1-Fc fusion protein. (B) Volcano plots showing sgRNAs enriched or depleted in cells binding with SARS-CoV-2 spike S1-Fc or human IgG isotype control. Results from 2 biologically independent replicates are shown. For underlying data, see S1 Data. ACE2, angiotensin-converting enzyme 2; CRISPRa, CRISPR activation; dCas9, “dead” Cas9; IgG, immunoglobulin G; LRRC15, leucin-rich repeat-containing 15; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, single guide RNA. https://doi.org/10.1371/journal.pbio.3001805.g001

LRRC15 directly interacts with the spike via the receptor-binding domain To validate the screening results, we utilized 2 different human cell lines, A375 and HeLa. These 2 cell lines do not express endogenous ACE2 and are not susceptible to SARS-CoV-2 without ectopic expression of ACE2 [21,30]. A375 and HeLa cells were transduced with 2 individual sgRNAs for LRRC15 and a single sgRNA for ACE2 to induce gene expression (S2A and S2B Fig). LRRC15-induced and ACE2-induced cells bound to the S1-Fc protein. The signal intensity in ACE2-induced cells was stronger than that of the LRRC15-induced cells. (Fig 2A). A similar pattern of protein interaction was observed in HeLa cells (Figs 2B and S2B). Trimeric full-length recombinant spike protein also bound to LRRC15-induced HeLa cells with the weaker signal intensity than ACE2-expressing cells (S2C Fig). PPT PowerPoint slide

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TIFF original image Download: Fig 2. LRRC15 binds with SARS-CoV-2 spike protein at the RBD. (A) A375 cells were transduced with indicated activating sgRNAs and incubated with SARS-CoV-2 spike S1-Fc fusion protein. Protein binding was measured by flow cytometry with MFI shown. (B) HeLa cells were transduced with indicated activating sgRNAs and incubated with SARS-CoV-2 spike S1-Fc fusion protein. Protein binding was measured by flow cytometry with MFI shown. (C) Dose-dependent binding of SARS-CoV-2 spike protein (Wuhan-Hu-1) to both ACE2 and LRRC15 with an Fc tag was determined by ELISA. Human IgG1 was included as a negative control. Dots indicate means of duplicates. (D) HeLa cells were transduced with indicated activating sgRNAs and incubated with SARS-CoV-2 spike NTD-Fc or RBD-Fc fusion protein. Protein binding was measured by flow cytometry with MFI shown. (E) The binding of the SARS-CoV-2 RBD and NTD to LRRC15 was measured by ELISA. For underlying data, see S3 Data. ACE2, angiotensin-converting enzyme 2; LRRC15, leucin-rich repeat-containing 15; MFI, mean fluorescence intensity; NTD, N-terminal domain; RBD, receptor-binding domain; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, single guide RNA. https://doi.org/10.1371/journal.pbio.3001805.g002 The interaction between LRRC15 and spike was further examined in a cell-free interaction model using recombinant proteins. An ELISA assay using recombinant LRRC15 and full-length spike indicated that LRRC15 directly interacts with the spike protein (K D = 109 nM). The affinity between LRRC15 and the spike seems to be weaker than that of ACE2 and spike (Fig 2C). Interaction with spike proteins of different SARS-CoV-2 variants was confirmed. Recombinant full-length spike proteins of α (B.1.1.7), β (B.1.351), ɣ (P.1), δ (B.1.617.2), and ι (B.1.526) variants were tested and LRRC15 interacted with all of these spike proteins with similar affinity (S2D Fig). ACE2 interacts with the spike protein via the RBD but not the NTD [31]. Interestingly, we identified that LRRC15 interacts with the spike in a similar way. Interaction assays in cells and in a cell-free assay using ELISA indicated that the RBD is sufficient to recapitulate the interaction between LRRC15 and spike with a similar affinity compared to full-length S1 (Fig 2D and 2E). Next, we examined whether this interaction is specific to SARS-CoV-2 or conserved in other β coronaviruses. The ELISA assay using recombinant RBD protein of SARS-CoV-1 and MERS-CoV showed that LRRC15 binds to spike of SARS-CoV-1 with similar affinity but does not interact with spike of MERS-CoV (S2E Fig). These results indicate that LRRC15 is a novel cellular binding protein for the spike protein of SARS-CoV-1 and SARS-CoV-2 and directly interacts with the spike via the RBD.

LRRC15 accumulates cell-attached viruses on the membrane and does not compete with ACE2 As SARS-CoV-2 entry is primarily dependent on ACE2, we assessed whether LRRC15 alters protein expression of ACE2. The level of ACE2 surface expression was unaltered or marginally decreased by sgRNA-mediated gene induction in HeLa-ACE2 cells and was slightly increased in LRRC15-induced Huh7.5 cells (Figs 4A and S4A). Although surface ACE2 expression was slightly different, they all showed the decreased entry of SARS-CoV-2 pseudotyped virus and authentic virus, indicating that the inhibitory effect of LRRC15 does not require the regulation of surface ACE2 levels. PPT PowerPoint slide

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TIFF original image Download: Fig 4. LRRC15 enhances SARS-CoV-2 attachment to the surface of ACE2-expressing cells. (A) HeLa-ACE2 cells were transduced with indicated activating sgRNAs or a LRRC15-expressing vector. Cell surface expression of ACE2 was measured by flow cytometry and calculated as MFI (n = 3). (B) HeLa or HeLa-ACE2 cells transduced with indicated activating sgRNAs were incubated with VSVΔG-S-SARS2 for 1 h on ice and washed 3 times with cold cell culture media. Viral genome copies were quantified by RT-qPCR and normalized to HeLa-ACE2 cells (n = 3). (C) Representative images of immunofluorescence staining of SARS-CoV-2 spike (green), LRRC15 (red), Actin (blue), and DAPI (cyan). Cells were inoculated with VSVΔG-S-SARS2 for 1 h on ice and incubated at 37°C for 1 h to allow internalization, followed by staining. The white arrowheads indicate spikes. The scale bar indicates 5 μm. (D) Quantification was performed by calculating the number of spikes on cells from multiple images per sample. (E) VSVΔG-S-SARS2 were incubated with ACE2-Fc, LRRC15-Fc, or IgG control for 1 h, prior to inoculating HeLa-ACE2 cells. Viral infectivity was quantified by measuring GFP signal at 20 hpi by flow cytometry and normalized to no antibody control (n = 6). Statistical significance was determined compared to IgG control at each dilution. (F, G) Competition assays between ACE2 and LRRC15 for immobilized His-tagged SARS-CoV-2 spike protein. Premixture of His-tagged protein at 4 different concentrations with a dilution series of Fc-tagged protein was added, and antihuman HRP determined the amount of Fc-tagged proteins remaining in the presence of competitor through a colorimetric readout. A combination of LRRC15-His and ACE2-Fc (F) or ACE2-His and LRRC15-Fc (G) was used. Data represent means ± SD (B, D, E). Data were analyzed by one-way ANOVA (B, D) or two-way ANOVA (E) with Dunnett multiple comparisons test. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. For underlying data, see S3 Data. ACE2, angiotensin-converting enzyme 2; GFP, green fluorescent protein; hpi, hours postinfection; HRP, horseradish peroxidase; IgG, immunoglobulin G; LRRC15, leucin-rich repeat-containing 15; MFI, mean fluorescence intensity; RT-qPCR, quantitative reverse transcription PCR; SARS-CoV-2, Severe Acute Respiratory Syndrome Coronavirus 2; sgRNA, single guide RNA. https://doi.org/10.1371/journal.pbio.3001805.g004 Interestingly, we found that spike-coated viruses were sequestered on the cellular surface of LRRC15-expressing cells. In the attachment assay, we measured viral attachment to cells by incubating spike-pseudotyped viruses and cells on ice, allowing attachment on the cell membrane and preventing internalization of viruses. As expected, SARS-CoV-2 spike-pseudotyped viruses bound to ACE2-expressing cells and the binding was not altered by inducing a control gene, CD45. Importantly, viruses were highly accumulated on LRRC15-induced cells, both in the absence and presence of ACE2 (i.e., approximately 3-fold increases in viral copies) compared to their controls (Fig 4B). The enhanced virus binding by LRRC15 was observed independent of expression of ACE2. Immunofluorescence staining of spike proteins confirmed the enhanced binding of pseudoviruses to LRRC15- but not to DC-SIGN-induced cells (Figs 4C, 4D and S4B). While few spikes were detected in HeLa-ACE2 cells after 1 h of internalization, significant number of spikes were detected in LRRC15-induced cells, implying that LRRC15 may inhibit the internalization of the virus (Fig 4D). Preincubation of soluble LRRC15 protein with spike-pseudotyped viruses partially blocked viral entry in HeLa-ACE2 cells at high concentrations, while preincubation with soluble ACE2 completely blocked viral entry (Fig 4E). The difference in blocking efficacy is likely due to the differing spike binding affinities of LRRC15 and ACE2. In summary, these results suggest that LRRC15 inhibits SARS-CoV-2 entry potentially by restricting the internalization of virions into the cell through binding to the spike protein. To test whether LRRC15 directly binds ACE2, we utilized His-tagged-LRRC15, SARS-CoV-2 spike protein, and MERS-CoV spike protein and assessed their interactions with Fc-tagged ACE2. Recombinant ACE2 did not show detectable binding to recombinant LRRC15 protein or the MERS-CoV spike whereas binding to SARS-CoV-2 spike was confirmed with high affinity (S4C Fig) [39]. Since both ACE2 and LRRC15 bind to the RBD of SARS-CoV-2 spike, we investigated whether LRRC15 competes with ACE2 for binding on the spike protein. The interaction between ACE2 and SARS-CoV-2 spike was measured in the presence of recombinant LRRC15 protein. Even at high concentrations, LRRC15 did not affect the spike-ACE2 binding (Fig 4F). Conversely, spike-LRRC15 interaction was not affected by excess ACE2, demonstrating LRRC15 and ACE2 do not share the same binding epitope within the RBD (Fig 4G). We confirmed that SARS-CoV-2 spike S1-Fc binding to Hela-ACE2 cells was not altered by gene induction or ectopic expression of LRRC15 (S4D Fig). Taken together, ACE2-mediated viral entry of SARS-CoV-2 is suppressed by LRRC15 on the cell membrane through its direct binding to the RBD without competition between LRRC15 and ACE2.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001805

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