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MHC class II transactivator CIITA induces cell resistance to Ebola virus and SARS-like coronaviruses

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The CIITAdel keeps viruses at bay

A better understanding of cellular mechanisms involved in viral resistance is needed for the next generation of antiviral therapies. Bruchez et al. used a transposon-mediated gene-activation screen to search for previously unreported host restriction factors for Ebola virus (see the Perspective by Wells and Coyne). The authors found that a transcription factor, major histocompatibility complex class II transactivator (CIITA), induces resistance in human cell lines by directing the expression of the p41 isoform of the invariant chain (CD74). CD74 p41 then disrupts cathepsin-mediated Ebola glycoprotein processing, which prevents viral fusion and entry. CD74 p41 can also stymie the endosomal entry of coronaviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This work should inform future treatments against cathepsin-dependent viruses such as filoviruses and coronaviruses. Additionally, the screening strategy used may serve as a blueprint for uncovering resistance mechanisms against other dangerous pathogens.

Science, this issue p. 241 see also p. 167

Abstract

Recent outbreaks of Ebola virus (EBOV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have exposed our limited therapeutic options for such diseases and our poor understanding of the cellular mechanisms that block viral infections. Using a transposon-mediated gene-activation screen in human cells, we identify that the major histocompatibility complex (MHC) class II transactivator (CIITA) has antiviral activity against EBOV. CIITA induces resistance by activating expression of the p41 isoform of invariant chain CD74, which inhibits viral entry by blocking cathepsin-mediated processing of the Ebola glycoprotein. We further show that CD74 p41 can block the endosomal entry pathway of coronaviruses, including SARS-CoV-2. These data therefore implicate CIITA and CD74 in host defense against a range of viruses, and they identify an additional function of these proteins beyond their canonical roles in antigen presentation.

Recent and ongoing outbreaks of Ebola virus (EBOV) in Africa (1) and the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic highlight the need to identify additional treatment strategies for viral infections, including approaches that might complement traditional antivirals. Of particular interest is the identification of host-directed therapies that target common vulnerabilities and may be efficacious against multiple viruses, including those that may emerge in the future.

We set out to identify host pathways of cellular resistance to pathogens with pandemic potential, using a transposon-mutagenesis–forward genetic approach. We used a modified PiggyBac (PB) transposon (Fig. 1A), which stimulates or disrupts the expression of neighboring genes, thereby allowing an interrogation of both gene activation and inactivation in a single screen (2). Transposon-mutagenized libraries were treated with Ebola glycoprotein (EboGP)–expressing recombinant vesicular stomatitis virus (referred to as EboGP-VSV). Susceptible wild-type U2OS cells died after 3 to 4 days of treatment, whereas surviving cells could be expanded from mutagenized libraries and exhibited stable resistance to rechallenge with EboGP-VSV (Fig. 1B). These cells showed no cross-resistance to vesicular stomatitis virus (VSV) containing the VSV glycoprotein (VSVg-VSV) (Fig. 1C), which suggests that most of the resistance mechanisms selected in this screen targeted EboGP-mediated entry.

Fig. 1 Transposon-mediated activation tagging generates mutant cells resistant to Ebola.

(A) Modified PB transposon. SV-Puro-pA, puromycin selection cassette; CMV, cytomegalovirus promoter; SD, splice donor. (B and C) Resistance of selected cells to EboGP-VSV (B) and VSVg-VSV (C). Data are means ± SD of n = 3 replicates for one representative pool. Student’s t test; **P < 0.01. (D) Distribution of transposon insertions. Inner rings show insertions per 1 Mb for individual libraries (black histograms) and CISs (P < 107). Outer ring shows combined insertions for all libraries (black histogram) and lowest P value for CISs (red bubble plot). Point size represents the number of libraries with the CIS. freq, frequency. (E and F) Cumulative independent insertions from all eight libraries mapping to NPC1 (E) and CIITA (F).

We identified candidate resistance genes by identifying genomic regions with high numbers of transposon insertions [referred to as common insertion sites (CISs)] (3). Combining data from eight independent screens revealed seven genomic loci with highly statistically significant (P < 10−8) CISs that occurred in more than one screen, representing high-confidence candidate-resistance mutations (Fig. 1D, outer ring). Likely target genes of transposon insertions were identified on the basis of transposon insertion position and orientation (Fig. 1D and table S1). We focused on the two genes that were found in all eight screens using the most stringent criteria.

The first of these was NPC1, located on chromosome 18. All transposon insertions at this site were intragenic in both sense and antisense orientations, and all were predicted to disrupt NPC1 expression (Fig. 1E). This is consistent with the role of NPC1 as the EBOV receptor (4, 5) and validates our screening approach. Notably, U2OS cells are haploid at the NPC1 locus (6), and these transposon insertions are therefore predicted to generate NPC1-null cells, which explains why NPC1 was the only predicted gene-disruption mutant identified as a high-stringency candidate gene.

All transposon insertions at the second CIS—located on chromosome 16—were upstream of the gene CIITA and were oriented in the sense orientation, consistent with activation of expression (Fig. 1F and fig. S1). CIITA overexpression in wild-type U2OS cells increased cell survival, reduced green fluorescent protein (GFP) reporter expression, and completely inhibited plaque formation, which confirms that CIITA increases resistance to EboGP-VSV 100- to 1000-fold (Fig. 2, A to E, and fig. S2). CIITA-overexpressing cells were also resistant to EboGP-pseudotyped single cycle viruses (Fig. 2, F and G), which strongly suggests that CIITA inhibits viral entry rather than targeting viral transactivators as suggested for HIV and human T cell leukemia virus (HTLV) (7, 8). Furthermore, using EboGP virus–like particles (EboGP-VLPs) carrying β-lactamase (9), we found that CIITA did not affect the internalization of EboGP-VLPs into cells (Fig. 2H), but it blocked viral fusion, which occurs in the endosome (10) (Fig. 2I). CIITA-expressing U2OS cells were also highly resistant to infection by high titers of native EBOV, showing reduced reporter gene expression, cell death, and plaque formation (Fig. 2, J to M). CIITA expression did not inhibit replication of an EBOV minigenome, which indicates that CIITA does not act on the viral replication complex (fig. S3). Furthermore, CIITA inhibited infection mediated by glycoproteins (GPs) from a range of EBOV species—including Sudan, Zaire, and Reston—as well as by those from the distantly related filovirus Marburg virus (Fig. 2G). Thus, CIITA induces broad antiviral activity against EBOV and other pathogenic filoviruses through the inhibition of viral GP-mediated entry.

Fig. 2 Identification of CIITA as an Ebola restriction factor.

(A) Resistance of CIITA-overexpressing and control (Cntrl) U2OS cells EboGP-VSV. MOI, multiplicity of infection. (B and C) Plaque formation assays (B) and effective viral titer (C) for control and CIITA-overexpressing U2OS cells infected with VSVg-VSV (VSV) and EboGP-VSV (Ebo). undil, undiluted; PFU, plaque-forming units. (D) Representative images of CIITA-transfected (CIITA), control-transfected (Cntrl), and unmanipulated U2OS cells (U2OS) infected with mCherry-expressing EboGP-VSV (red) and stained with Hoechst 33342 to resolve cell nuclei (blue). (E to G) Infection of control and CIITA-expressing U2OS cells by recombinant VSV pseudotyped with EboGP, LFVGP (Lassa virus GP), or VSVg (E); single cycle murine leukemia virus (MLV) pseudotyped with VSVg and EboGP (F); or single cycle HIV pseudotyped with VSVg or GP from EBOV, Taï Forest virus (TAFV), Bundibugyo virus (BDBV), Sudan virus (SUDV), Reston virus (RESTV), or Marburg virus (MARV) (G). (H and I) Internalization (H) and fusion (I) of EboGP-VLPs by control and CIITA-overexpressing U2OS cells. No env, nonenveloped control VLPs. (J to M) Infection of control and CIITA-overexpressing U2OS cells by infectious EBOV measured by imaging of GFP reporter (green) and cell nuclei (blue) (J), cell survival (K), infected cells (L), or plaque formation (M). Data are means ± SEM of three independent experiments [(A) to (I)] or experiments with three independent cell clones [(K) to (M)]. Student’s t test [(A), (C), and (K) to (M)] or analysis of variance (ANOVA) with Tukey’s multiple comparison test [(E) to (I)]; *P < 0.05; **P < 0.01; ND, not detected. Scale bars, 100 μm.

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Fig. 2 Identification of CIITA as an Ebola restriction factor.

(A) Resistance of CIITA-overexpressing and control (Cntrl) U2OS cells EboGP-VSV. MOI, multiplicity of infection. (B and C) Plaque formation assays (B) and effective viral titer (C) for control and CIITA-overexpressing U2OS cells infected with VSVg-VSV (VSV) and EboGP-VSV (Ebo). undil, undiluted; PFU, plaque-forming units. (D) Representative images of CIITA-transfected (CIITA), control-transfected (Cntrl), and unmanipulated U2OS cells (U2OS) infected with mCherry-expressing EboGP-VSV (red) and stained with Hoechst 33342 to resolve cell nuclei (blue). (E to G) Infection of control and CIITA-expressing U2OS cells by recombinant VSV pseudotyped with EboGP, LFVGP (Lassa virus GP), or VSVg (E); single cycle murine leukemia virus (MLV) pseudotyped with VSVg and EboGP (F); or single cycle HIV pseudotyped with VSVg or GP from EBOV, Taï Forest virus (TAFV), Bundibugyo virus (BDBV), Sudan virus (SUDV), Reston virus (RESTV), or Marburg virus (MARV) (G). (H and I) Internalization (H) and fusion (I) of EboGP-VLPs by control and CIITA-overexpressing U2OS cells. No env, nonenveloped control VLPs. (J to M) Infection of control and CIITA-overexpressing U2OS cells by infectious EBOV measured by imaging of GFP reporter (green) and cell nuclei (blue) (J), cell survival (K), infected cells (L), or plaque formation (M). Data are means ± SEM of three independent experiments [(A) to (I)] or experiments with three independent cell clones [(K) to (M)]. Student’s t test [(A), (C), and (K) to (M)] or analysis of variance (ANOVA) with Tukey’s multiple comparison test [(E) to (I)]; *P < 0.05; **P < 0.01; ND, not detected. Scale bars, 100 μm.

CIITA, also known as NLRA, is a nucleotide-binding oligomerization domain (Nod)–like receptor (NLR) (11), but unlike most other NLRs, which function as cytosolic sensors, CIITA is a transcription factor (12). We therefore hypothesized that its antiviral activity occurred through the altered expression of host target genes. Supporting this hypothesis, mutation of domains required for transcriptional activity completely ablated CIITA antiviral activity (fig. S4). Resistance also required NF-Y, a component of the enhanceosome multiprotein complex, which mediates transcriptional activation by CIITA (13), but resistance was independent of another enhanceosome protein, RFX5 (figs. S4 and S5). Antiviral activity was therefore mediated by a subset of NF-Y–dependent, RFX5-independent CIITA target genes, which includes genes associated with antiviral immunity (14). Systematic knockdown of all CIITA target genes identified a single gene, CD74, required for CIITA-mediated resistance (Fig. 3, A and B). This was confirmed by CRISPR knockout of CD74 expression and function in CIITA-overexpressing cells (Fig. 3C).

Fig. 3 Transcriptional activity of CIITA and enhanceosome components are required for resistance.

(A) Genes regulated by CIITA in U2OS cells, with strongest induced genes identified. Mean of three independent CIITA-expressing clones and controls. (B) EboGP-VSV infection of CIITA-expressing cells treated with small interfering RNA (siRNA) against CIITA transcriptional targets. Data are from two siRNAs per gene, N = 3 independent screens, and bars indicate means with 95% confidence intervals (CIs). One-way ANOVA with Bonferroni’s multiple comparisons; *P < 0.05; **P < 0.01. Dotted lines indicate 99% CIs from no siRNA control. (C) CD74 CRISPR-targeting in CIITA-overexpressing U2OS cells was verified by immunoblot, and infection and survival were measured after EboGP-VSV challenge. Data are means ± SEM of N = 3 experiments using two independent cell clones. (D and E) Ciita and Cd74 expression in wild-type (wt) or Ciita−/− mouse BMDMs with or without priming by IFN-γ and LPS. ko, knockout; NS, not significant. (F and G) Fusion of EboGP-VLPs in unprimed (F) or primed (G) mouse BMDM from Ciita−/− and Cd74−/− mice, measured as geometric mean fluorescence (GMFI) of cleaved CCF2. BLAM, β-lactamase. Data are means ± SEM for independent cultures from three mice per group. Student’s t test; *P < 0.05; **P < 0.01. Similar results were observed in three independent experiments.

Both CIITA and CD74 are expressed at high levels by macrophages and dendritic cells (DCs), which are early targets of EBOV (15, 16). To test whether CIITA has antiviral activity in immune cells, we used primary bone marrow–derived macrophages (BMDMs) from Ciita−/− and Cd74−/− mice. Naïve BMDMs did not express high levels of CIITA or CD74, and they showed no difference in viral fusion. Treatment with interferon-γ (IFN-γ) and lipopolysaccharide (LPS) induced expression of CIITA and CD74, and Ciita−/− and Cd74−/− BMDMs primed with IFN-γ and LPS had higher levels of EboGP-VLP fusion than those observed in equivalent wild-type cells (Fig. 3, D to G, and fig. S6). Similar results were seen in Cd74−/− bone marrow–derived DCs and in a CD74−/− human macrophage-like cell line (differentiated THP-1) (figs. S7 and S8). Thus, endogenous CIITA and CD74 have antiviral activity in primary immune cells, which can be induced by exposure to IFN-γ and LPS.

CD74 is the major histocompatibility complex class II (MHC-II) invariant chain, and human cells express four main isoforms of CD74, which differ in the presence of an N-terminal endoplasmic reticulum (ER) retention signal and an internal thyroglobulin domain (Fig. 4A) (17). Only one CD74 isoform, p41, was able to fully rescue resistance to EboGP-VSV infection in CIITA-expressing, CD74-knockout cells (Fig. 4B and fig. S9). p41 conferred resistance independently of CIITA expression (Fig. 4C), which demonstrates that CD74 p41 expression was sufficient to induce antiviral activity. This property of CD74 was not limited to U2OS cells, as CD74 p41 similarly inhibited fusion when expressed in THP-1 cells (Fig. 4D). The p41 isoform contains the thyroglobulin domain, lacks the ER retention signal, and normally accumulates in endosomes. Mutant constructs of CD74 revealed that only the thyroglobulin domain is essential for antiviral activity, but dissociation from the membrane—either by addition of a furin cleavage site (labeled furin in Fig. 4E) or deletion of the transmembrane sequence (No TM in Fig. 4E)—or delivery to the cell surface by fusion to a heterologous cytoplasmic and transmembrane sequence from tetherin (tetherin in Fig. 4E) almost completely removed antiviral activity (Fig. 4E and fig. S10). Thus, antiviral activity required delivery of the thyroglobulin domain to the endosomal membrane. Electron microscopy showed that EboGP-VSV virions accumulated in late endosomal multivesicular bodies (MVBs) of CIITA- and CD74 p41–expressing cells, with some virions within intraluminal vesicles (Fig. 4F and fig. S11). Confocal microscopy confirmed that virus-like particles (VLPs) localized proximal to CD63 and the ESCRT component Hrs, which mark MVBs (18, 19) (Fig. 4, G and H). Thus, CIITA and CD74 p41 inhibit fusion by arresting viral particles in MVB compartments.

Fig. 4 CD74 p41 inhibits cathepsin-mediated cleavage of EboGP.

(A) Human CD74 isoforms with ER retention signal (ER), CLIP, acidic, and p41 thyroglobulin (Thyro) domains. (B and C) EboGP-VSV infection and survival of Cd74−/− CIITA-expressing (B) or wt (C) U2OS cells expressing CD74 isoforms. (D) EboGP-VLP fusion in THP-1 macrophage-like cells expressing CD74 p33 and p41. (E) EboGP-VSV infection of U2OS cells expressing CD74 mutant constructs. Cyto, cytoplasmic domain; TM, transmembrane domain; Thyro, thyroglobulin domain; CT del, carboxy-terminus deletion; No TM, deletion of the transmembrane sequence. (F) Transmission electron micrographs of control, CIITA-expressing, and CD74-expressing U2OS cells 3 hours after infection with EboGP-VSV. Dotted-line regions are enlarged in adjacent panels (as indicated by white arrows). Intraluminal vesicles (black arrowheads) and internalized EboGP-VSV (black arrows) are marked. Scale bars, 1 μm (left, center left, and right panels) and 200 nm (center right panels). (G) Confocal microscopy of control and p41-expressing U2OS cells showing EBOV-VLP (red), CD63, or Hrs (green), and nuclei (white). Scale bars, 10 μm. (H) VLPs associated with CD63 endosomes in U2OS cells expressing CIITA and CD74 as indicated. Each point represents a single cell, mean ± SD n ≥ 9. Mann-Whitney U test; **P < 0.01. Similar results were seen in three independent experiments. (I) Immunoblot of EboGP in EboGP-VSV–infected U2OS cells. EboGP-VSV preparation ± thermolysin (Therm) is shown for reference (left). Cells were treated with cathepsin inhibitors (Cat. inhib) E64D (E) or FYDMK (F), or expressed CIITA and CD74. EboGP in virus particles (arrow), after proteolysis (closed arrowhead), and after partial cleavage (open arrowhead) are indicated. (J) EboGP-VSV infection of U2OS cells expressing p41 with CTSL binding site mutations. (K) Infection of control, p33-, or p41-expressing U2OS cells by HIV-GFP pseudotyped with GPs from VSV, EBOV, SARS-CoV, or WIV1-CoV, measured as focus-forming units per milliliter of virus (FFU/ml). (L) Infection of control, p33-, or p41-expressing Vero cells by SARS-CoV-2, showing representative crystal violet-stained monolayers and infection measured as plaque-forming units per milliliter of virus (PFU/ml). Except where indicated, data are means ± SEM of data from ≥3 independent experiments. Student’s t test with Benjamini correction; *P < 0.05; **P < 0.01.

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Fig. 4 CD74 p41 inhibits cathepsin-mediated cleavage of EboGP.

(A) Human CD74 isoforms with ER retention signal (ER), CLIP, acidic, and p41 thyroglobulin (Thyro) domains. (B and C) EboGP-VSV infection and survival of Cd74−/− CIITA-expressing (B) or wt (C) U2OS cells expressing CD74 isoforms. (D) EboGP-VLP fusion in THP-1 macrophage-like cells expressing CD74 p33 and p41. (E) EboGP-VSV infection of U2OS cells expressing CD74 mutant constructs. Cyto, cytoplasmic domain; TM, transmembrane domain; Thyro, thyroglobulin domain; CT del, carboxy-terminus deletion; No TM, deletion of the transmembrane sequence. (F) Transmission electron micrographs of control, CIITA-expressing, and CD74-expressing U2OS cells 3 hours after infection with EboGP-VSV. Dotted-line regions are enlarged in adjacent panels (as indicated by white arrows). Intraluminal vesicles (black arrowheads) and internalized EboGP-VSV (black arrows) are marked. Scale bars, 1 μm (left, center left, and right panels) and 200 nm (center right panels). (G) Confocal microscopy of control and p41-expressing U2OS cells showing EBOV-VLP (red), CD63, or Hrs (green), and nuclei (white). Scale bars, 10 μm. (H) VLPs associated with CD63 endosomes in U2OS cells expressing CIITA and CD74 as indicated. Each point represents a single cell, mean ± SD n ≥ 9. Mann-Whitney U test; **P < 0.01. Similar results were seen in three independent experiments. (I) Immunoblot of EboGP in EboGP-VSV–infected U2OS cells. EboGP-VSV preparation ± thermolysin (Therm) is shown for reference (left). Cells were treated with cathepsin inhibitors (Cat. inhib) E64D (E) or FYDMK (F), or expressed CIITA and CD74. EboGP in virus particles (arrow), after proteolysis (closed arrowhead), and after partial cleavage (open arrowhead) are indicated. (J) EboGP-VSV infection of U2OS cells expressing p41 with CTSL binding site mutations. (K) Infection of control, p33-, or p41-expressing U2OS cells by HIV-GFP pseudotyped with GPs from VSV, EBOV, SARS-CoV, or WIV1-CoV, measured as focus-forming units per milliliter of virus (FFU/ml). (L) Infection of control, p33-, or p41-expressing Vero cells by SARS-CoV-2, showing representative crystal violet-stained monolayers and infection measured as plaque-forming units per milliliter of virus (PFU/ml). Except where indicated, data are means ± SEM of data from ≥3 independent experiments. Student’s t test with Benjamini correction; *P < 0.05; **P < 0.01.

EBOV entry requires endosomal cathepsins (4, 10, 20) (fig. S12), which sequentially process EboGP (Fig. 4I and fig. S13). The CD74 thyroglobulin domain inhibits cathepsins (21), which suggests that this may be the mechanism for antiviral activity. In support of this, CD74 inhibited EboGP processing, similar to the effects of the cathepsin L (CTSL) inhibitor FYDMK (Fig. 4I). Additionally, disruption of the p41 CTSL binding site (22, 23) by mutation completely inhibited antiviral activity (Fig. 4J and fig. S10). GP cleavage by endosomal proteases facilitates the entry of other viruses, including coronaviruses. SARS-CoV and SARS-CoV-2 S proteins can be processed by either endosomal cathepsin B and CTSL or alternatively by cell-surface serine proteases including TMPRSS2 (24, 25). In TMPRSS-expressing cells, such as lung epithelium, inhibition of both cathepsins and serine proteases is required to inhibit viral entry, whereas cathepsin inhibitors alone block infection in cell lines—such as U2OS and Vero cells—that lack TMPRSS2 (25). p41 inhibited the entry of viruses pseudotyped with S proteins from SARS-CoV and a related bat virus, WIV1-CoV, into U2OS cells, which demonstrates that p41 inhibited S protein processing (Fig. 4K). To determine whether p41 exhibited antiviral activity against authentic SARS coronavirus, we challenged p41-expressing Vero E6 cells with SARS-CoV-2. CD74 p41 expression completely inhibited plaque formation, which demonstrates that this antiviral activity extended beyond filoviruses (Fig. 4L).

Here, we identify the antiviral activity of CIITA and CD74. We show that CIITA induces resistance by up-regulation of the p41 isoform of CD74, which blocks cathepsin-mediated cleavage of viral GPs, thereby preventing viral fusion. This antiviral activity protects against a wide range of cathepsin-dependent viruses, including filoviruses and coronaviruses; functions in macrophages and DCs that are early targets of infection (15, 16); and is activated by IFN-γ. We demonstrate that CIITA and CD74 mediate the endosomal sequestration of certain viruses as a mechanism of cellular host defense. We speculate that this activity is evolutionarily ancient and precedes their better-known role in antigen processing. We anticipate that the application of this transposon screening approach to other models of infection will reveal additional mechanisms that have eluded conventional screening strategies.

Supplementary Materials

Acknowledgments: We thank M. Mason, M. Rosasco, S. Presnell, and the Bioinformatics Department at Benaroya Research Institute (BRI) for support in data analysis and V. Gersuk and the BRI genomics core for sequencing. We thank B. Schneider and S. MacFarlane from the Electron Microscopy Resource at Fred Hutch for help with transmission electron microscopy experiments and L. Eisenlohr and M. O’Mara at Children’s Hospital of Philadelphia for providing Cd74-knockout mouse bone marrow. Funding: This work was supported by National Institutes of Health grants R33AI102266, U01AI070330, and R33AI119341 (to A.L.-H. and L.M.S.); U19AI125378-04S1 (to A.L.-H.); and R21AI135912 (to E.M.). Work at NIAID Integrated Research Facility was funded by contract no. HHSN272200700016I to Battelle Memorial Institute (BMI). J.J. performed this work as an employee of BMI. SARS-CoV-2 work was performed in the BSL3 at Case Western Reserve University (CWRU), which is supported by the CWRU and University Hospitals Center for AIDS research grant P30AI36219. Author contributions: A.B. performed most of the experiments. Screen and data analysis tools were developed by K.S. BSL4 experiments were performed by J.J. and G.G.O., minigenome experiments were performed by A.J.H. and E.M., and K.A.M. designed all CD74 and CIITA mutations. H.M., R.P., and L.G. provided technical assistance. C.S. assisted with data analysis and visualization. G.G.O. and E.M. provided assistance with experimental planning and data interpretation. L.C., E.V.S., L.M.S., and A.L.-H. conceived the study. The manuscript was written by A.L.-H. and L.M.S. with assistance from A.B. and K.S. Competing interests: E.V.S. is presently an employee of Merck and Co., Inc., Kenilworth, NJ, and holds stock in Merck and Co. This work was conducted before E.V.S.’s affiliation with Merck. The authors declare no other competing interests. Data and materials availability: Full analysis of screen results is presented in the supplementary materials. DNA and RNA sequencing data are deposited at Gene Expression Omnibus (under accession nos. GSE156598 and GSE155204, respectively). The PB transposon was obtained under a material transfer agreement with the Wellcome Trust Sanger Institute. All other data are available in the manuscript or the supplementary materials. This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using such material.

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This 14-Year-Old’s Discovery Could Lead to a Cure for COVID-19

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With no clear end in sight to the pandemic, there is an urgent need for a cure to COVID-19. As scientists around the world work to develop possible vaccines, one 14-year-old girl from Texas has made a new discovery that could lead to a potential treatment.

On Wednesday, Anika Chebrolu from Frisco, Texas was named the winner of the 3M Young Scientist Challenge after discovering a molecule that can selectively bind to the spike protein of the SARS-COV-2 virus, which causes COVID-19.

The competition opened in December 2019 and invited students in grades five to eight to find a unique solution to an everyday problem. Anika won $25,000, a special destination trip, and the title of “America’s Top Young Scientist” for her achievement.

Her discovery could lead to important developments in COVID-19 research. By binding to the spike protein in the coronavirus, the molecule she found can potentially prevent virus entry into the host cell, and can be used in creating a potential drug to cure COVID-19.

Anika used in-silico methodology — methods and experiments that make use of computers — to screen millions of small molecules. She originally planned for her project to focus on the influenza virus, but pivoted once COVID-19 hit and she realized the severity of the pandemic. Anika was in eighth grade when she submitted the project.

She told CNN that she hopes to work with other scientists and researchers to develop her discovery into an actual cure for the virus.

At the moment, the World Health Organization (WHO) is tracking over 170 candidate vaccines around the world. However, since many of them are still in early development, the effectiveness of these vaccines are still unknown. Vaccines go through multiple stages of testing and experts predict that a vaccine will only be available to the public in 2021, at the earliest.

In August, Russia was the first country to claim to have developed a vaccine for COVID-19. However, many were skeptical as President Vladimir Putin had ordered to speed up clinical trials. The vaccine, which was not subject to the extensive Phase III testing, was registered after less than two months of human testing. Experts have said that the vaccine is based on a common cold virus, which many people have been exposed to, potentially limiting its effectiveness. Other countries working on vaccines include the United Kingdom, Germany, and China.

At the moment, there are no specific vaccines or drugs for COVID-19. Developing one to prevent or cure infection from the novel coronavirus could help decrease the number of fatalities and help hospitals manage patients better.

As of posting, there have been a total of over 40 million cases of COVID-19 and 1.1 million deaths around the world. The United States and India have the most number of cases, with 8.1 million and 7.5 million total COVID-19 cases respectively. Few places across the globe have successfully managed  the virus, including New Zealand, Taiwan, and Singapore.

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Mammalian lipid droplets are innate immune hubs integrating cell metabolism and host defense

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Cells drop a bomb on pathogens

Lipid droplets (LDs) accumulate in cells to serve as lipid storage organelles. They are also an attractive source of nutrients for many pathogens. Bosch et al. show that various proteins involved in innate immunity form complexes on LDs in response to bacterial lipopolysaccharide (see the Perspective by Green). Upon activation, LDs became physically uncoupled from mitochondria, driving a shift in cells from oxidative phosphorylation to aerobic glycolysis. This work highlights the ability of LDs both to kill pathogens directly and to establish a metabolic environment conducive to host defense. This may inform future antimicrobial strategies in the age of antibiotic resistance.

Science, this issue p. eaay8085; see also p. 294

Structured Abstract

INTRODUCTION

In all eukaryotic cells, lipid droplets (LDs) store and supply essential lipids to produce signaling molecules, membrane building blocks, and metabolic energy. The LD monolayer also accommodates proteins not obviously related to lipids, such as transcription factors, chromatin components, and toxic proteins.

Common parasites (such as trypanosomes and Plasmodium falciparum), bacteria (such as mycobacteria and Chlamydia), and viruses (such as hepatitis C and dengue) induce and target LDs during their life cycles. The current view is that LDs support infection, providing microorganisms with substrates for effective growth.

RATIONALE

Successful innate defense is critical for survival, and host species have efficiently coevolved with pathogens to develop a plethora of immune responses. Multiple cues, including cellular stress and danger-associated molecular patterns such as lipopolysaccharide (LPS), induce LD formation. Thus, LD localization and dynamics may potentially be advantageous for organizing an intracellular host defense. We have investigated the possibility that mammalian LDs have a direct and regulated role in innate immunity.

RESULTS

We show that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated during polymicrobial sepsis and by LPS. Light and electron microscopy demonstrated specific association of LDs and bacteria in human macrophages, suggesting the existence of docking mechanisms that facilitate the engagement of antibacterial LD proteins with bacteria.

A comparative mass spectrometry profiling of proteins differentially associated with LDs in response to LPS (LPS-LDs) revealed the profound remodeling of the organelle proteome. A stringent evaluation identified 689 proteins differentially regulated on LPS-LDs (317 enriched and 372 reduced). Ingenuity Pathway Analysis revealed an enrichment of innate immune system–related components and reduction of metabolism-related LD-resident proteins. Additional analyses suggested that LDs serve as innate immune hubs, integrating major intra- and extracellular immune responses.

Among the five members of the perilipin family of LD surface proteins (PLINs), PLIN5 was the only one down-regulated on LPS-LDs. PLIN5 reduction promoted physical and functional disconnection of LPS-LDs and mitochondria, with a concomitant reduction of oxidative metabolism and ketogenesis. Forced PLIN5 reexpression increased the number of LD-mitochondria contacts, reducing LD-bacteria interactions and compromising the antimicrobial capacity of cells.

By contrast, PLIN2 was the most up-regulated PLIN on LPS-LDs. Gene interaction analysis revealed that multiple immune proteins nucleated around PLIN2 in response to LPS. LPS-LDs accrued several interferon-inducible proteins such as viperin, IGTP, IIGP1, TGTP1, and IFI47. Furthermore, LPS-LDs also accumulated cathelicidin (CAMP), a broad-spectrum antimicrobial peptide with chemotactic properties. Cells overexpressing a LD-associated CAMP were more resistant to different bacterial species, including Escherichia coli, methicillin-resistant Staphylococcus aureus, and Listeria monocytogenes.

CONCLUSION

These results demonstrate that LDs form a first-line intracellular defense. They act as a molecular switch in innate immunity, responding to danger signals by both reprogramming cell metabolism and eliciting protein-mediated antimicrobial mechanisms. Mechanisms of LD trafficking and docking with phagocytic and parasitophorous membranes, observed here and described for several pathogens, may facilitate the delivery of immune proteins located on the LD surface. Intracellular LDs can provide infected cells with several biological benefits, serving as a location to attract pathogens as well as coordinating different immune systems that operate simultaneously against different classes of pathogens. LDs may also sequester cytotoxic compounds (such as antimicrobial peptides), reducing damage to other cellular organelles. In view of the widespread resistance to current antibiotics, this study helps decipher molecular mechanisms involved in antimicrobial defense that could be exploited for development of new anti-infective agents.

LDs mediate innate immune defense.

Serial blockface scanning electron microscopy data reconstruction showing an infected macrophage. Bacteria (blue) and LDs (green) in the three-dimensional dataset have been colored and projected onto a single image. LDs associate with the bacteria surface (black square). This interaction is proposed to bring a specific set of antipathogenic proteins in contact with the membrane-enclosing bacteria (inset).

Abstract

Lipid droplets (LDs) are the major lipid storage organelles of eukaryotic cells and a source of nutrients for intracellular pathogens. We demonstrate that mammalian LDs are endowed with a protein-mediated antimicrobial capacity, which is up-regulated by danger signals. In response to lipopolysaccharide (LPS), multiple host defense proteins, including interferon-inducible guanosine triphosphatases and the antimicrobial cathelicidin, assemble into complex clusters on LDs. LPS additionally promotes the physical and functional uncoupling of LDs from mitochondria, reducing fatty acid metabolism while increasing LD-bacterial contacts. Thus, LDs actively participate in mammalian innate immunity at two levels: They are both cell-autonomous organelles that organize and use immune proteins to kill intracellular pathogens as well as central players in the local and systemic metabolic adaptation to infection.

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The nucleus acts as a ruler tailoring cell responses to spatial constraints

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The nucleus makes the rules

Single cells continuously experience and react to mechanical challenges in three-dimensional tissues. Spatial constraints in dense tissues, physical activity, and injury all impose changes in cell shape. How cells can measure shape deformations to ensure correct tissue development and homeostasis remains largely unknown (see the Perspective by Shen and Niethammer). Working independently, Venturini et al. and Lomakin et al. now show that the nucleus can act as an intracellular ruler to measure cellular shape variations. The nuclear envelope provides a gauge of cell deformation and activates a mechanotransduction pathway that controls actomyosin contractility and migration plasticity. The cell nucleus thereby allows cells to adapt their behavior to the local tissue microenvironment.

Science, this issue p. eaba2644, p. eaba2894; see also p. 295

Structured Abstract

INTRODUCTION

The human body is a crowded place. This crowding is even more acute when the regulation of cell growth and proliferation fails during the formation of a tumor. Dealing with the lack of space in crowded environments presents cells with a challenge. This is especially true for immune cells, whose task is to patrol tissues, causing them to experience both acute and sustained deformation as they move. Although changes in tissue crowding and associated cell shape alterations have been known by pathologists to be key diagnostic traits of late-stage tumors since the 19th century, the impact of these changes on the biology of cancer and immune cells remains unclear. Moreover, it is not known whether cells can detect and adaptively respond to deformations in densely packed spaces.

RATIONALE

To test the hypothesis that cells possess an ability to detect and respond to environmentally induced changes in their shape, we fabricated artificial microenvironments that mimic the conditions experienced by tumor and immune cells in a crowded tissue. By combining dynamic confinement, force measurements, and live cell imaging, we were able to quantify cell responses to precisely controlled physical perturbations of their shape.

RESULTS

Our results show that, although cells are surprisingly resistant to compressive forces, they monitor their own shape and develop an active contractile response when deformed below a specific height. Notably, we find that this is achieved by cells monitoring the deformation of their largest internal compartment: the nucleus. We establish that the nucleus provides cells with a precise measure of the extent of their deformation. Once cell compression exceeds the size of the nucleus, it causes the bounding nuclear envelope (NE) to unfold and stretch. The onset of the contractile response occurs when the NE reaches a fully unfolded state. This transition in the mechanical state of the NE and its membranes permits calcium release from internal membrane stores and activates the calcium-dependent phospholipase cPLA2, an enzyme known to operate as a molecular sensor of nuclear membrane tension and a critical regulator of signaling and metabolism. Activated cPLA2 catalyzes the formation of arachidonic acid, an omega-6 fatty acid that, among other processes, potentiates the adenosine triphosphatase activity of myosin II. This induces contractility of the actomyosin cortex, which produces pushing forces to resist physical compression and to rapidly squeeze the cell out of its compressive microenvironment in an “evasion reflex” mechanism.

CONCLUSION

Although the nucleus has traditionally been considered a passive storehouse for genetic material, our work identifies it as an active compartment that rapidly convers mechanical inputs into signaling outputs, with a critical role of its envelope in this sensing function. The nucleus is able to detect environmentally imposed compression and respond to it by generating a signal that is used to change cell behaviors. This phenomenon plays a critical role in ensuring that cells, such as the immune cells within a tumor, can adapt, survive, and efficiently move through a crowded and mechanically heterogeneous microenvironment. Characterizing the full spectrum of signals triggered by nuclear compression has the potential to elucidate mechanisms underlying signaling, epigenetic, and metabolic adaptations of cells to their mechanoenvironment and is thus an exciting avenue for future research.

The nuclear ruler and its contribution to the “life cycle” of a confined cell.

(1) Cell confinement below resting nucleus size, leading to nuclear deformation and to unfolding, and stretching of the nuclear envelope. (2) Nuclear membrane tension increase, which triggers calcium release, cPLA2 activation, and arachidonic acid (ARA) production. (3) Actomyosin force (F) generation. (4) Increased cell migratory capacity and escape from confinement.

Abstract

The microscopic environment inside a metazoan organism is highly crowded. Whether individual cells can tailor their behavior to the limited space remains unclear. In this study, we found that cells measure the degree of spatial confinement by using their largest and stiffest organelle, the nucleus. Cell confinement below a resting nucleus size deforms the nucleus, which expands and stretches its envelope. This activates signaling to the actomyosin cortex via nuclear envelope stretch-sensitive proteins, up-regulating cell contractility. We established that the tailored contractile response constitutes a nuclear ruler–based signaling pathway involved in migratory cell behaviors. Cells rely on the nuclear ruler to modulate the motive force that enables their passage through restrictive pores in complex three-dimensional environments, a process relevant to cancer cell invasion, immune responses, and embryonic development.

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