SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosisстатья из журнала
Аннотация: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of COVID-19, so understanding its biology and infection mechanisms is critical to facing this major medical challenge. SARS-CoV-2 is known to use its spike glycoprotein to interact with the cell surface as a first step in the infection process. As for other coronaviruses, it is likely that SARS-CoV-2 next undergoes endocytosis, but whether or not this is required for infectivity and the precise endocytic mechanism used are unknown. Using purified spike glycoprotein and lentivirus pseudotyped with spike glycoprotein, a common model of SARS-CoV-2 infectivity, we now demonstrate that after engagement with the plasma membrane, SARS-CoV-2 undergoes rapid, clathrin-mediated endocytosis. This suggests that transfer of viral RNA to the cell cytosol occurs from the lumen of the endosomal system. Importantly, we further demonstrate that knockdown of clathrin heavy chain, which blocks clathrin-mediated endocytosis, reduces viral infectivity. These discoveries reveal that SARS-CoV-2 uses clathrin-mediated endocytosis to gain access into cells and suggests that this process is a key aspect of virus infectivity. The internal membrane system represents a major evolutionary advance associated with the emergence of the eukaryotic cell. Its dynamics are integral to basic cellular function and underlie much of the novel capability of the cell, including the almost infinite range of physiological responsiveness required for the complexity of eukaryotic organisms. Unfortunately, viruses subvert this complexity to gain access into cells and to propagate in a manner that allows them to limit immune surveillance. Antiviral strategies targeting the earliest steps of infection, such as cellular entry, are appealing, as interference through a common early step can provide robust efficacy. Thus, understanding the mechanisms of viral cellular entry is crucial. Since the beginning of the 21st century, 3 coronaviruses have crossed the species barrier to cause deadly types of pneumonia in humans: Middle-East respiratory syndrome coronavirus (MERS-CoV) (1Zaki A.M. van Boheemen S. Bestebroer T.M. Osterhaus A.D. Fouchier R.A. Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia.N. Engl. J. 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The S1 subdomain encodes the receptor binding domain and is responsible for binding to host cells, whereas the S2 subdomain encodes the transmembrane portion of the spike protein and is responsible for fusion of the viral membrane with cellular membranes. The receptor for the spike glycoprotein is angiotensin-converting enzyme 2 (ACE2) (9Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280Abstract Full Text Full Text PDF PubMed Scopus (12600) Google Scholar, 10Letko M. Marzi A. Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses.Nat. Microbiol. 2020; 5: 562-569Crossref PubMed Scopus (1954) Google Scholar, 11Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. 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Pathol. 2004; 203: 631-637Crossref PubMed Scopus (4013) Google Scholar). The receptor binding domain of the spike protein binds ACE2 with low nM affinity, allowing the virus to stably associate with the plasma membrane (12Walls A.C. Xiong X. Park Y.J. Tortorici M.A. Snijder J. Quispe J. Cameroni E. Gopal R. Dai M. Lanzavecchia A. Zambon M. Rey F.A. Corti D. Veesler D. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion.Cell. 2019; 176: 1026-1039Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar). Cleavage of the spike glycoprotein between the S1 and S2 domains, mediated by the type II transmembrane serine protease TMPRSS2 (9Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280Abstract Full Text Full Text PDF PubMed Scopus (12600) Google Scholar), and perhaps by furin, activates the S2 subdomain. The S2 subdomain then mediates the fusion of the viral membrane and the cellular membrane in a process that is a molecular mimic of SNARE-mediated fusion of cellular membranes (12Walls A.C. Xiong X. Park Y.J. Tortorici M.A. Snijder J. Quispe J. Cameroni E. Gopal R. Dai M. Lanzavecchia A. Zambon M. Rey F.A. Corti D. Veesler D. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion.Cell. 2019; 176: 1026-1039Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 16Hamming I. Timens W. Bulthuis M.L. Lely A.T. Navis G. van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis.J. 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Molecular basis of binding between novel human coronavirus MERS-CoV and its receptor CD26.Nature. 2013; 500: 227-231Crossref PubMed Scopus (539) Google Scholar) and a second stating the opposite that viral entry before infectivity uses a clathrin-independent process (27Inoue Y. Tanaka N. Tanaka Y. Inoue S. Morita K. Zhuang M. Hattori T. Sugamara T. Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic Tail Deleted.J. Virol. 2007; 81: 8722-8729Crossref PubMed Scopus (270) Google Scholar). Here we demonstrate rapid, clathrin-mediated endocytosis of SARS-CoV-2 and provide evidence that this process is critical for infectivity. HEK-293T and A549 cell lines were obtained from the American Type Culture Collection (CRL-1573, CCL-185). The adherent Vero-SF-ACF cell line is from the American Type Culture Collection (CCL-81.5) (29Kiesslich S. Losa J.P.V.-C. Gelinas J.-F. Kamen A.A. Serum-free production of rVSV-ZEBOV in Vero cells: Microcarrier bioreactor versus scale-X hydron fixed bed.J. Biotech. 2020; 310: 32-39Crossref PubMed Scopus (18) Google Scholar). The HEK-293T-ACE2 stable cell line was a gift from Dr Jesse D. Bloom (30Crawford H.D.K. Eguia R. Dingens A.S. Loes A.N. Malone K.D. Wolf C.R. Chu H.Y. Tortorici M.A. Veesler D. Murphy M. Pettie D. King N.P. Balazs A.B. Bloom J.D. Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays.Viruses. 2020; 12513Crossref PubMed Scopus (410) Google Scholar). All cells were cultured in Dulbecco's modified Eagle's medium (DMEM) high-glucose (GE Healthcare cat# SH30081.01) containing 10% bovine calf serum (GE Healthcare cat# SH30072.03), 2-mM L-glutamate (Wisent cat# 609065), 100 IU penicillin, and 100 μg/ml streptomycin (Wisent cat# 450201). Cell lines were checked monthly for Mycoplasma contamination using the Mycoplasma detection kit (biotool cat# B39038). Cells were collected in Hepes lysis buffer (20-mM Hepes, 150-mM sodium chloride, 1% Triton X-100, pH 7.4) supplemented with protease inhibitors. Cells in the lysis buffer were gently rocked for 30 min (4 °C). Lysates were spun at 238,700g for 15 min at 4 °C and equal protein aliquots of the supernatants were analyzed by SDS-PAGE and immunoblot. Lysates were run on large 5 to 16% gradient polyacrylamide gels and transferred to nitrocellulose membranes. Proteins on the blots were visualized by Ponceau staining. Blots were then blocked with 5% milk, and antibodies were incubated O/N at 4 °C with 5% bovine serum albumin in tris-buffered saline with 0.1% Tween 20. The peroxidase conjugated secondary antibody was incubated in a 1:5000 dilution in tris-buffered saline with 0.1% Tween 20 with 5% milk for 1 h at room temperature (RT) followed by washes. SARS-CoV-2 spike protein antibody is from GeneTex (GTX632604). ACE2 antibody is from GeneTex (GTX01160). 6x-His Tag antibody is from Thermo Fisher Scientific (MA1-21315-D550). GAPDH antibody is from OriGene (TA802519). HSC70 antibody is from Enzo (ADI-SPA-815-F). Clathrin heavy chain (CHC) antibody is from Cell Signaling Technology (4796S). Conjugated transferrin (Tf) antibody is from Thermo Fisher Scientific (T23366). Alexa Fluor 488, 568, and 647 conjugated secondary antibodies are from Invitrogen. Cells grown on poly-L-lysine–coated coverslips were fixed in 4% paraformaldehyde for 10 min and then washed 3 times with PBS. Cells were then permeabilized in 0.2% Triton X-100 in PBS and blocked with 5% bovine serum albumin in PBS for 1 h. The coverslips were incubated in a wet chamber with diluted antibody in a blocking buffer overnight at 4 °C. The following day, cells were washed 3 times and incubated with corresponding Alexa Fluorophore diluted in the blocking buffer for 1 h at RT. Cells were again washed 3 times with a blocking buffer and once with PBS. Nuclei were stained using 4′,6-diamidino-2-phenylindole (DAPI) (1 μg/ml diluted in a blocking buffer) for 10 min. Finally, coverslips were mounted on a slide using mounting media (DAKO, Cat# S3023). Imaging was performed using a Leica TCS SP8 confocal microscope, Zeiss LSM-880 confocal microscope, and Opera Phoenix High-Content Screening microscope. Cells were incubated at 37 °C with serum-free media (DMEM) for 3 h to enhance ACE2 receptor expression. Before the addition of spike protein, cells were cooled to 4 °C by being placed on ice. Spike protein was added to each well (3 μg per 200 μl of media) and incubated on ice for 30 min (to allow ligand attachment to the cell surface). Subsequently, cells were incubated at 37 °C (to allow internalization) for indicated time points (Tf was also added in some experiments in the same manner). Before fixation, cells were acid washed (washing off extracellular spike protein) or PBS washed for 1 min and rinsed with acid or PBS. Cells were then washed 3 times with PBS followed by fixation for 10 min at 4 °C. Various cell types at 60% confluency were transfected with siRNA targeted against CHC (Dharmacon; SMARTpool: ON-TARGETplus; L-004001–01–0010) or control siRNA (Dharmacon; ON-TARGETplus CONTROL) using the jetPRIME reagent. On day 3, cells were processed for immunoblot to investigate the effect of siRNA, and in parallel, cells were infected with pseudovirus or used for spike endocytosis assays. Purified SARS-CoV-2 spike protein prefusion-stabilized ectodomain (C-term His tag, with furin cleavage site removed, trimerization stabilized) was produced by LakePharma (#46328). Lentivirus pseudotyped with SARS-CoV-2 spike protein was supplied by Creative Diagnostics (catalog number COV-PS02). Cells were washed five times with serum-free media (DMEM) to remove any traces of serum. Furthermore, cells were incubated with Dynasore (80 μM; Abcam, Ab120192) or Pitstop 2 (15 μM; Abcam, ab120687) in serum-free media for 30 or 20 min, respectively. DMSO was used as a control for Dynasore and Pitstop 2 negative control (Abcam, ab120688). After incubation, drug-containing media was replaced with spike protein (3 μg per 200 μl of media) and further incubated for 5 min to allow internalization. Finally, cells were processed for immunofluorescence labeling. Cells were transfected with control siRNA or siRNA targeted against CHC (Dharmacon) using jetPRIME. Cells were split on day 2, and 10,000 cells were seeded in each well (96-well plate; CellCarrier-96 Ultra Microplates, PerkinElmer). At 12 to 14 h after cell seeding, cells were incubated with concentrated pseudovirus SARS-Cov-2 for 12 h. After this incubation, pseudovirus containing media was replaced with regular DMEM media and incubated further for 48 h. Cells were fixed with 4% paraformaldehyde for 10 min and stained with DAPI. Purified spike protein and Tf (internalized or on the plasma membrane): ImageJ was used to measure the fluorescence intensity. The DAPI channel was used to count the number of cells per image. This was performed by calculating the maxima using the "Find Maxima" function. The threshold was set at 150. This automated the way in which to count the cells. Then, spike protein or Tf fluorescence corresponding to the cell count was calculated using the "measure" function. Pearson Correlation Coefficient: Using ImageJ Coloc2 plugin, colocalization was calculated after background subtraction. The colocalization was calculated for 144 × 144 μm. Fluorescence was then divided by cell count to calculate the fluorescence per cell in each condition. Infection of cells using pseudovirus SARS-Cov-2: GFP-expressing cells in CellCarrier-96 Ultra Microplate were imaged using Opera Phenix HCS (40× objective). The total number of GFP expressing cells in each condition was normalized to the total number of cells in the respective wells. Graphs were prepared using GraphPad Prism 6 software. For all data, comparisons were made using Student's t test or one-way ANOVA. All data are shown as the mean ± SEM with p < 0.05 considered statistically significant. The spike glycoprotein is critical for binding ACE2 and allowing for SARS-CoV-2 infectivity (6Tortorici M.A. Veesler D. Structural insights into coronavirus entry.Adv. Virus Res. 2019; 105: 93-116Crossref PubMed Scopus (516) Google Scholar, 8Wrapp D. Wang N. Corbett K.S. Goldsmith J.A. Hsieh C.L. Abiona O. Graham B.S. McLellan J.S. Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation.Science. 2020; 367: 1260-1263Crossref PubMed Scopus (85) Google Scholar, 9Hoffmann M. Kleine-Weber H. Schroeder S. Krüger N. Herrler T. Erichsen S. Schiergens T.S. Herrler G. Wu N.H. Nitsche A. Müller M.A. Drosten C. Pöhlmann S. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor.Cell. 2020; 181: 271-280Abstract Full Text Full Text PDF PubMed Scopus (12600) Google Scholar, 10Letko M. Marzi A. Munster V. Functional assessment of cell entry and receptor usage for SARS-CoV-2 and other lineage B betacoronaviruses.Nat. Microbiol. 2020; 5: 562-569Crossref PubMed Scopus (1954) Google Scholar, 11Zhou P. Yang X.L. Wang X.G. Hu B. Zhang L. Zhang W. Si H.R. Zhu Y. Li B. Huang C.L. Chen H.D. Chen J. Luo Y. Guo H. Jiang R.D. et al.A pneumonia outbreak associated with a new coronavirus of probable bat origin.Nature. 2020; 579: 270-273Crossref PubMed Scopus (13649) Google Scholar, 12Walls A.C. Xiong X. Park Y.J. Tortorici M.A. Snijder J. Quispe J. Cameroni E. Gopal R. Dai M. Lanzavecchia A. Zambon M. Rey F.A. Corti D. Veesler D. Unexpected receptor functional mimicry elucidates activation of coronavirus fusion.Cell. 2019; 176: 1026-1039Abstract Full Text Full Text PDF PubMed Scopus (392) Google Scholar, 16Hamming I. Timens W. Bulthuis M.L. Lely A.T. Navis G. van Goor H. Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis.J. Pathol. 2004; 203: 631-637Crossref PubMed Scopus (4013) Google Scholar, 17Weber T. Zemelman B.V. McNew J.A. Westermann B. Gmachl M. Parlati F. Söllner T.H. Rothman J.E. SNAREpins: Minimal machinery for membrane fusion.Cell. 1998; 92: 759-772Abstract Full Text Full Text PDF PubMed Scopus (2042) Google Scholar). WT HEK-293T cells or HEK-293T cells stably expressing ACE2 (30Crawford H.D.K. Eguia R. Dingens A.S. Loes A.N. Malone K.D. Wolf C.R. Chu H.Y. Tortorici M.A. Veesler D. Murphy M. Pettie D. King N.P. Balazs A.B. Bloom J.D. Protocol and reagents for pseudotyping lentiviral particles with SARS-CoV-2 Spike protein for neutralization assays.Viruses. 2020; 12513Crossref PubMed Scopus (410) Google Scholar) were incubated for 30 min on ice with purified spike protein containing a His6 tag. After a PBS wash, spike protein binds to the plasma membrane of cells expressing ACE2 but not to the control HEK-293T cells (Fig. 1, A and B). In contrast, Tf, which binds to the Tf receptor (TfR), is detected on the surface of the cells independent of their ACE2 status (Fig. 1, A and B). After a brief acid wash, both spike protein and Tf are stripped from the surface of cells (Fig. 1, C and D). Thus, HEK-293T cells lacking or expressing ACE2 are a functional model system for examining potential SARS-CoV-2 endocytosis. We first tested for endocytosis using purified spike glycoprotein. Although a reductionist approach, it is unlikely that receptor binding and initial membrane trafficking depends on whether or not the spike protein is on a viral particle. For example, epidermal growth factor and insulin both undergo similar cell surface binding and clathrin-mediated endocytosis whether they are purified proteins or attached to magnetic beads (31Li H.-S. Stolz D.B. Romero G. Characterization of endocytic vesicles using magnetic microbeads coated with signaling ligands.Traffic. 2005; 6: 324-334Crossref PubMed Scopus (32) Google Scholar). We thus added purified spike protein to HEK-293T cells, WT or expressing ACE2. After 30 min on ice, the cells were transferred to 37 °C for an additional 30 min. After this treatment, cells were washed briefly with acid to strip off any surface-bound spike protein. Spike protein was internalized into the cells (as revealed by its resistance to acid wash) in an ACE2-dependent manner (Fig. 2, A and B). In contrast, Tf was internalized independent of ACE2 (Fig. 2, A and B). We next examined the time course of internalization. Spike protein is internalized into cells rapidly and is detected in cells within 5 min (Fig. 3, A and B), a hallmark of endocytosis. The amount of spike protein in cells continues to increase for up to 30 min (Fig. 3, A and B). Thus, SARS-CoV-2 spike protein enters cells via endocytosis. There are conflicting reports about endocytosis of coronaviruses in general and SARS-CoV specifically (27Inoue Y. Tanaka N. Tanaka Y. Inoue S. Morita K. Zhuang M. Hattori T. Sugamara T. Clathrin-dependent entry of severe acute respiratory syndrome coronavirus into target cells expressing ACE2 with the cytoplasmic Tail Deleted.J. Virol. 2007; 81: 8722-8729Crossref PubMed Scopus (270) Google Scholar, 28Wang H. Yang P. Liu K. Guo Z. Zhang Y. Zhang G. Jiang C. SARS coronavirus entry into host cells through a novel clathrin- and caveolae-independent endocytic pathway.Cell Res. 2008; 18: 290-301Crossref PubMed Scopus (519) Google Scholar). The endocytic mechanisms of SARS-CoV-2 have yet to be examined. Clathrin-mediated endocytosis is a major mechanism for cellular internalization. We thus used HEK-293T cells stably expressing ACE2 to examine the internalization of SARS-CoV-2 in the presence of two drugs that are known to block clathrin-mediated endocytosis, Dynasore and Pitstop 2 (32Marcia E. Ehrlich M. Massol R. Boucrot E. Brunner C. Kirchhausen T. Dynasore, a cell-permeable inhibitor of dynamin.Dev. Cell. 2006; 10: 839-850Abstract Full Text Full Text PDF PubMed Scopus (1558) Google Scholar, 33von Kleist L. Stahlschmidt W. Bulut H. Gromova K. Puchkov D. Robertson M.J. MacGregor K.A. Tomilin N. Pechstein A. Chau N. Chircop M. Sakoff J. von Kries J.P. Saenger W. Kräusslich H.G. et al.Role of the clathrin terminal domain in regulating coated pit dynamics revealed by small molecule inhibition.Cell. 2011; 146: 471-484Abstract Full Text Full Tex
Год издания: 2021
Авторы: Armin Bayati, Rahul Kumar, Vincent Francis, Peter S. McPherson
Издательство: Elsevier BV
Источник: Journal of Biological Chemistry
Ключевые слова: SARS-CoV-2 and COVID-19 Research, Cellular transport and secretion, Lipid Membrane Structure and Behavior
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Том: 296
Страницы: 100306–100306