are enveloped, negative single-strand RNA viruses of the Filoviridae
]. Since the discovery of Marburg virus (MARV) in 1967 [2
] and Ebola virus (EBOV) in 1976 [3
], the US Centre of Disease Control has reported several epidemic outbreaks in humans and nonhuman primates [4
]. Despite intense world-wide research efforts, no antiviral treatments or vaccines have yet been licensed. In addition to primates, filoviruses infect pigs, dogs, duikers, and fruit bats in nature, and rodents and ferrets can be infected experimentally [6
The viral glycoprotein (GP), the only viral surface protein, exclusively mediates the entry and internalization of filoviruses into cells. The precursor protein GP0 is synthesized on the endoplasmic reticulum, and cleaved in the constitutive secretory pathway into the surface unit GP1, which binds to host cell factors, and the transmembrane unit GP2, which mediates fusion of viral envelopes with endosomal membranes. Filoviruses display a broad cell tropism [13
]. Almost any cell type with the notable exception of lymphocytes is susceptible to infection by authentic filoviruses in vitro [14
], or to transduction by retrovirus particles pseudotyped with GP [16
]. Moreover, immortalized cell lines cultured in suspension are resistant to filovirus entry, while cell adhesion enhances susceptibility to infection [18
]. Thus, the broad cell tropism observed in infected primates, where virus can be isolated from all organs but not from lymphocytes [14
], is also recapitulated in vitro.
The availability of host factors on the cell surface that interact with viral envelope GP or with envelope lipids such as phosphatidylserine (PtdSer) often determines viral cell tropism. Such virus–host interactions mediate virus attachment, and are a necessary prerequisite for virus internalization, viral fusion with host membranes, and viral genome release into the cytosol for transcription and replication [16
]. Several plasma membrane proteins have been implicated in filovirus attachment: cellular lectins such as asialoglycoprotein receptor (ASGR-R), dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), liver/lymph node-specific intercellular adhesion molecule-3-grabbing non-integrin (L-SIGN), human macrophage C-type lectin specific for galactose and N-acetylglucosamine (hMGL), or liver and lymph node sinusoidal endothelial cell C-type lectin (LSECtin) [24
], T-cell immunoglobulin and mucin domain 1 and 4 (TIM-1, TIM-4) [29
], members of the TAM family (Tyro3, Axl, Mer) of receptor tyrosine kinases [31
], integrin αVβ1 [32
], and scavenger receptor A. However, none of these factors seems to be essential for filoviral infection across cell lines. Rather, their role in cell entry is considered to be cell type dependent, and some of them may promote entry indirectly by regulating downstream processes such as macropinocytosis or GP proteolytic cleavage [34
]. In contrast, several intracellular proteins are essential for filovirus infection in all cell types studied thus far. The endosomal and lysosomal cysteine proteases cathepsin B and cathepsin L cleave GP and thereby expose its receptor binding domain [38
], and the two-pore channel 1 (TPC1) and two-pore channel 2 (TPC2) mediate endolysosomal Ca2+
]. Finally, the endolysosomal cholesterol transporter Niemann–Pick C1 (NPC1) [40
] binds to processed GP1 [42
The remarkable diversity of plasma membrane proteins implicated in filovirus cell entry prompted us to analyze twelve cell lines for a potential correlation of host factor expression to filovirus susceptibility. We could show that the neuroblastoma SH-SY5Y cell line is specifically resistant to filovirus infection although all intracellular proteins known to be essential were expressed, and although its overall transcriptome was very similar to that of susceptible cell lines. Heterokaryon assays revealed that SH-SY5Y cells did not express a dominant restriction factor that inhibited filovirus GP-driven cell entry, but recombinant GP could not bind to their plasma membrane. By individual overexpression of a wide range of different filovirus attachment-promoting proteins, SH-SY5Y cells became susceptible to filovirus GP-driven transduction. Our findings demonstrate that filoviruses—in contrast to many other viruses—can highjack a diverse range of different plasma membrane proteins for the critical cell attachment step, which is a mandatory prerequisite for productive infection of any cell and thus any host.
2. Material and Methods
2.1. Cell Lines
HepG2, Huh-7.5, SW13, HEK293T, EA.hy926, A549, and 786-O cells were a kind gift from Charles M. Rice (Rockefeller University, NY, USA). HPMEC and hTERT-BJ1 cells were generously provided by Andreas Tiede (Department of Hematology, Hemostasis, Oncology, and Stem Cell Transplantation, Hannover Medical School, Hannover, Germany). SK-N-BE(2)-C, SK-N-MC, and SH-SY5Y were kindly provided by Herbert Hildebrant (Department of Cellular Chemistry, Hannover Medical School, Hannover, Germany). Adherent immortalized cell lines were cultured in Dulbecco´s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Calf Serum (FCS) (Sigma-Aldrich, San Luis, MS, USA), penicillin/streptomycin (100 U/mL), nonessential amino acids, and L-glutamine (2 mM) at 37 °C and 5% CO2. The suspension cell line Jurkat was a kind gift of Abel Viejo-Borbolla (Institute of Virology, Hannover Medical School, Hannover) and was cultured with Roswell Park Memorial Institute (RPMI) 1640 medium plus 25 mM HEPES with the same supplements and conditions as adherent cell lines. Primary human hepatocytes (PHH) were provided by Florian Vondran (Department of Surgery, Hannover Medical School, Hannover).
The cell lines Huh-7.5/Tet3G and SH-SY5Y/Tet3G were engineered to stably express Tet-On 3G transactivator protein, and the cell line HEK293T/ZsGreen1 was developed to stably express ZsGreen1 fluorescent reporter protein under the control of Tet-On 3G-inducible PTREG3V promoter. Genetically modified cell lines were generated by stable transduction of parental cell lines either with lentiviral particles of the pLVX-Tet3G plasmid vector (Clontech) for transactivator expression or pLVX-TRE3G-ZsGreen1 for reporter expression. 0.6 mg/mL G418 or 2 µg/mL puromycin were added in the media of transduced cell lines to select for transactivator or reporter expressing cells, respectively. Clone HEK293T-H6/ZsGreen1 was isolated by cell subcloning to obtain a permanent cell line with homogenous reporter expression. Stable cell lines were maintained in culture as parental cell lines with 10% Tet-Free FBS (Clontech, Takara Bio, Mountain View, CA, USA) in instead of FCS.
2.2. DNA Plasmid Constructs
Surrogate HIV-based pseudovirion packaging plasmid HIV Gag-pol, transfer plasmids CSLucW2 (Firefly luciferase expression) or CSGW (hrGFP), and envelope plasmid for VSV-G protein and its backbone plasmid pcDNA3.1 have been described previously [43
]. The pWPI-mCherry-BSD bicistronic expression vector was generated by introduction of the mCherry DNA sequence into a pWPI-BSD empty vector [44
]. Both plasmids were digested with restriction enzymes BamHI and SpeI. The mCherry encoding fragment was gel isolated, purified and ligated into the new lentivirus expressing vector. mCherry expression was confirmed by pseudovirion generation and fluorescence-activated cell sorting (FACS) analysis. Lenti-XTM
inducible expression system plasmids pLVX-Tet3G and pLVX-TRE3G-ZsGreen1 were purchased from Takara Bio. (Mountain View, CA, USA).
GP encoding plasmids pVR1012-GP(Z) (EBOV Mayinga), pVR1012-GP(R) (RESTV Pennsylvania), pVR1012-GP(S) (SUDV Boniface), and pVR1012-GP(IC) (TAFV Cote d´Ivoire) were provided by Drs. Anthony Sanchez and Gary Nabel (NHI, Bethesda, USA). pCAGGS-MARV, pCAGGS-BDBV or pCAGGS-LLOV encoding for MARV Musoke BDBV Bundibugyo and Cuevavirus
LLOV were a gift from Drs. Heinz Feldmann (MARV and BDBV) (NIH, Hamilton, USA) and Ayato Takada (Research Center for Zoonosis Control, Hokkaido, Japan), respectively. EBOV Makona Kissidougou-C15 and mutant variant A82V GP expression plasmids were a donation of Jonathan K. Ball (School of Life Science, University, Nottingham, UK). pCAGGS-SARS-S expressing spike protein S of SARS-CoV strain Frankfurt has previously been described [45
]. pCMV-LassaGPC (expressing LASV strain AV GP precursor) was provided by Dr. Francois-Loic Cosset (INSERM U758, Lyon, France). pMD.RVG.CVS24-B2c encoding for the GP of the rabies virus variant B2c (RABV B2c) was a gift from Manfred Schubert (Addgene plasmid # 19713; http://n2t.net/addgene:19713
; RRID:Addgene_19713). pIRES-EGFP-CHIKV E3-E1 was a kind gift of Dr. Barbara Schnierle (Department of Virology, Paul-Elrich-Institut, Langen, Germany). EBOV Mayinga GP1-human Fc fusion protein expression plasmid pAB61-ZEBOV-GP-Fc and pAB61 are published [46
Overexpressing vectors for tyrosine Axl (pLV[Exp]-Neo-EF1A>hAXL[NM_021913.4]), Mer (pLV[Exp]-Neo-EF1A>hMERTK[NM_006343.2]) or NPC1 (pLV[Exp]-Bsd-EF1A>hNPC1[NM_000271.4]*) were commercially purchased (VectorBuilder Inc, Shenandoah, United States). MLV-based DC-SIGN overexpressing plasmid pQCXIP_hDC-SIGN-AU1 has been described [47
]. For construction of pWPI-HAVCR1-BSD vector, the coding sequence of human TIM-1 (hTIM-1) was synthesized (Integrated DNA Technologies) and amplified by PCR using the forward primer 5’CCTGCAGGCGCGCCGGATCCGCCACC ATGCATCCTC AAGTGGTCAT CTTAAGCCTC ATCCTACATC TGGCAGATTC TGTAGCTGGTTCTGTAAAGG TTGGTGG3′ and the reverse primer 5′GATATCCGGAGCCGCCTCCTCCATCCGTGGCATAAAGACTATTC3′ and cloned into the lentiviral expression vector pWPI-BSD [44
] by Gibson assembly. The hTIM-1 insert was confirmed by sequencing.
2.4. Reagents and Antibodies
The drugs Z-Phe-Tyr(tBu)-diazomethylketone (FYdmk) (219427, Calbiochem, San Diego, CA, USA), U18666A (662015, Calbiochem, San Diego, CA, USA) and tetrandrine (sc-201492, Santa Cruz Biotechnology, Santa Cruz, CA, USA) were dissolved in DMSO according to supplier’s instructions. Working solutions were prepared by dilution of the stock solution with culture medium. Doxycycline hyclate (DOX) (D9891, Sigma-Aldrich, San Luis, MS, USA) for cell–cell fusion reporter expression activation was prepared as 50 mg/mL stock solution in DMSO and stored at −80 °C. The stock solution was diluted in culture media supplemented with Tet-free approved FBS (Clontech, Takara Bio, Mountain View, CA, USA) to 500 ng/mL. Recombinant conjugated protein EGF-AlexaFluor555 for EGF intravesicular accumulation analysis was purchased from Invitrogen™ (E35350) (Carlsbad, CA, USA). Filipin III (F4767) for unesterified cholesterol detection was obtained from Sigma-Aldrich (San Luis, MS, USA).
Mouse monoclonal allophycocyanin(APC)-conjugated antibodies (mAbs) against human surface entry factors Axl (FAB154A), human integrin β1 (FAB17783A), Mer (FAB8912A), or DC-SIGN (FAB161A) and their respective isotype controls, IgG1 (IC002A) and IgG2B (IC0041A) as well as AlexaFluor488-conjugated mAb against human integrin αV (FAB1219G-025) and its IgG1 isotype control (IC002G) were purchased from R&D systems (Minneapolis, MN, USA). Mouse APC mAb against human TIM-1 (353905) or APC-mIgG1 κ isotype control (400121) were obtained from Biolegend Inc. (San Diego, CA, USA). For immunoblot detection of intracellular factors rabbit polyclonal antibodies (pAbs) directed against human procathepsin L + cathepsin L ab (ab200738) or mouse antihuman Niemann Pick-C1 mAb (ab55706, ab134113) were purchased from Abcam (Cambridge, UK). Antihuman procathepsin B and cathepsin B rabbit mAb (31718) was acquired from Cell Signaling Technology Inc. (Danvers, MA, USA). Rabbit pAbs directed against human two pore channel segment 1 (TPC1) (LS-C110014) was purchased from LifeSpan Bioscience Inc. (Seattle, WA, USA) or against human two pore channel segment 2 (TPC2) (ACC-072) from Alomone labs (Jerusalem, Israel). Mouse mAb against human β-tubulin (T7816) or GAPDH (G9545) were purchased from Sigma-Aldrich (San Luis, MS, USA). Horseradish peroxidase-coupled (HRP) goat pAbs directed against mouse (A4416) or against rabbit (A6154) IgGs were purchased from Sigma-Aldrich (San Luis, MS, USA). Mouse pAb conjugated with Fluorescein isothiocyanate (FITC) and directed against human IgG, Fcγ fragment specific ab (209-095-098) for fusion protein cell binding assays was purchased from Jackson ImmunoResearch Europe Ltd. (Newmarket, UK).
2.5. Pseudovirion Production and Transduction
HIV-based pseudotype viral particles were generated as described previously [43
]. In brief, 90% HEK293T cells were cotransfected in 6-well plates with 1 µg total DNA using polyethylenimine (PEI) (Sigma-Aldrich, San Luis, MS, USA) according to manufacturer’s instructions. DNA ratios were 1 part transfer plasmid:1 part packaging plasmid:4 parts envelope plasmid for reporter pseudotype virus generation or 7:7:1 for transgene delivery. Cell supernatants were collected at 48 and 72 h post-transfection and passed through a 0.45 µm pore filter. Viral stocks were stored at 4 °C for short time (less than 7 days) or aliquoted and frozen at −80 °C for long-term storage.
For transduction assays, viral supernatants were first mixed with polybrene (H9268-5G, Sigma-Aldrich, San Luis, MS, USA) at a final concentration of 4 µg/mL and 500 µL or 1 mL was added to 30–50% confluent target cells (80–90% for heterokaryon transductions) in 12 or 6-well plate format, respectively, for 6 h. Seventy-two hours post transduction, cells were lysed for luciferase quantification or processed for fluorescence detection. For virus–cell specificity studies, GFP-based pseudoparticles were titrated by limiting serial dilution and subsequent transduction of the susceptible cell line Huh-7.5. Transducing units per ml (TU/mL) were calculated as [((% infected cells/100) * # transduced cells) / virus volume (mL)] by FACS analysis. For pharmacological inhibition studies, target cells were preincubated with drugs in DMSO or DMSO alone for 1 h prior to addition of pseudoparticles and further incubation.
Luciferase-based GP-driven entry was measured as previously described [48
]. For filovirus GP-driven susceptibility cell screening, nonspecific (NoEnvpp), EBOV (EBOVpp), or MARV (MARVpp) GP-mediated entry relative light units (RLU) values were normalized to positive control VSV GP-driven (VSV-Gpp) values for cross-experimental comparison [(EBOVpp or MARVpp RLUs / VSV-Gpp RLUs) × 100] and displayed as luciferase activity arbitrary units. EBOV species susceptibility evaluation is expressed as absolute RLU values in 100 µl cell lysate. For fluorescence reporter expression, target cells were quantified by FACS and shown as the % positive cells from total.
2.6. Authentic Filovirus Infection
Fully infectious filovirus work was performed under Biosafety Level 4 (BSL4) conditions at the Institute of Virology, Phillips-Marburg University, Germany. Susceptible cell lines SK-N-BE(2)-C and HEK293T and resistant cell lines SK-N-MC and SH-SY5Y seeded on 6-well plates with 1 coverslip per well were mock treated, or infected with EBOV (Mayinga isolate) (GenBank accession number NC002549) or MARV (Musoke isolate) (GenBank accession number NC001608) at a MOI of 0.1 TCID50/mL for 1 h at 37°C. After inoculation, virus was removed, and cells were further incubated for 72 h in DMEM with 3% FCS. Three days later, the cells were fixed with 4% PFA for 48 h. For immunofluorescence analysis, free aldehyde groups were quenched with 0.1 mM glycine in PBS, and the cells were permeabilized with 1% Triton-X 100 (TX100). Permeabilized cells were incubated with goat α-EBOV or α-MARV antisera (α-EBOV serum was used for mock infection) against virus Nucleoprotein (NP) as primary Ab. α-goat AlexaFluor488 was used as secondary Ab with DAPI for nuclear staining. NP staining was visualized using a Zeiss Axiophot microscope with a 40x objective with an illumination of 1000 ms for FITC channel and 500 ms for DAPI.
Remnant cells were scraped from the wells into 1% SDS/PBS for immunoblot analysis. 3% lysates were run through an SDS/PAGE gel and transferred onto a nitrocellulose membrane. NP or tubulin protein detection was carried out by the same goat EBOV and MARV antisera as used for IF and a tubulin specific mab, respectively, followed by antigoat IRDye680-conjugated secondary ab incubation. Immuno-labelled proteins were detected with and odyssey infrared-imaging system (LI-COR).
2.7. rVSVΔG-EBOVGP Infection
Briefly, HEK293T and SH-SY5Y cells were infected with 500 µL/well of recombinant VSV bearing EBOV Mayinga strain GP (rVSVΔG-EBOVGP) at a MOI of 1 (3.3 × 105
TCID50) or mock infected for 1 h at 37 °C in DMEM without FCS. At 1 hpi, 2.5 mL DMEM with 3% FCS was added to the virus inoculum and further incubated for 24 h (2 replicates) or 48 h (1 replicate). Cell supernatant was collected each day, and viral titers were determined by TCID50 limiting-dilution assay 48–72 h after starting of assay [49
]. Simultaneously, bright-field images were acquired at 24 h (2 replicates) and 48 hpi (1 replicate) with a Nikon TS100 microscope using a 10× objective.
2.8. Flow Cytometry
For cell surface protein detection immunostaining and subsequent fluorescence quantification was performed. In brief, adherent cells were detached with versene (15040066, Gibco, Gaithersburg, MD, USA), a nonenzymatic cell dissociation reagent. The cell suspensions were spun at 700 rpm for 5 min, and the cells were resuspended in 200 µL 2% FCS in PBS (FACS buffer) solution per staining. Fc receptors were blocked with 1 µL human FcR blocking reagent (Miltenyi Biotec Gmbh, Bergisch Gladbach, Germany) for 10 min. Subsequently, saturating concentrations of conjugated specific or isotype control Abs were added and incubated for at least 15 min. Stained cells were washed with FACS buffer and resuspended in 400 µL FACS buffer for fluorescence detection. Cells were kept at 4 °C and in the dark throughout the staining. Protein expression was evaluated by APC or FITC channel intensity in a BD FACSCanto cytometer (BD, Frankin Lakes, NJ, USA). Protein expression was calculated as the difference of geometric mean fluorescence intensity (ΔMFI) between specific Ab and isotype control staining. Cell surface detection of bound GP1-Fc fusion protein was detected as described before [50
]. Briefly, adherent cells were detached with versene. 5 × 105
cells/well were washed with 1× DPBS containing Ca2+
(14040133, Gibco, Gaithersburg, MD, USA) and Fc receptors blocked with human FcR blocking reagent. Subsequently, cells were incubated with 100 nM GP1-Fc or Fc for 1.5 h, washed with FACS Buffer++
(2% FCS in PBS with Ca2+
) and incubated with FITC-conjugated mouse Abs directed against human Fc fragment ab (1:200) for 45 min. Cells were washed twice with FACS Buffer++
and fixed in 1 part FACS Buffer++
/3 parts 3% PFA for 10 min. Cells were maintained at 4 °C throughout the processing. Bound protein was quantified by FITC channel intensity in a BD FACSCanto cytometer (BD, Frankin Lakes, NJ, USA) and subtracted from MFI of secondary Ab control. To monitor GFP, cells were detached with trypsin, washed with 1× PBS and fixed with 3% PFA for 10 min at 4 °C and analyzed with a BD FACSCanto cytometer. Cell–cell fusion/transduction fluorescence quantification analysis was performed at the Hannover Medical School Cell Sorting Core Facility with a BD FACSAria Fusion (BD, Frankin Lakes, NJ, USA). Cells were prepared as described for GFP quantification. The gating strategy for heterokaryon detection and transduction is depicted in Figure S2
. 10,000 events were recorded for single cell line controls, 50,000 for the PBS treatment control, and 10,000 ZsGreen1 positive events (heterokaryon cells) for PEG treatment. Data was analyzed with FlowJo7 software (Tree Star, Ashland, OR, USA).
2.9. Expression of EBOV GP1-Fc Fusion Protein
Plasmids encoding soluble EBOV Gp1-Fc or Fc alone were transfected by polyethylenimine into HEK293Tcells. Cell supernatants with EBOV GP1-Fc and Fc, respectively, were collected 48 and 72 h post transfection, concentrated using a tangential flow cassette (Vivaflow 200) (Sartorius, Gottingen, Germany) and diluted with an equal amount of 20 mM phosphate buffer (pH 7). Proteins were purified by affinity chromatography using a HiTrap Protein G HP 1 mL column (GE Healthcare, Braunschweig, Germany) according to the manufacturer’s instructions followed by concentration and size exclusion chromatography using a Superose 6 Increase 10/300 GL column (GE Healthcare, Braunschweig, Germany) equilibrated with 10 mM Tris pH 8, 150 mM NaCl.
2.10. Western Blot
To detect intracellular filovirus entry factors immunoblotting was conducted. Cells were lysed in Cell Lytic M (C2978, Sigma-Aldrich, San Luis, MS, USA) plus protease inhibitors (11836145001, Roche, Basel, Switzerland). Protein concentrations were determined by Bradford assay (23200, Thermo Fischer Scientific, Waltham, MA, USA) using a Nanodrop™ 2000 spectrophotometer (ND-2000, Thermo Fischer Scientific, Waltham, MA, USA). 1.5 µg of protein was solubilized with 6× DTT sample buffer (Tris-HCl, Glycerin, SDS, DTT, Bromophenol), boiled for 5 min at 95 °C, cooled down and resolved by electrophoresis. Proteins were blotted to a PVDF membrane (GE Healthcare, Braunschweig, Germany) by Mini Trans-Blot® (Bio Rad, Hercules, CA, USA) wet transfer system. Membranes were blocked and incubated with ab in 5% milk powder with 0.1% PBS-Tween20 either for 1 h at RT or o/n at 4 °C with constant shaking. Protein was detected using the HRP-based ECL chemiluminescence system (GE Healthcare, Braunschweig, Germany) and blots were documented with a LAS-4000 Mini (Fujifilm, Düsseldorf, Germany).
2.11. RNA Extraction and Microarray Analysis
Cells were grown to about 90% confluency on 10 cm dishes prior to extraction. Total RNA was extracted as instructed in the RNeasy Midi kit (Qiagen, Hilden, Germany) guidelines.
Microarray analysis was carried out at the Research Core Unit Transcriptomics of Hannover Medical School. The whole Human Genome Oligo Microarray Kit 4x44K v2 (G4845A, design ID 026652, Agilent Technologies, Santa Clara, CA, USA), covering 44495 probes and about 26,000 transcripts, was used to analyze mRNA levels. Quick Amp Labeling Kit, two-color (5190-0444, Agilent Technologies, Santa Clara, CA, United States) was used for the generation of Cy3- or Cy5- labelled cRNA. Raw data was acquired as Relative Light Units (RLU). Microarray data has been deposited in public genomic data repository Gene Expression Omnibus (GEO) under the accession number GSE104008.
2.12. Hierarchical Clustering Analysis (HCA)
Microarray data was compiled for gene expression clustering analysis among studied cell lines. Briefly, matrices were prepared by assigning attributing individual expression values of each gene (row) versus the corresponding cell line (column). Gene probes that yielded no detectable signal in any of the studied cell lines were excluded from the analysis. An unbiased heatmap was generated from these matrixes through the R software heatmpap.2 gplot package [51
] by applying the “euclidean” distance method and the “average” (UPGMA) clustering method. Color scales represent values between the maximum and minimum expression values of the individual cell lines.
2.13. Cathepsin B and L Activity Assay
Cysteine proteases cathepsin B and L proteolytic activities were measured using the fluorometric cathepsin L (ab65300) and cathepsin B (ab65306) activity assay kit (Abcam, Cambridge, UK) according to the manufacturer´s protocol with the modification of the starting material. 1 × 106 cells were spun down, washed and lysed in 150 µl chilled CL lysis buffer. Lysates were split into 3 wells with equal volumes (≈3.3 × 105 cells/well) for intra-assay variation evaluation. Fluorometric values were recorded with a microplate reader using a Bio-tek FLx 800 Multifunction Fluorescence Luminescence Microplate Reader (Bio-Tek, Winooski, USA) with a 528/20 emission filter.
2.14. Cholesterol and EGF Endosomal Accumulation Assays
NPC1 and TPC1/2 functionality was evaluated by unesterified cholesterol and EGF endolysosomal accumulation, respectively. 8 × 104 HEK293T or 2 × 105 SH-SY5Y cells were seeded onto poly-L-lysine coated coverslips in a 24-well plate. On the next day, cells were treated with vehicle (DMSO) control or target-specific drugs for 24 h. Twenty-four hours post-treatment, cells for NPC1-mediated cholesterol shuttling analysis were washed and fixed with 3% PFA in PBS for 20 min at RT. Furthermore, cells for TPC1/2 functionality assessment were incubated for 30 min with 1 µg/mL EGF-AlexaFluor555 conjugated protein in serum-free DMEM without or with 2 µg/mL tetrandrine. Subsequently, cells were washed with serum-free DMEM and treatments applied for extra 3.5 h in normal culture medium. Finally, cells were washed and fixed as for cholesterol accumulation.
2.15. Light Microscopy
Fluorescence microscopy was used to assess NPC1 or TPC1/2 functionality or heterokaryon formation and the susceptibility of heterokaryons to filovirus GP-mediated transduction. Sample preparation was performed as described previously [52
]. For functionality assays, cells were fixed with 3% paraformaldehyde in PBS for 20 min followed by quenching of the remaining fixative with 50 mM NH4
Cl for 10 min and permeabilization with 0.1% TX100 for exactly 5 min. The cells were washed in PBS and blocked with 0.5% BSA in PBS for 30 min. To assess the functionality of NPC1 or TPC1/2, samples were then stained with 0.5% BSA/PBS solution containing 50 µg/mL Filipin III (unesterified cholesterol) or 0.05 mg/mL DAPI, respectively, for 30 min followed by extensive washing. All incubations were done at RT. Lastly, coverslips were washed with H2
O and mounted with Mowiol containing 2.5% (wt/vol) 1,4-diazabicyclo-[2.2.2]octane on glass slides. For cell–cell fusion/transduction assays, coverslips were processed identically as for TPC1/2 samples. EGF accumulation (TPC1/2) and heterokaryon formation and transduction were analyzed at a confocal fluorescence microscope (TCS SP8, Leica microsystems, Wetzlar, Germany) with plan-apochromat 63×/1.40 oil immersion objectives, and 405-, 488-, 561-, and 633-nm lasers. Cholesterol accumulation (NPC1) was analyzed at a Leica DM5000 B epifluorescence microscope (Leica microsystems, Wetzlar, Germany) coupled to a UV light filter. Pictures were acquired with a 40x magnification objective. Documentation was performed with LAS AF Lite software (Leica microsystems, Wetzlar, Germany) and image processing with ImageJ2/Fiji package [53
] or Adobe Photoshop CS4 (Adobe Systems Inc., Mountain View, CA, USA).
2.16. Polyethylene Glycol (PEG) Mediated Cell–Cell Fusion
Cells expressing either DOX-inducible Tet-On 3G transactivator (Huh-7.5, SH-SY5Y) or ZsGreen1 under the control of the PTREG3V
promoter (HEK293T-H6) were chemically fused. Cell–cell fusion methodology has been previously described [54
]. 5 × 105
Huh-7.5 or 5 × 105
SH-SY5Y cells were coseeded with 3 × 105
HEK293T-H6 cells in 6-well plates. Twenty-four hours after coculture, the cells had reached about 80–90% confluency increasing the chance of cell–cell contacts and hence fusion efficiency. Cocultured cells were either treated with 500 µl PBS as fusion control or with pre-warmed 40% PEG1500 (10783641001 Roche, Sigma-Aldrich, San Luis, MS, USA) as fusogenic agent for 5 min at 37 °C. After incubation, any trace of PEG was removed by extensive washing with PBS. Finally, cells were allowed to recover for at least 1 h at 37 °C with fresh new media prior to further experimentation.
2.17. Statistical Analysis
Unless stated otherwise, experiments were carried out as 3 biological replicates with 3 technical replicates for intra-assay control and quantified as the mean of the 9 data points with error bars representing standard deviation (SD). Viral transductions were performed with 3 different viral stocks. Microscopy experiments were performed twice for functionality assays. Cell–cell fusion/filovirus and transduction was documented once by microscopy and quantified by flow cytometry in three independent experiments. Data analysis and representation was done with Graph Pad Prism 7 statistical software (La Jolla, CA, USA). Multiple t tests was performed for statistical significance discovery correcting for multiple comparison with the Holm–Sidak method. P-value significance was represented as: n.s. P > 0.05; * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001; **** P ≤ 0.0001
The large number of host proteins reported to promote filoviral internalization and thus infection prompted us to analyze systematically the cellular requirements for filovirus susceptibility. We tested a panel of immortalized cell lines for EBOV and MARV GP-mediated transduction to correlate susceptibility to the presence of plasma membrane proteins or endosomal proteins required during the early phases of infection. As previously reported, fibroblast, epithelial, and endothelial cells were susceptible to filovirus GP-driven transduction, whereas the lymphocytic cell line Jurkat was fully resistant to pseudoparticle mediated reporter expression [16
]. However, we identified two neuroblastoma cell lines, SK-N-MC and SH-SY5Y, which were resistant to filovirus GP-driven transduction. This was unexpected, as most human cell lines previously described to be resistant to filovirus infection are nonadherent or lymphocytic cell lines [14
SK-N-MC was isolated from metastatic neuroblastoma tissue of a 14 years old Caucasian female [56
]. These cells were not susceptible to EBOV-GP transduction or EBOV infection but supported a low level of MARV-GP transduction and MARV infection. This disparity might be due to different receptor usage among filoviruses, to different internalization mechanisms, or even indicate a host factor specifically required for EBOV but not for MARV [17
]. On the other hand, SH-SY5Y cells, a thrice cloned subline of the neuroblastic SK-N-SH cell line [56
], were highly resistant to both EBOV and MARV by all assays (Figure 1
and Figure 2
). These cells are susceptible to chikungunya virus, dengue virus, varicella-zoster virus, and neurovirulent poliovirus [71
]. Our study revealed that SH-SY5Y cells are also susceptible to transduction driven by the G protein of rabies virus (Figue2B). Together, these results suggest that the entry defect observed is largely filovirus specific.
According to the current model on filovirus cell entry, one or several proteins on the cell surface facilitate infection in addition to the essential intracellular endosomal proteins. At the plasma membrane, Ca2+
-dependent carbohydrate binding C-type lectins interact with N-glycans attached to filoviral GP [75
] and function as attachment factors [34
]. Furthermore, members of the TAM (Tyro3, Axl, Mer) and TIM-1 PtdSer binding protein families enhance filovirus cell entry, TAMs likely by enhancing macropinocytosis [37
] and TIM-1 by a process depending on GP or PtdSer [30
]. Finally, the intracellular proteins cathepsin B and cathepsin L, TPC1 and TPC2, and the bona fide receptor NPC1 are essential for productive filovirus infection in vitro and in vivo [38
]. Our panel of cell lines was largely lacking expression of lectins at the mRNA level with the exception of ASGR1 and ASGR2, which were expressed on Huh-7.5 cells, as reported previously [28
]. The absence of expression of other entry promoting lectins is in line with the tissue distribution of DC-SIGN, hMGL (dendritic cells and macrophages), L-SIGN, and LSEctin (endothelial cells of the liver and lymph nodes) [24
]. Therefore, although lectins might contribute to the cell tropism of filoviruses [20
], clearly they are not essential for filovirus infection. In line with previous studies [30
], TAM and TIM-1 mRNA expression was heterogeneous among the cell lines studied here, suggesting that they are not essential but may facilitate filovirus attachment and internalization. Indeed, studies on the role of Axl in filovirus cell entry have shown a cell-type-specific effect with antibodies against Axl reducing Axl-dependent filoviral GP-driven transduction in some Axl-positive cell lines but not in others [77
]. Similarly, overexpression of DC-SIGN but not of TIM-1 renders Jurkat cells susceptible to filovirus virus-like particles (VLP) [76
] and infection studies with TIM-1-/-
mice also suggest a dispensable role of TIM-1 since EBOV viral loads are similar in KO and WT mice [78
]. Our data clearly demonstrate that TIM-1, Axl, Mer, and DC-SIGN have important yet redundant roles during filovirus entry. This is in line with published studies supporting the function of C-type lectins, TAM tyrosine kinases, and TIM proteins in cell entry [23
]. Prospective further attachment factor protein expression profiling among susceptible and resistant cell lines could provide even a clearer picture on the cellular requirements for productive filovirus cell entry. In sum, expression of TIM-1, Axl, Mer, or DC-SIGN is an important determinant of susceptibility to filoviruses.
Finally, the required intracellular endosomal proteins were expressed and functional in all cell lines studied here including the resistant SH-SY5Y cells, irrespective of their susceptibility to EBOV infection, suggesting that they are not the determinants of resistance in SH-SY5Y cells. A similar scenario has been observed in resistant lymphocytic cell lines, which clearly express NPC1 yet they are resistant to infection [68
]. In fact, the Jurkat cells analyzed here had higher NPC1
mRNA levels than primary human hepatocytes (432 over 308 RLU) (Table S2
) and expressed NPC1 protein (Figure 5
B). Thus, resistance to filoviral cell entry is more common than previously thought and not restricted to either nonadherent or lymphocytic cells or to the presence of intracellular host factors.
The disparity between susceptibility to infection and surface protein expression could be attributed to different reasons. First, resistant cell lines might express a dominant restriction factor that blocks productive internalization or viral fusion (reviewed in [61
]). However, this was not the case for the SH-SY5Y cells reported here, as heterokaryons of susceptible HEK293T-H6 and SH-SY5Y cells were also susceptible to filovirus GP-driven transduction. Second, filoviruses might rely on different cofactors in different cell types. In Jurkat cells, this might be conceivable. An overexpression of DC-SIGN but not of TIM-1 renders otherwise resistant Jurkat cells susceptible to EBOV-GP virus-like particles [76
]. Moreover, EBOV virions bind to resistant Jurkat and activated CD4+ T-cells in a TIM-1 dependent-manner without productive infection [78
]. However, ectopic expression of Axl, an inhibitor of the type I interferon signaling [69
], renders Jurkat cells susceptible. This suggests that Jurkat cells might be able to resist filovirus infection because they have an intrinsic antiviral innate immune mechanism activated. This hypothesis could be tested in the future by determining whether heterokaryons of resistant Jurkats with susceptible cells will be susceptible or resistant to filovirus infection. In SH-SY5Y cells, however, an ectopic expression of any potential attachment-promoting plasma membrane protein of filoviruses, such as DC-SIGN, Axl, Mer, or TIM-1, conferred susceptibility to filovirus GP-driven transduction or infection. Interestingly, the enabling potency was different among attachment factors. These differences may be related to the mechanism by which the attachment factors promote susceptibility in SH-SY5Y cells. For instance, TAM receptors may confer susceptibility by enhancing micropinocytosis, whereas TIM-1 or DC-SIGN could bind to the virus GP [26
]. Further mechanistic studies on the role of the studied attachment factors could shed light on the differences observed in GP-mediated filovirus susceptibility in SH-SY5Y cells. Nevertheless, these data highlight the concept that virus–receptor interactions at the cell surface are required for productive entry but several alternative host cell factors can be utilized. To our knowledge, this is the first study which directly shows that several unrelated proteins can be utilized alternatively to initiate filoviral cell entry in one cellular context. These findings may help to explain the broad cell tropism of filoviruses.
In summary, we have characterized SH-SY5Y cells as a novel adherent nonlymphocytic cell line exhibiting a specific resistance to filoviral cell entry, and show that GP-mediated filovirus attachment to cells is impaired. This lack of susceptibility could be circumvented by expression of any one of several unrelated cell surface proteins that had previously been implicated in filoviral cell entry using other cellular backgrounds and experimental settings. We thus provide the most direct experimental evidence to support the current model of filoviral cell entry with alternative unspecific interactions between filoviral particles and plasma membrane host proteins mediating attachment and subsequent infection of target cells. Moreover, the filovirus-specific resistance of SH-SY5Y cells makes this cell line an interesting new tool for studying filoviral cell entry and for potentially identifying new cellular factors in the filoviral entry puzzle.