Antibodies, mouse immune serum, and media.
A human serum from a recovered SARS patient was kindly provided by William Bellini (Centers for Disease Control and Prevention [CDC], Atlanta, Ga.). Antibodies to CD11c, CD14, CD40, CD80, CD86, and HLA-DR were purchased from BD Pharmingen. A fluorescein isothiocyanate (FITC)-conjugated mouse antibody against the C-terminal His tag was purchased from Invitrogen (Carlsbad, Calif.). Media for human primary cell culture were purchased from Cambrex (East Rutherford, N.J.). RPMI 1640 medium and Dulbecco's modified Eagle medium were purchased from Invitrogen. A mouse immune serum against the SARS-CoV S protein was generated by vaccinating 10-week-old BALB/c mice with CMV/R plasmid DNA expression vectors, described below, encoding the S protein (mice were vaccinated with 25 μg, three times, at 3-week intervals, and were bled after 2 months). Negative-control antisera were obtained in a similar fashion by injecting the same plasmids with no insert.
Human primary cell lines from renal proximal tubule epithelial cells (RPTEC), renal epithelial cells (HRE), renal cortex epithelial cells (HRCE), small airway epithelial cells (SAEC), bronchial epithelial cells (NHBE), lung fibroblasts (NHLF), lung microvascular endothelial cells (HMVEC-L), umbilical vein endothelial cells (HUVEC), microvascular endothelial cells (HMVEC), mammary epithelial cells (NHMEC), and keratinocytes (NHEK), as well as hepatocytes, were purchased from Cambrex. The following human and animal cell lines were purchased from the American Type Culture Collection: ACHN (human kidney adenocarcinoma), 293 (human embryonic kidney cells), 786-O (human kidney adenocarcinoma), A549 (human lung carcinoma), HeLa (human cervical adenocarcinoma), Colo205 (human colon adenocarcinoma), Jurkat (human T cells), CEM (human acute lymphoblast leukemia), M8166 (human CD4+
lymphoid cells), HL60 (human promyelocytic leukemia cells), THP-1 (human acute monocytic leukemia), Vero (African green monkey kidney epithelial cells), CRFK (cat kidney cortex epithelial cells), OK (opossum kidney cortex epithelial cells), M-1 (mouse kidney cortex epithelial cells), FC2.Lu and FC28.lu (cat lung fibroblasts), AK-D (cat lung epithelial cells), MLE12 (mouse lung epithelial cells), MM14.lu (mouse lung), LA-4 (mouse lung adenoma), LH4 (guinea pig lung fibroblasts), and CHL-11 (Chinese hamster lung fibroblasts). Human peripheral blood mononuclear cells were prepared from whole blood by Ficoll gradient centrifugation. The THP-1, THP-DC-SIGN (THP-1 cells expressing human DC-SIGN), and THP-DC-SIGNΔ35 (THP-1 cells expressing DC-SIGN with a 35-amino-acid [35-aa] cytoplasmic domain deleted) cell lines were kindly provided by D. R. Littman (19
). Human T-cell leukemia cell lines A3R5 (a subline of CEM expressing both CCR5 and CXCR4), MT-2 (expressing CXCR4), and 293T were gifts from John Mascola.
Gene synthesis and construction of expression vectors.
Genes encoding the SARS-CoV S, M, and E proteins were synthesized by using human-preferred codons. To synthesize these genes, protein sequences obtained from GenBank (SARS-CoV strain Urbani, accession no. AY278741) were reverse translated by using human-preferred codons. Sets of 75-bp oligonucleotides with 25-bp overlaps covering each fragment were synthesized and gel purified. The oligonucleotides were assembled into DNA fragments by using Pfu Turbo Hotstart DNA polymerase (Stratagene, La Jolla, Calif.) at a 50-to-65°C gradient annealing temperature. DNA fragments were cloned into the pCR-Blunt II-Topo vector (Invitrogen) and sequenced. Clones with the fewest mutations were picked and were further corrected by using the QuikChange kit (Stratagene) according to the manufacturer's protocol. Fully corrected DNA fragments for each gene were finally cloned into the mammalian expression vector CMV/R-mcs. COOH-terminal deletion mutants were generated by using the QuikChange kit (Stratagene) and were cloned into the CMV/R-mcs expression vector, which contains the cytomegalovirus (CMV) enhancer/promoter and splice donor and the human T-cell leukemia virus type 1 R region (W. Akahata, Z.-Y. Yang, and G. J. Nabel, unpublished data). These mutants include (i) SΔCD, in which the cytoplasmic domain was truncated (terminated at aa 1229), (ii) SΔTM2, in which the transmembrane and cytoplasmic domains were deleted (terminated at aa 1190), and (iii) SΔHR1, in which the transmembrane, cytoplasmic, and heptad-2 domains were removed (terminated at aa 1153). For S(1190)-Myc-His, the S protein was truncated at aa 1190 to remove the transmembrane and cytoplasmic domains and was tagged with a Myc and a His epitope at the COOH-terminus. The expression vectors were sequenced on both strands to ensure that each gene was correct and were further confirmed by Western blot analysis.
Purification and differentiation of human mDC.
Myeloid dendritic cells (mDC) were purified from elutriated monocytes from healthy adult donors by a two-step procedure consisting of automated leukapheresis and counterflow centrifugal elutriation at the Transfusion Medicine Department of the Warren Grant Magnuson Clinical Center, National Institutes of Health, Bethesda, Md. (1
). mDC were isolated from the elutriated monocyte fraction with negative selection by removing cells expressing BDCA-4 and CD9 with microbeads (Miltenyi Biotec, Auburn, Calif.), followed by positive selection using antibodies to CD1c (Miltenyi Biotec). mDC were then cultured in a medium containing granulocyte-macrophage colony-stimulating factor (10 ng/ml; PeproTech) and induced to differentiate to mature mDC by using poly(I
C) (50 ng/ml; Sigma, St. Louis, Mo.) for 48 h (5
). Antibodies to CD11c and CD14 (BD Pharmingen) were used to assess the purity of DC, and antibodies to CD40, CD80, CD86, and HLA-DR (BD Pharmingen) were used to characterize the differentiation of DC by flow cytometry.
Production of pseudotyped lentiviruses and retroviruses.
Recombinant lentiviruses and retroviruses expressing a luciferase reporter gene were produced as described previously (17
). Briefly, 5 × 106
293T cells were plated in 10-cm-diameter tissue culture dishes the day before transfection. The cells were transfected the next day by using calcium phosphate reagent (Invitrogen). The amount of plasmid DNA used for making different pseudotyped vectors was as follows: for lentiviral vectors, 7 μg of pCMVΔR8.2 plus 7 μg of pHR′CMV-Luc and either 400 ng of CMV/R-SARS-S or 2 μg of pNGVL-4070A (Ampho); for retroviral vectors, 7 μg of pNGVL-GagPol (MLV) plus 7 μg of pLZR-Luc and 400 ng of CMV/R-SARS-S or 2 μg of pNGVL-4070A (amphotroic MLV gp70), respectively. Cells were transfected overnight, washed, and replenished with fresh medium. Forty-eight hours later, supernatants were harvested, filtered through a 0.45-μm-pore-size syringe filter, and stored in aliquots at −80°C. p24 levels were measured from different viral stocks (4
) by using the Coulter HIV-1 p24 Antigen Assay kit (Beckman Coulter, Somerset, N.J.). One-tenth the amount of plasmids CMV/R-M and CMV/R-E were used with CMV/R-S for making combinational pseudoviruses.
Production of a GFP-Vpr-labeled SARS-CoV S-pseudotyped lentivirus.
A green fluorescent protein (GFP)-Vpr-labeled SARS-CoV S-pseudotyped lentivirus was produced by transfection of human embryonic kidney 293T cells with a pLAI provirus from which env
had been deleted (10 μg), CMV/R-SARS-S (1 μg), and plasmid pEGFP-C3 (Clontech, Palo Alto, Calif.), containing the entire Vpr coding region fused to the carboxy terminus of eGFP (GFP-Vpr; 15 μg) (27
). Cells were washed at 16 to 20 h posttransfection and replenished with fresh medium. Forty-eight hours later, supernatants were harvested, filtered through a 0.45-μm-pore-size syringe filter, and concentrated. Briefly, 32 ml of supernatant was layered onto 5 ml of Optiprep (Iodoxinal) medium (Invitrogen) and centrifuged at 50,000 × g
for 1.5 h with a Surespin 630 rotor (Sorvall, Newtown, Conn.). The last 3 ml of supernatant remaining above the Optiprep interface was collected and frozen at −80°C in 500-μl aliquots.
Infection of cells with SARS-CoV and titration of SARS-CoV.
Cells in six-well dishes were infected with 100 μl of a 1:10 dilution of SARS-CoV strain Urbani (106.25
50% tissue culture infective doses/ml; CDC) per well under appropriate containment in a BSL3 laboratory. After 1 h of adsorption, the cells were washed three times with medium, replenished with 3 ml of fresh medium, and maintained at 37°C in a 5% CO2
incubator. Seventy-two hours after infection, 0.5 ml of tissue culture medium was harvested and incubated with Vero cells in a 96-well plate; viral titers in the medium were calculated in 50% tissue culture infective doses per milliliter 4 days after infection of Vero cells (38
). The viral cytopathic effect was determined on days 3 and 4. Mature and immature mDC were infected in 96-well plates (10,000 cells/well) with 50 μl of 1:10-diluted viral stock and were titered similarly 3 days later.
Infection of cells with pseudovirus.
A total of 30,000 cells were plated into each well of a 48-well dish the day before infection. Cells were infected with 150 μl of viral supernatant for 16 to 18 h for SARS-CoV S-pseudotyped viruses and for 3 to 4 h for Ampho Env and Ebola glycoprotein-pseudotyped viruses. The viral supernatant was replaced with fresh medium at the end of infection. Forty-eight hours after infection, cells were lysed in “mammalian cell lysis buffer” (Promega, Madison, Wis.). The same amount of cell lysate was used in a luciferase assay with “Luciferase assay reagent” (Promega) according to the manufacturer's suggestions.
Transfection and Western blot analysis.
293T cells were transfected by using calcium phosphate (Invitrogen). Transfected cells were harvested 48 h after transfection. Cell lysates were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Bio-Rad, Hercules, Calif.). The membrane was incubated with convalescent-phase human sera from a SARS patient (dilution, 1:2,500; kindly provided by William Bellini of the CDC) for 1 h at room temperature in blocking buffer (Tris-buffered saline, 1% bovine serum albumin, 5% skim milk, 0.3% Tween 20), followed by three washes in washing buffer (Tris-buffered saline-0.3% Tween 20). The blot was further incubated in blocking buffer with horseradish peroxidase-conjugated donkey anti-human immunoglobulin G (IgG) (dilution, 1:5,000; Chemicon, Temecula, Calif.) for 30 min and then washed four times in washing buffer. Detection was performed with the ECL reagent (Amersham, Piscataway, N.J.).
Cell staining with S(1190)-Myc-His.
A 500-ng portion of purified S(1190)-Myc-His glycoprotein was incubated with 106 cells in 100 μl of phosphate-buffered saline (PBS) containing 2% fetal bovine serum for 20 min on ice and then washed once with 1 ml of cold PBS. Each cell line was split into two aliquots, which were stained either with an FITC-conjugated mouse anti-His tag monoclonal antibody (dilution, 1:100; Invitrogen) or with an FITC-labeled isotype control, followed by flow cytometric analysis.
mDC (105) isolated from human elutriated monocytes were plated onto a 12-well-dish. Twenty-four to 48 h later, the cells were infected with 100 μl of a Vpr-GFP-labeled SARS-CoV S pseudolentivirus for 30 min. Cells were washed, detached with trypsin-EDTA, washed again, and added to human renal epithelial cells (786-O; 3 × 104 cells/well) plated onto 8-well coverslip slides (Nalge Nunc, Naperville, Ill.). Sequential images of live cells were recorded every 3 min by confocal microscopy (SP2-AOBS; Leica Microsystems), and uptake, polarization, and transfer were assessed with representative cells.
pH-dependent entry of SARS-CoV-S-pseudotyped lentiviral vectors.
Vero cells were plated in a 48-well-dish (30,000 cells/well) the day before infection. Cells were preincubated with the indicated amounts of ammonium chloride or bafilomycin A (Sigma) for 1 h. Pseudoviruses were mixed with the same concentrations of reagents in tubes and added to cells. Eight hours later, viruses were removed and replaced with fresh medium. Cells were harvested 48 h after infection, and a luciferase assay was performed.
Cell-mediated transfer of SARS-CoV and the SARS-CoV S pseudotyped lentiviral vector.
THP, THP-DC-SIGN, or THP-DC-SIGNΔ35 (30,000 cells/well) was incubated with the SARS-CoV S-pseudotyped lentiviral vector for 2 h and then washed three times with tissue culture medium. Cells were then added to Vero, A3R5, or MT2 cells (30,000 cells/well) plated in 24-well dishes. These cells were harvested 72 h later for a luciferase assay to assess THP, THP-DC-SIGN, or THP-DC-SIGNΔ35 cell-mediated transfer of pseudovirus to the respective cells.
To measure the transfer of SARS-CoV by mature mDC and to assess whether an anti-SARS-CoV S immune serum can block the transfer of SARS-CoV by mDC, mature mDC were incubated with SARS-CoV for 1 h, washed, detached with trypsin, and replated onto Vero cells in a 96-well-plate (10,000 cells/well) in the presence of a control or an S-specific mouse antiserum (dilution, 1:100). Cell culture medium (Dulbecco's modified Eagle medium-10% FBS) was collected 72 h later, and SARS-CoV titers in the cell culture medium were measured as described above.