GoKawiilCell lines and plasmids
HEK293T and Huh7 cells (American Type Culture Collection (ATCC)) were maintained in Dulbecco’s modified eagle medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. HUVEC cells (ATCC) were cultured in vascular cell basal medium supplemented with the Endothelial Cell Growth Kit-BBE (ATCC), 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. THP-1 cells (ATCC) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum, 0.05 mM 2-mercaptoethanol (Gibco), 100 U ml−1 penicillin and 100 µg ml−1 streptomycin. To induce macrophage-like differentiation, THP-1 cells were treated with 30 nM phorbol 12-myristate 13-acetate (PMA; Sigma-Aldrich) for 24 h, followed by a 24-h incubation in PMA-free medium31. Expi293F cells (Thermo Fisher) were grown in Expi293 Expression Medium (Thermo Fisher). ss320 Escherichia coli (Lucigen) and TG1 E. coli (Lucigen) were cultured in 2YT medium. HEK293T, Huh7 and THP-1 cells were authenticated by the vendors using short tandem repeat (STR) profiling. Authentication documentation for Expi293F and HUVEC cells was not available on the vendors’ websites. HEK293T cells were tested for mycoplasma contamination in our laboratory and by the vendor and were negative in both cases. Huh7, HUVEC, THP-1 and Expi293F cells were tested and confirmed negative for mycoplasma by the vendors. No commonly misidentified cell lines were used.
Genes encoding RAVV GP (GenBank: ACD13005.1), Musoke MARV GP (NCBI Reference Sequence: YP_001531156.1), Angola MARV GP (GenBank: APQ46224.1), EBOV GP (NCBI RefSeq protein: NP_066246.1) and human NPC1 (UniProt: O15118) were synthesized (GenScript). For full-length GP pseudovirus production, GP genes were cloned into the pcDNA3.1(+) vector with or without a C-terminal C9 tag (the tag-free version was defined as wild type), as previously described32. For GP-ΔM pseudovirus production, the RAVV GP-ΔM gene (residues 1–636, lacking residues 257–425 corresponding to the MLD) was cloned into pcDNA3.1(+) with a C-terminal C9 tag. For protein expression, the RAVV GP-ΔM gene (residues 1–636, lacking residues 257–425 and containing a K589I mutation to stabilize GP2) and the EBOV GP-ΔM gene (residues 1–632, lacking residues 313–463 corresponding to the MLD) were each fused to a C-terminal foldon trimerization motif and His6 tag, and cloned into the Lenti-CMV vector, as previously described25,33. The human NPC1-C gene (residues 374–620, wild type or containing introduced mutations) was fused to a C-terminal His6 tag, and the gene encoding Nanosota-MB1 was fused to a C-terminal human IgG1 Fc tag (GenBank: AEV43323.1); both were cloned into the Lenti-CMV vector, as previously described25,32.
Preparation of GP and NPC1
RAVV GP-ΔM and human NPC1-C proteins were expressed and purified as previously described19,25,32. Plasmids (500 μg) encoding GP-ΔM or NPC1-C were transiently transfected into 500 ml Expi293F cells using 1.5 ml polyethylenimine (Polysciences). Three days post-transfection, supernatants were collected, and proteins were purified on a Ni-NTA column (Cytiva) with an imidazole gradient in PBS. Further purification was performed by size-exclusion chromatography: GP-ΔM on a Superose 6 column (Cytiva) and NPC1-C on a Superdex 200 column (Cytiva), both in buffer containing 20 mM Tris (pH 7.4) and 200 mM NaCl. Purified proteins were flash-frozen in liquid nitrogen and stored at –80 °C. To generate RAVV GPcl, 3 mg of RAVV GP-ΔM was treated with 60 μg of trypsin (Sigma-Aldrich) for 1 h, followed by purification on a Superose 200 column (Cytiva). To generate EBOV GPcl, 3 mg of EBOV GP-ΔM was treated overnight with 15 μg of thermolysin L (Sigma-Aldrich)25, followed by purification on a Superose 200 column (Cytiva).
Preparation of nanobodies
RAVV GP-targeting nanobodies were generated as previously described25,34,35. An alpaca was immunized subcutaneously with RAVV GP-ΔM at 2-week intervals for a total of seven immunizations (Turkey Creek Biotechnology; animal protocol 18-03, in accordance with institutional and national guidelines for the care and use of research animals). Following immunization, blood was collected, and peripheral blood mononuclear cells were isolated (Vanderbilt Antibody and Protein Resource Core). A cDNA library was constructed from peripheral blood mononuclear cell RNA using oligo(dT) primers and Superscript IV reverse transcriptase (Thermo Fisher). Nanobody genes were amplified by nested PCR and cloned into a modified pADL22 vector (Antibody Design Labs). Ligation products were electroporated into E. coli TG1 to generate the nanobody phage display library, following the manufacturer’s protocol (Antibody Design Labs).
Screening of the nanobody phage display library was performed as previously described25,34,35. Three rounds of bio-panning were carried out to enrich for nanobodies binding to RAVV GP-ΔM. For each round, 20 μg of purified GP-ΔM was coated onto an immune tube overnight. The tube was blocked with 5% milk, incubated with 500 μl of phages for 1 h, and washed; the retained phages were eluted and used to infect E. coli TG1. Amplified phages were used for subsequent rounds. After the third round, eluted phages were used to infect E. coli ss320, which were plated on 2YT agar. Single colonies were picked, and nanobody expression was induced with 1 mM IPTG. Supernatants were screened by ELISA to identify GP-ΔM-binding nanobodies.
ELISA was performed as previously described25,34,35. In brief, plates were coated with 100 ng of RAVV GP-ΔM overnight and blocked with 2% BSA. Plates were then sequentially incubated with supernatants from E. coli ss320 expressing haemagglutinin (HA)-tagged nanobodies and with horseradish peroxidase-conjugated anti-HA antibody (Sigma-Aldrich). ELISA substrate (Invitrogen) was added, and reactions were stopped with 1 N H 2 SO 4 . Absorbance at 450 nm (A 450 ) was measured using a Synergy LX Multi-Mode Reader (BioTek).
His-tagged nanobodies were expressed and purified from the periplasm of E. coli ss320 as previously described25,34,35. Expression was induced with 1 mM IPTG. Cell pellets were collected, resuspended in 15 ml TES buffer (0.2 M Tris, pH 8.0, 0.5 mM EDTA and 0.5 M sucrose) and shaken on ice for 1 h. The suspension was then diluted with 40 ml of one-quarter of TES buffer (each component at one-quarter concentration) and shaken on ice for another hour. Nanobodies in the supernatant were purified sequentially on a Ni-NTA column (Cytiva) followed by a Superdex 200 column (Cytiva).
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