Protein purification
Soluble spike proteins
In an effort to create soluble spikes for ANDV, synthetic genes for the Gn and Gc proteins from ANDV (strain Chile-9717869, UniProt: Q9E006) were introduced into a bicistronic plasmid, p-sSpike, optimized for expression in Drosophila cells. The hetero-octamerization domain was based on HALC4_136, a synthetic homo-tetramerization structure consisting of three antiparallel helices (PDB: 8D09). We first cloned the ectodomain of Gn (amino acids 21–483) into the first cistron, adding the first two helices of HALC4_136, now termed HALC4_136N. The second cistron contains the entire ectodomain of Gc (amino acids 652–1107), followed by the third α-helix of HALC4_136, which we call HALC4_136C. To prevent aggregation, mutations were introduced into Gc (F1089A, F1093A, L1100A, L1104A). We also designed a disulfide bond for stability between the C terminus of HALC4_136N and the N terminus of HALC4_136C. For purification, a double strep-tag was added to the C terminus of HALC4_136C. Additionally, we cloned the extracellular cadherin domains EC1–4 of human PCDH1 (amino acids 58–503, UniProt: Q08174) into a pMT/BiP plasmid along with a His and double-strep tag, creating plasmid p-sEC1–4. These plasmids were used to create stable transfectants in Drosophila S2 cells, along with the pCoPuro plasmid (in a 1:20 ratio) for puromycin selection. Stable cell lines with p-sSpike alone and those co-transfected with p-sSpike and p-sEC1–4 were established and kept in LM-SFM4 medium with puromycin. Cultures were grown in spinner flasks with antibiotics to about 107 cells per ml, and protein expression was induced with 4 μM CdCl2. After five days, the S2 medium was concentrated and purified using Strep-Tactin affinity chromatography, followed by His affinity chromatography and size-exclusion chromatography (SEC). The protein purity and presence of (Gn–Gc)4 spikes and PCDH1 in the SEC fractions were evaluated with SDS–PAGE.
ANDV-VLPs
To acquire the PCDH1–EC1 complex with ANDV-VLPs, we optimized the nucleic acid sequence for the first extracellular domain (amino acids 58–165) of human protocadherin 1, adding a C-terminal 5 × GGGS linker, which was synthesized and inserted into the pCAG-ANDV-DM-VLP plasmid. This insertion was placed between the ANDV native signal peptide (1–18) and Gn’s N-terminal residue. The resulting plasmid, pCAG-PCDH1-EC1-GS5-ANDV-DM-VLP, was then mixed with 25 kDa polyethyleneimine and transfected into FreeStyle 293-F cells. These cells were incubated under specific conditions for 72 hours following transfection. Afterward, we collected the supernatants by centrifugation and filtered them. The concentrated supernatant underwent purification using ultracentrifugation through a discontinuous sucrose density gradient, and the relevant fractions were further concentrated for cryo-EM grid preparation.
PCDH1 fragments
To obtain His-tagged versions of PCDH1 domains (sEC1–4, sEC1–2, and sEC1), we cloned the codon-optimized fragments for humans into various plasmids in alignment with a histidine tag. The plasmids sEC1–4 and sEC1 were used to transfect Expi293 cells, while the sEC1–2 plasmid, which has a FLAG tag, was used in FreeStyle 293-FS cells. Proteins were purified through various chromatography methods, including nickel-chelation chromatography for sEC1–2 and TALON affinity chromatography followed by SEC for the others.
Antibodies
We developed full-length IgG versions of the monoclonal antibodies by cloning the synthetic variable regions into pCAGGS plasmids linked to corresponding human IgG1 constant regions. These were expressed in Expi293 cells and then purified using protein G affinity chromatography. Furthermore, we obtained scFv versions by linking the variable regions of the heavy and light chains and transfecting them into Drosophila S2 cells. The fragments were purified similarly through Strep-Tactin affinity chromatography and SEC.
Cryo-EM grid preparation and data collection
Soluble spike proteins
For cryo-EM observation, the purified soluble spikes, either alone or with PCDH1 EC1–4, were diluted and deposited onto specifically prepared copper grids. The next steps involved blotting, freezing and imaging with a sophisticated system equipped for super-resolution at an elevated magnification. A detailed description of the data collection parameters can be located in associated tables.
ANDV-VLPs
For VLP preparation, the procedure of grid conditioning and plunge freezing was similar to that used for the spikes. The collected grids were screened prior to transferring to different facilities for high-resolution imaging. Data collection was executed on an advanced imaging system, resulting in a significant number of videos that would be processed for further analysis.
EM data processing and model building
Soluble spikes
For processing the EM data, multiple computational techniques, including motion correction and refinement, were employed using cryoSPARC. The model was initially built with high-resolution structures as reference points, followed by extensive manual adjustments and iterative refinements to finalize the structure.
ANDV-VLPs
The VLP data was processed similarly, following established methodologies, beginning with motion correction and CTF estimation. The resulting models were classified and refined to obtain high-quality structures for analysis.
ELISA
Filtration plates were coated with the recombinant PCDH1, incubated, and washed before blocking. Binding experiments with ANDV spikes involved serial dilutions, followed by detection through various antibody incubations and measurement of absorbance. The binding interactions of other proteins were also evaluated using established protocols, including competition and structural ELISA assays leveraging reporter vectors.
BLI measurements
To measure binding interactions, a series of experiments using BLI were set up with sensors that were first activitated and then exposed to His-tagged proteins before evaluations took place, recording rates of interactions under specific conditions. Control interactions were also monitored to ensure specificity in the assays.
Crystallization and structure determination of the complex GnH–MIB22
The crystallization of the complex was initiated through careful preparation and screening. Crystals formed under controlled environments were then preserved and processed for data collection, where structural determinations were made using sophisticated software and analysis techniques.
Cloning of pSHV-ANDV Gn libraries
A system was developed allowing for the expression of ANDV proteins in a replicon system aimed at studying hantavirus entry. The coding sequence was segmented and cloned into a vector, resulting in libraries containing various mutations for further exploration, with sequences verified to ensure quality.
Generation and amplification of SHV libraries
Transfections of the sublibrary pools were performed to generate viral stocks, with steps taken to prevent any contamination from non-mutant variants. Isolation and concentration of viruses followed by cleaning procedures ensured high-quality virus samples for further experiments.
Pull-down of PCDH1-bound SHV particles
SHV particles were enriched through ELISA techniques, involving careful coating and blocking strategies to isolate PCDH1-binding variants. Subsequent analyses would focus on RNA isolation and PCR to evaluate the presence of specific variants within the samples.
Illumina sequencing of SHV genomes
RNA from PCDH1-bound SHVs was isolated and sequenced to assess various mutations and confirm their characteristics against a reference sequence. Detailed protocols ensured high coverage during sequencing, with follow-up analyses performed for comprehensive genetic insights.
Analysis of the SHV sequencing data
Sequencing data was processed meticulously, aligning reads to references and extracting mutations of interest. A range of metrics guided the evaluation of enrichment and reproducibility across samples, establishing robust findings for further investigations.
rVSVs
Replication-competent rVSVs were produced bearing specific hantavirus proteins, utilizing established methods in cell lines. These constructs underwent verification processes to confirm their expressions and were subsequently propagated for in-depth studies.
Infection inhibition and neutralization assays
The capacity of PCDH1 to inhibit rVSV infections was assessed. An array of dilution experiments helped establish the neutralizing prowess of full-length antibodies against the rVSV variants while infection parameters were thoroughly recorded.
Immunoblotting
To quantify glycoprotein levels within rVSVs, immunoblotting procedures were used. Different antibodies targeted specific viral proteins, allowing for detailed examination of glycoprotein incorporation into viral structures, ensuring reliable normalization for assays.
Detection of soluble PCDH1 bound to Gn–Gc-expressing cells by flow cytometry
The binding of sEC1 to ANDV glycoproteins displayed on cell membranes was scrutinized through flow cytometry. Cells infected with rVSVs were treated with blocking agents and incubated with the probe, enabling the identification of specific interactions within cell populations.
Figure preparation and visualization
Molecular representations and figures were crafted using advanced visualization tools, facilitating the presentation of findings. Various software aids were employed for comprehensive data visualization across graphical displays.
Reporting summary
Further research design information is accessible through linked summaries to this article.
