Human lung epithelial A549 cells (CCL-185, ATCC) were maintained in F-12 medium (Thermo Fisher) supplemented with 10% fetal bovine serum. A549 cells stably expressing hACE2 (hACE2-A549 cells) were kindly gifted by Dr. Chia-Yi Yu (National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes, Taiwan). African green monkey kidney Vero E6 cells (CRL-1586, ATCC) were maintained in Eagle’s minimum essential medium (Thermo Fisher) supplemented with 10% fetal bovine serum.

The expression of exogenous genes was achieved using a lentivirus system. hACE2-A549 cells were transduced with RFP-expressing lentiviruses from RNA Technology Platform and Gene Manipulation Core (RNAi Core, Taiwan) to generate hACE2-RFP-A549 cells. A549 cells were transduced with mCherry-expressing lentiviruses (RNAi Core) to generate mCherry-A549 cells. To generate hACE2-A549 clones, single cell was sorted from the population of hACE2-A549 cells by using a FACSJazz-6 color cell sorter (BD Biosciences). These clones were cultured in 96-well plates for 1 week and then transferred to 6-well plates for 4 days to obtain sufficient cells for further experiments. The hACE2 expression level of each hACE2-A549 clone was analyzed using IFA and then confirmed through Western blotting with anti-ACE2 antibody.

SARS-CoV-2 (hCoV-19/Taiwan/4/2020, GISAID accession ID: EPI_ISL_411927) isolated from a patient with COVID-19 was obtained from the Taiwan Centers of Disease Control. The virus was amplified in Vero E6 cells and the virus titer was determined using a tissue culture infective dose assay.

We used the following primary antibodies: anti-tubulin rabbit monoclonal antibody (mAb) (#2128, Cell Signaling), anti-ACE2 rabbit mAb (GTX01160), anti-SARS-CoV-2 S protein mouse mAb (GTX632604), anti-SARS-CoV-2 nucleocapsid protein (N) mouse mAb (GTX632269), anti-SARS-CoV-2 nonstructural protein 3 (NSP3) rabbit polyclonal antibody (GTX135589) (GTX all from GeneTex), and anti-SARS-CoV-2 S protein humanized monoclonal antibody (hmAb; kindly gifted by Dr. An-Suei Yang, Genomics Research Center, Academia Sinica, Taiwan). Secondary antibodies included goat Alexa Fluor 488-conjugated anti-mouse, Alexa Fluor 488-conjugated anti-human, and Alexa Fluor 568-conjugated anti-rabbit IgG antibodies (Thermo Fisher).

Neutralizing hmAbs against the RBD of SARS-CoV-2 S protein were kindly gifted by Dr. Han-Chung Wu [12] (Institute of Cellular and Organism Biology, Academia Sinica, Taiwan). The control hmAb was kindly gifted by Dr. Kuo-I Lin (Genomics Research Center, Academia Sinica, Taiwan).

For cell-free infection, hACE2-A549 cells were infected with SARS-CoV-2, which was premixed with antibodies or plasma at 37 °C for 1 h, at a multiplicity of infection (MOI) of 0.2. At 1 h post infection (hpi), additional antibody- or plasma-containing medium was added. The supernatant was harvested for the virus infectivity assay, and cells were fixed for IFA with anti-NSP3 antibody and high-content image analysis at 24 hpi.

For the coculture system, hACE2-A549 cells were infected with SARS-CoV-2 at an MOI of 0.2 for 24 h for obtaining the virus donor cells. The virus donor cells were trypsinized and cocultured with virus recipient cells (hACE2-RFP-A549 cells) at a 1:1 ratio in antibody- or plasma-containing medium. The supernatant was harvested for the virus infectivity assay, and at 24 h after coculture, the cells were fixed and subjected to IFA with anti-NSP3 antibody and high-content image analysis.

For temporal viral spread analysis, hACE2-A549 clones at approximately 90% confluence seeded in 96-well plates were infected with SARS-CoV-2 at an MOI of 0.01. At 1 hpi, the medium was replaced with antibody-containing medium. At 24, 48, and 72 hpi, the supernatant was harvested for the virus infectivity assay, and the cells were fixed and subjected to IFA with anti-NSP3 antibody and high-content image analysis.

For the virus infectivity assay, hACE2-A549 cells with approximately 50% confluence were seeded in 96-well plates and then incubated with the supernatant harvested from the virus transmission assay. At 24 h after culture, the cells were fixed and subjected to IFA with anti-NSP3 antibody.

For the viral shedding assay, hACE2-A549 clones were adsorbed with SARS-CoV-2 at an MOI of 0.2 for 1 h. After the removal of the viral inoculant, the cells were washed twice with phosphate-buffered saline (PBS) and incubated in fresh medium. At 24 hpi, the supernatant was harvested and viral RNA was extracted using the RNeasy Mini Kit (Qiagen) and was quantified using real-time reverse transcription–polymerase chain reaction (RT-PCR) with primers targeting the E gene of SARS-CoV-2, as described previously [13]. The viral RNA copy number was determined using a real-time RT-PCR standard generated from a synthetic oligonucleotide fragment of E gene (Genomics BioSci and Tech).

All experiments involving SARS-CoV-2 were conducted in a biosafety level 3 laboratory in accordance with the guidelines established by the Biosafety Level 3 Facility of the Institute of Biomedical Sciences (IBMS), Academia Sinica, Taipei, Taiwan.

Cells were seeded in 96-well plates or on coverslips for 24 h. After infection, the cells were fixed with 4% paraformaldehyde at room temperature for 20 min, permeated with 0.5% Triton X-100 buffer for 2 min, and blocked with 3% BSA for 30 min. The cells were stained with primary antibodies at room temperature for 1 h, washed three times with PBS, and incubated with secondary antibodies, DAPI (Sigma) for nuclear staining, or Alexa Fluor 647 phalloidin (Thermo Fisher) for F-actin staining at room temperature for 1 h. Viral RNA (vRNA) was stained using the RNAscope Multiplex Fluorescent V2 Assay kit (ACD) with a SARS-CoV-2-specific probe (NC_045512.2, ACD).

For high-content image analysis, immunofluorescence images were acquired using the ImageXpress Micro XLS Widefield high-Content Analysis System (Molecular Devices), and the virus infection rate was measured using MetaXpress Software (Molecular Devices), as described previously [14]. To obtain super-resolution immunofluorescent images, images were acquired using a Zeiss LSM880 confocal microscope equipped with Airyscan [15].

First, 5 × 105 hACE2-A549 cells were infected with SARS-CoV-2 at an MOI of 0.2 for 24 h and fixed with 4% paraformaldehyde and 0.2% glutaraldehyde in PBS at room temperature for 1 h. The fixed cells were washed twice with PBS for 5 min before immunofluorescence staining as described above. The immunofluorescence images were acquired using a Zeiss LSM700 microscope. Next, the same sample was incubated with 0.1% OsO4 in 0.1 M PBS for postfixation, dehydrated, and embedded in Spurr’s resin for further transmission electron microscopy (TEM) examination. Ultrathin Sects. (100 nm) were examined using Tecnai G2 Spirit TWIN TEM (Thermo Fisher). Subsequently, for precise interpretation of CLEM data, both immunofluorescence and TEM images were aligned using the ec-CLEM plugin on the Icy platform.

Data are presented as mean ± SD from three independent samples. Statistical significance was set at p < 0.05, and the data were analyzed using two-tailed Student’s t test.