Dual-Fluorescence Labeling Pseudovirus for Real-Time Imaging of Single SARS-CoV-2 Entry in Respiratory Epithelial Cells.

The pseudovirus strategy makes studies of highly pathogenic viruses feasible without the restriction of high-level biosafety facility, thus greatly contributing to virology and is used in the research studies of SARS-CoV-2. Here, we generated a dual-color pseudo-SARS-CoV-2 virus using a human immunodeficiency virus-1 pseudovirus production system and the SARS-CoV-2 spike (S) glycoprotein, of which the membrane was labeled with a lipophilic dye (DiO) and the genomic RNA-related viral protein R (Vpr) of the viral core was fused with mCherry. With this dual-color labeling strategy, not only the movement of the whole virus but also the fate of the labeled components can be traced. The pseudovirions were applied to track the viral entry at a single-particle level in four types of the human respiratory cells: nasal epithelial cells (HNEpC), pulmonary alveolar epithelial cells (HPAEpiC), bronchial epithelial cells (BEP-2D), and oral epithelial cells (HOEC). Pseudo-SARS-CoV-2 entered into the host cell and released the viral core into the cytoplasm, which clearly indicates that the host entry mainly occurred through endocytosis. The infection efficiency was found to be correlated with the expression of the known receptor of SARS-CoV-2, angiotensin-converting 2 (ACE2) on the host cell surface. We believe that the dual-color fluorescently labeled pseudovirus system created in this study can be applied as a useful tool for many purposes in SARS-CoV-2/COVID-19.

Introduction

Since 2020, COVID-19 has raged globally, posing a huge threat to human health and severely damaging the world economy.[1−3] Understanding the infection mechanism has become one of the top priorities. While determining that the pathogen was a new coronavirus, later named SARS-CoV-2, scientists immediately confirmed that SARS-CoV-2 uses the same cell entry receptor-angiotensin converting enzyme II (ACE2)-as SARS-CoV by its spike (S) protein.[4−6] SARS-CoV-2 enters host cells mainly through endocytosis after binding to the ACE2 receptor, and the proposed model is based on lysosomotropic inhibitor treatment of the infected cells.[7] However, how the virus enters the cell after binding to the receptor has never been visualized through live imaging. In addition, how sensitive are different parts of the respiratory tract to the virus remains undocumented. Our interest is to conduct systematic investigations of these issues.

Due to the high pathogenicity of SARS-CoV-2, it needs to be operated in a biosafety level-3 laboratory (BSL-3), which restricts many routine research studies.[8−11] The pseudovirus strategy can provide a safe manner to study highly pathogenic viruses without high-level biosafety facility. They usually refer to a retrovirus that can integrate with the envelope glycoprotein of another virus to form an envelope from the foreign virus, and the genome retains the characteristics of the retrovirus itself.[12−14] The pseudovirus has a part of the functional structure of the target virus but has lost the function of reproducing in host cells, and hence, they can be used to identify the function of the viral components.[15,16] SARS-CoV-2 pseudovirus has been successfully applied to determine the receptor and infection pathway, evaluate neutralizing antibodies, and construct recombinant vaccine.[7,17,18] In our previous studies, we have developed multicolor labeling methods for viruses to visualize key molecular processes of viral infection and to elucidate the basic virological issues.[19−23]

In this study, a dual-color pseudo-SARS-CoV-2 was generated by incorporating the SARS-CoV-2 S protein into human immunodeficiency virus (HIV)-1 pseudovirus. The viral lipid envelope and the Vpr protein were labeled with a lipophilic membrane dye (DiO) and mCherry fluorescence protein, respectively. The dual-labeled virus was used to infect the human airway epithelium cultures to image the entry pathway and infection efficiency at a single-particle level in four different respiratory epithelial cells– human nasal epithelial cells (HNEpC), human oral epithelial cells (HOEC), human bronchial epithelial cells (BEP-2D), and human pulmonary alveolar epithelial cells (HPAEpiC). The entry pathway and cell-type susceptibility of SARS-CoV-2 to respiratory tract cells were first identified and systematically investigated by single-virus tracking.

Results

Construction and Characterization of Pseudo-SARS-CoV-2 Virus

The SARS-CoV-2 S plasmid, encoding the S protein, was synthesized and transfected into the 293T cells. Western blot was used to analyze the expression of the SARS-CoV-2 S protein. The major band, reflecting the full-length S protein (180 kDa), was detected using the rabbit anti-S antibody. S proteins were incorporated into HIV particles to generate a highly infectious SARS-CoV-2 pseudovirions, and the efficiency was evaluated using a monoclonal mouse anti-S1 antibody. The full-length S protein was incorporated into the pseudotyped virus; however, the majority of S proteins in pseudovirions were cleaved (Figure A). Pseudovirions were further characterized by negative staining in transmission electron microscopy (TEM). The round-shaped virus particles had an average size of 100 nm, and the cobbled surface structure of SARS-CoV-2 S proteins had an average size of 15 nm (Figure B).

Figure 1

(A) Western Blot analysis of the SARS-CoV-2 S protein in the cell lysates (left). Western Blot analysis of the SARS-CoV-2 S1 protein in pseudovirions (right). (B) TEM images of the negatively stained pseudo-SARS-CoV-2 (scale bar: 50 nm). (C) Titers of pseudo-SARS-CoV-2 with or without fluorescence tested using RT-PCR. (D) Co-localization of DiO and anti-S antibody (TRITC) signals in pseudo-SARS-CoV-2; co-localization of anti-S antibody (FITC) and mCherry fluorescent protein signals in pseudo-SARS-CoV-2; and co-localization of DiO and mCherry fluorescent protein signals in pseudo-SARS-CoV-2. The inset (right) is a zoomed view of the co-localized dots (scale bar: 2 μm).

The pseudo-SARS-CoV-2 was overlaid onto coverslips and stained for immunofluorescence (IF) with an anti-S antibody. The lipid envelope of the pseudotyped virus was labeled using DiO, and the majority of DiO (approximately 70%) was co-localized with the S protein (Figure D). The capsid protein (p24) of pseudovirions were also stained with an anti-p24 antibody and DiO, and we found that they were well co-localized. Then, pseudo-SARS-CoV-2 were exposed to the combined anti-S and anti-p24 immunofluorescence staining, and the S protein was found co-localized with the p24 protein (Figure S1). These results demonstrated that SARS-CoV-2 S proteins were successfully incorporated into the pseudovirions.

Dual-Labeled Fluorescent Infectious Pseudovirions

We generated dual-labeled fluorescent infectious pseudovirions to visualize the dynamic entry of the SARS-CoV-2 virus into the host cells. The Vpr protein of the viral core was labeled by fusing with the mCherry fluorescence protein, and the Vpr-mCherry complex was encapsulated into the pseudo-SARS-CoV-2 during virus assembly. Immunofluorescence staining with an anti-S antibody or anti-p24 antibody verified the successful labeling of the Vpr proteins with mCherry fluorescence protein (Figures D and S1). The second color was obtained by labeling the lipid envelope with DiO, and fluorescence co-localization of Vpr-mCherry and DiO verified the successful construction of dual-fluorescent pseudo-SARS-CoV-2 (Figure D).

Pseudo-SARS-CoV-2 with/without fluorescence were analyzed using RT-PCR to determine whether fluorescent labeling affected the viral infectivity. The results showed that the titer activity of single-labeled particles (Vpr-mCherry or DiO) and dual-labeled particles was similar to that of unlabeled pseudovirions, demonstrating that the labeling did not impair virus transmission (Figure C).

Real-Time Imaging of Pseudo-SARS-CoV-2 Entry into Upper Respiratory Cells, Human Nasal Epithelial Cells (HNEpC)

Pseudo-SARS-CoV-2 was used to infect different respiratory epithelial cells to conduct real-time imaging of the viral entry process. The dynamic entry of dual-labeled pseudo-SARS-CoV-2 was first tracked in the upper respiratory cells, human nasal epithelial cells (HNEpC). Only the co-localized signals of Vpr-mCherry and DiO were considered as single virus. A virus particle was observed on the surface of the HNEpC cell membrane, exhibiting the entry of pseudo-SARS-CoV-2 into HNEpC cells. Figure A,B and Movie S1 show the trajectory of the pseudovirions. The virus particle was first attached to the HNEpC cell membrane and was rapidly transported into the cytoplasm (Figure C). The results of mean square displacement (MSD) indicated that the pseudovirions were actively transported into HNEpC cells (Figure D).

Figure 2

(A) Sequential snapshots of pseudo-SARS-CoV-2 entry into an HNEpC cell (scale bar: 1 μm). (B) Differential interference contrast (DIC) image of the host cell. The blue line shows the trajectory of the virus. (C,D) Analysis of mean velocity (C) and MSD plot (D) of the virus particles shown in (A). (E) Sequential images of the separation of mCherry-Vpr and DiO of pseudo-SARS-CoV-2 in the HNEpC cell (scale bar: 1 μm). (F) DIC image of the host cell. The red spot indicates the separation site. (G–I) Trajectories [(G) scale bar: 0.5 μm], mean velocities (H), and MSD plots (I) of mCherry-Vpr and DiO.

A common virus, vesicular stomatitis virus glycoprotein (VSV-G), was incorporated into the HIV particles to generate VSV-G pseudovirions and used as the control virus. We chose the VSV-G envelope because it fused with the target cells and entered through endocytosis at a high efficiency. The control virus was also labeled with DiO and Vpr-mCherry and used to infect HNEpC cells. We observed similar endocytic patterns in HNEpC cells (Figure S1A–D and Movie S9). The results suggested that pseudo-SARS-CoV-2 entered HNEpC cells through endocytosis.

Release of Viral Core from the Envelope During Endocytic Entry Revealed by Single-Particle Tracking

During the endocytic entry of pseudovirions, we visualized the release of the viral core from the envelope into the cytoplasm in various respiratory epithelial cells. The release was first observed in HNEpC cells by temporal tracking of the dual-labeled pseudo-SARS-CoV-2. The virus particles with co-localized signals of Vpr-mCherry and DiO were imaged in the cytoplasm of HNEpC cells. During virus transportation, Vpr-mCherry (red) was separated from DiO (green), indicating the release of the viral core from the envelope (Figure E,F and Movie S2). Figure G–I shows the dynamic trajectories, velocities, and MSD of Vpr-mCherry and the envelope with DiO. Both the fluorescent dots had different trajectories, velocities, and MSD after their separation. The results suggested that the viral core successfully escaped from the endosomes and was released into the cytoplasm of HNEpC cells, and the dynamic process was necessary for productive infection.

The release process was further confirmed by using VSV-G pseudovirions as the control virus to infect HNEpC cells. While tracking the dual-labeled control virus, Vpr-mCherry (red) separated from DiO (green) in the host cells (Figure S1E–I and Movie S10). These results suggested that the viral core of pseudo-SARS-CoV-2 escaped from the envelope and were released into the cytoplasm of HNEpC cells.

Entry Process of Pseudo-SARS-CoV-2 into Lower Respiratory Cells, Human Pulmonary Alveolar Epithelial Cells

The entry process of dual-labeled pseudo-SARS-CoV-2 was tracked in HPAEpiC cells, and the dynamic behavior of the virus was visualized through real-time imaging in the lower respiratory cells. Figure A,B and Movie S3 show the trajectories of virus particles in HPAEpiC cells. Initially, the virus particles attached to the cell membrane and then were rapidly transported into the cytosol (Figure C). The MSD results suggested that the pseudovirions were endocytosed into the host cells through active transport (Figure D).

Figure 3

(A) Sequential snapshots of the entry of pseudo-SARS-CoV-2 into an HPAEpiC cell (scale bar: 1 μm). (B) DIC image of the host cell. The blue line shows the trajectory of the virus. (C,D) Analysis of mean velocity (C) and MSD plot (D) of the virus particle shown in (A). (E) Sequential images of the separation of mCherry-Vpr and DiO of pseudo-SARS-CoV-2 in an HPAEpiC cell (scale bar: 1 μm). (F) DIC image of the host cell. The red spot indicates the separation site. (G–I) Trajectories [(G) scale bar: 0.5 μm], mean velocities (H), and MSD plots (I) of mCherry-Vpr and DiO.

Then, the release process of pseudo-SARS-CoV-2 was imaged in human HPAEpiC cells. We visualized the separation of the Vpr-mCherry signal (red) from the DiO signal (green) during virus transportation in the cytoplasm, indicating that the viral core was released from the envelope (Figure E,F and Movie S4). The different dynamic trajectories, velocities, and MSD for these two parts during the separation behavior in these cells are shown in Figure G–I, demonstrating the dynamic release of the viral core into the cytoplasm.

The control virus, DiO/Vpr-mCherry dual-labeled VSV-G pseudovirions, was used to infect HPAEpiC cells and showed similar entry and release processes (Figure S3 and Movies S11, S12). The results suggested that the pseudo-SARS-CoV-2 entered HPAEpiC cells through endocytosis.

Single-Particle Tracking of Pseudo-SARS-CoV-2 in the Other Two Respiratory Epithelial Cells

The dual-labeled pseudo-SARS-CoV-2 was also used to infect human bronchial epithelial (BEP-2D) cells, a type of lower respiratory cells, and HOECs, a type of upper respiratory cells. The endocytic patterns of pseudo-SARS-CoV-2 in these two cell lines were visualized through single-particle tracking. The virus particles were attached to the cell membrane, rapidly transported into the cytosol, followed by the sequential release of the viral core from the envelope (Figures , 5 and Movies S5, S6, S7, S8). The control group captured similar phenomena through real-time imaging of the dual-labeled VSV-G pseudotyped virus in both BEP-2D cells and HOECs (Figures S4–S5 and Movies S13, S14, S15, S16). The results suggested that the pseudo-SARS-CoV-2 entered BEP-2D cells and HOEC cells through endocytosis. The endocytic entry of pseudo-SARS-CoV-2 in different types of respiratory epithelial cells was visualized and it was found to exhibit similar sequential patterns.

Figure 4

(A) Sequential snapshots of the entry of pseudo-SARS-CoV-2 into a BEP-2D cell (scale bar: 1 μm). (B) DIC image of the host cell. The blue line shows the trajectory of the virus. (C,D) Analysis of mean velocity (C) and MSD plot (D) of the virus particle shown in (A). (E) Sequential images of the separation of mCherry-Vpr and DiO of pseudo-SARS-CoV-2 in a BEP-2D cell (scale bar: 1 μm). (F) DIC image of the host cell. The red spot indicates the separation site. (G–I) Trajectories [(G) scale bar: 0.5 μm], mean velocities (H), and MSD plots (I) of mCherry-Vpr and DiO.

Figure 5

(A) Sequential snapshots of the entry of pseudo-SARS-CoV-2 into a HOEC cell (scale bar: 1 μm). (B) DIC image of the host cell. The blue line shows the trajectory of the virus. (C,D) Analysis of mean velocity (C) and MSD plot (D) of the virus particle shown in (A). (E) Sequential images of the separation of mCherry-Vpr and DiO of pseudo-SARS-CoV-2 in a HOEC cell (scale bar: 1 μm). (F) DIC image of the host cell. The red spot indicates the separation site. (G–I) Trajectories [(G) scale bar: 0.5 μm], mean velocities (H), and MSD plots (I) of mCherry-Vpr and DiO.

Receptor ACE2 was Critical for the Efficiency of SARS-CoV-2 Productive Infection

We performed a high-throughput analysis of dual-labeled pseudo-SARS-CoV-2 in different respiratory epithelial cells to study the efficiency of viral productive infection. The infected cells were fixed at different time points, and viral entry efficiency was analyzed in HNEpC, HPAEpiC, BEP-2D, and HOEC, respectively. At each time point, 500 host cells were randomly selected for statistical analysis, and the results were collected during 0–180 min post-infection window. In the cytoplasm, the obvious increase in the co-localized signals of DiO and Vpr-mCherry at 0–60 min was due to the entry of the virus particles into the host cells. Also, there was a significant decrease in the co-localized dots at 60–90 min, indicating that the viral core was released from the envelope during this period. The productive infection of pseudo-SARS-CoV-2 showed a noticeable difference in HNEpCs, HPAEpiCs, BEP-2D cells, and HOECs (Figure A). However, similar levels in the productive infection were observed among the four cell types infected with VSV-G pseudotyped virus (control) (Figure B). The results suggested that the differences between the pseudo-SARS-CoV-2 and VSV-G pseudovirions were probably mediated by the interaction between S proteins and the SARS-CoV-2 receptors on these respiratory epithelial cells.

Figure 6

(A) Statistical analysis of the dual-fluorescent pseudo-SARS-CoV-2 in respiratory epithelial cells which were fixed for 0–180 min post-infection. (B) Statistical analysis of the dual-fluorescent VSV-G pseudovirions in respiratory epithelial cells which were fixed for 0–180 min post-infection. (C) Western blot analysis of ACE2 receptor in the BHK-21 cells as well as different respiratory epithelial cells. (D) RT-PCR analysis of the intracellular viral RNA copies, representing SARS-CoV-2 entry.

ACE2, the receptor for SARS-CoV-2, in these respiratory epithelial cells was tested using western blot. In Figure C, the expression of the ACE2 receptor on these cells was consistent with SARS-CoV-2 productive infection based on statistical analysis. Next, pseudo-SARS-CoV-2 with equal numbers of RNA copies was used to infect respiratory epithelial cells to further determine the efficiency of the virus entry (Figure D). At 2 h post-infection, total RNA was extracted from the host cells, and the intracellular viral RNA copy number was analyzed by RT-PCR. The intracellular viral RNA copy number of pseudo-SARS-CoV-2 in each host was positively correlated with the expression of the ACE2 receptor, indicating that the ACE2 receptor was critical for the efficiency of SARS-CoV-2 productive infection.

Discussion

In this work, we constructed dual-color pseudo-SARS-CoV-2 to investigate the entry pathway of the virus as well as the infection efficiency in respiratory epithelial cells under the conditions of BSL-2. Western blot and TEM results showed that the SARS-CoV-2 S protein had been incorporated into the pseudovirions and formed the cobbled surface structure in the HIV-1 pseudovirus system. Additionally, the lipid envelope staining or immunofluorescence staining was performed to verify the fluorescence labeling of the virus particles. The results suggested that the constructed pseudo-SARS-CoV-2 possessed a high fluorescence co-localization efficiency. These particles also owned high infectivity, and the labeling strategies exerted no significant interference to the viral structure and function. Thus, the dual-fluorescent pseudo-SARS-CoV-2 provides a suitable tool for imaging the dynamic process of the virus entry and capture the details at the single-particle level.

As an air-transmitted virus, SARS-CoV-2 infection must happen through interactions between the virus and respiratory cells. Whether it only infects particular types of respiratory cells remains elusive. By observing the patterns of the virus entry among the four types of respiratory cells (HNEpC, HOEC, BEP-2D cells, and HPAEpiC), we found that about 20–25 similar events were captured by tracking 20,000 virus particles, and pseudo-SARS-CoV-2 exhibited identical sequential process, suggesting there is either none or little preference among these cells. Meanwhile, we showed that SARS-CoV-2 productive infection in different types of respiratory epithelial cells occurred through endocytosis. This result is consistent with the report from Ou et al. who showed that drugs blocking endocytosis can prevent the entry of SARS-CoV-2.[7]

Statistical analysis and RT-PCR results showed that there was a significant difference in the infection efficiency on various respiratory epithelial cells, and pseudo-SARS-CoV-2 preferentially infected HNEpC. This could be attributed to the different levels of expression of SARS-CoV-2 receptors on these cells, because pseudo-SARS-CoV-2 infection required interaction between the S protein and ACE2. In contrast, VSV-G-pseudotyped virus failed to differentiate cells with different amounts of ACE2 receptors. The results were consistent with the earlier reports that the nose contained the highest percentage of ACE2-expression cells in the airways, and the nasal surfaces were the dominant initial site for SARS-CoV-2 respiratory tract infection.[2,16,24]

Our study provides an excellent tool to systematically visualize the progression of SARS-CoV-2 infection in live cells. In the future, other viral components could be included in the pseudovirions system to investigate additional processes of the viral life cycle at the single-particle level. On the other hand, although we found that endocytosis via interaction between SARS-CoV-2 S protein and ACEs is the major driving force of SARS-CoV-2 infection, we have not evaluated the effects of endocytosis inhibitors, neutralizing antibody, or mutations of S protein during virus entry into cells. These relevant studies should be performed using live SARS-CoV-2 virus under the conditions of BSL-3.

Conclusions

We constructed a dual-color fluorescence-labeled pseudo-SARS-CoV-2 to study virus entry and cell susceptibility under BSL-2 conditions. The dynamic process of virus entry was visualized, imaged, and analyzed in live cells at the single-virion level. We observed similar patterns of entry pathways in different respiratory epithelial cells through endocytosis. There was a positive correlation between the infection efficiency of the SARS-CoV-2 and the expression level of the ACE2 receptor. The method may be further developed to elucidate the life cycle of SARS-CoV-2 at a single-particle level.

Materials and Methods

Plasmids

The pcDNA3.1(+)-Vpr-mCherry was produced to express the Vpr-mCherry protein. pNL4-3-KFS (△env), pSARS-CoV-2-S, and pVSV-G were used to assemble the pseudovirions. The SARS-CoV-2 spike protein: MN908947.3 (https://www.ncbi.nlm.nih.gov/nuccore/MN908947.3).

Cell Culture

Immortalized human bronchial epithelial (BEP-2D) cells and human alveolar epithelial cells (HPAEpiCs) were obtained from BioDee (Beijing, China). Human oral epithelial cells (HOECs) were obtained from BlueBio (Shanghai, China). Human nasal epithelial cell line (HNEpC) was obtained from Tongpai Biotechnology (Shanghai, China). All cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS (Gibco) at 37 °C in a 5% CO2 humidified atmosphere.

Pseudovirions Preparation

The virus-related experiments were conducted in the BSL-2 laboratories. The 293T cells (5 × 106) were co-transfected with 10 μg of pNL4-3-KFS (△env) and 10 μg of pSARS-CoV-2-S using lipofectamine 3000 (Invitrogen) to construct the pseudo-SARS-CoV-2. Also, 293T cells were co-transfected with 10 μg of pNL4-3-KFS (△env) and 10 μg of pVSV-G to generate VSV-G pseudovirions. The virus particles were harvested at 24 h post-transfection.

Fluorescent Labeling and Purification

Cells were transfected with 10 μg of pNL4-3-KFS (△env), 10 μg of pSARS-CoV-2-S, and 2 μg of pcDNA3.1 (+)-Vpr-mCherry to produce pseudo-SARS-CoV-2 (Vpr-mCherry). Next, 10 mL of the virus particles were stained with 0.5 μL of DiO (Invitrogen) for 1 h at 37 °C to label the lipid envelope. The DiO solution contained dimethylformamide, and the concentration was 1 mM. Equal volumes of PEG 20,000 solution [PEG 20,000 (20% (w/v)] were dissolved in 0.9% NaCl solution) and the virus was mixed at 4 °C overnight, and the solution was centrifuged at 9000 rpm for 20 min. The purified virus was resuspended in 1 mL of Opti-MEM. The cells were transfected with 20 μg of pNL4-3-KFS (△env), 10 μg of pVSV-G, and 2 μg of pcDNA3.1 (+)-Vpr-mCherry to prepare VSV-G pseudovirions (Vpr-mCherry). DiO was also used to prepare VSV-G pseudovirions (DiO).

Immunofluorescence Staining

Pseudo-SARS-CoV-2 was fixed in 4% paraformaldehyde, permeabilized using 0.1% Triton-X100, and blocked with FBS containing 10% bovine serum albumin. Next, they were incubated overnight with primary antibody at 4 °C, followed by incubation with a secondary antibody for 2 h at 37 °C. Pseudo-SARS-CoV-2 was stained using the rabbit anti-SARS-CoV-2 S antibody (1:200, Sinobio) and/or mouse anti-p24 antibody (1:200, Abcam). Goat anti-mouse IgG conjugated with Alexa Fluor 488 (1:500, CST), rabbit anti-mouse IgG conjugated with Alexa Fluor 488 (1:500, CST), goat anti-rabbit IgG conjugated with Alexa Fluor 488 (1:500, CST), and goat anti-rabbit IgG conjugated with Alexa Fluor 555 (1:500, CST) were used as secondary antibodies.

Virus Infection and Fluorescence Imaging

Respiratory epithelial cells were plated into a confocal dish and incubated with pseudo-SARS-CoV-2 (DiO-mCherry) for 30 min at 4 °C. The unbound particles were removed and replaced with fresh media before incubation at 37 °C in a 5% CO2 atmosphere. Infected cells were imaged under an UltraView Vox spinning disk confocal laser scanning system using a Nikon Ti-e microscope with 60× objective. DiO and mCherry were excited at 488 and 561 nm, respectively. Real-time imaging was usually performed at an interval of 10 s for 15 min to minimize photobleaching.

Statistical Analysis

Respiratory epithelial cells were incubated with dual-labeled fluorescent pseudo-SARS-CoV-2 (DiO-mCherry) for 30 min at 4 °C and removed and incubated at 37 °C for different time intervals (0, 30, 60, 90, 120, and 180 min). Finally, the cells were fixed in 4% formaldehyde for imaging. The viral entry efficiency was analyzed in the fixed cells, and 500 host cells were randomly selected for statistical analysis at each time point.

Western Blot

The samples were boiled for 10 min, separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a PVDF membrane. After blocking the non-specific sites with 5% (w/v) skim milk, the membranes were incubated with the primary antibody, followed by the HRP-conjugated secondary antibody. The enhanced chemiluminescence detection kit (Beyotime Biotechnology) was used to detect the bands, which were visualized using a ChemStudio Imaging System (Analytik Jena AG). Rabbit anti-SARS-CoV-2 S antibody (1:1000; ABclone), mouse anti-SARS-CoV-2 S1 antibody (1:1000; Sinobio), rabbit anti-ACE2 antibody (1:1000; Abcam), mouse anti-GAPDH antibody (1:5000; TransGen Biotech), mouse anti-β-actin antibody (1:2000; CST), and mouse anti-p24 antibody (1:2000; Abcam) were used as the primary antibodies. HRP-linked horse anti-mouse IgG and HRP-linked horse anti-rabbit IgG (1:2000; CST) were used as the secondary antibodies.

Transmission Electron Microscopy Analysis

Pseudo-SARS-CoV-2 was concentrated using the PEG 20000 solution, followed by ultrafiltration using an Amicon Ultra-4 centrifugal filter device (50 kDa, Millipore). The carbon support copper grid was placed on the purified pseudovirions and incubated for 10 min. Then, the pseudovirions loaded on the carbon support copper grid were negatively stained with 2% phosphotungstate for 30 s. The samples were observed on a 200 kV JEM-F200 transmission electron microscope.

Real-Time PCR

Cellular total RNA of the infected cells was extracted 24 h post-transfection, and the quantitative real-time PCR analysis was performed using a HiScript II One-Step qRT-PCR Probe Kit (Vazyme Biotech). To confirm the virus entry, cellular RNA was extracted 2 h post-transfection and analyzed using RT-PCR. The DNA sequences used were as follows: the primer HIV sense (TTT GAC TAG CGG AGG CTA GAA G), HIV antisense (CCC TGG CCT TAA CCG AAT TTT), and a specific TaqMan probe (FAM-CGC TTA ATA CCG ACG CTC TCG C-TAMRA).