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 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 the pseudovirions while assembling the virus, 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 (Fig. 1A). Pseudovirions were further characterized by negative staining in 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 (Fig. 1B).
Pseudo-SARS-CoV-2 were 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 co-localized with the S protein (Fig. 1D). 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 combined anti-S and anti-p24 immunofluorescence staining, and S protein was found co-localized with p24 protein (Supplementary Fig. 1). 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 (Fig. 1D and Supplementary Fig. 1). 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 (Fig. 1D).
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 were similar to that of unlabeled pseudovirions, demonstrating that the labeling did not impair virus transmission (Fig. 1C).
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. Fig. 2A-2B and Supplementary Movie 1 show the trajectory of pseudovirions. The virus particle was first attached to the HNEpC cell membrane and was rapidly transported into the cytoplasm (Fig. 2C). The results of mean square displacement (MSD) indicated that the pseudovirions were endocytosed into HNEpC cells through active transport (Fig. 2D).
A common virus, vesicular stomatitis virus glycoprotein (VSV-G) pseudovirions, was used as the control virus. 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 (Supplementary Fig. 1A-1D and Supplementary Movie 9). The results suggested that pseudo-SARS-CoV-2 entered HNEpC cells through endocytosis.
The 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 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 dual-labeled pseudo-SARS-CoV-2. 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 (Fig. 2E-2F and Supplementary Movie 2). Fig. 2G-2I show the dynamic trajectories, velocities, and MSD of Vpr-mCherry and the envelope with DiO. Both 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 (Supplementary Fig. 1E-1I 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.
The entry process of pseudo-SARS-CoV-2 into lower respiratory cells, human pulmonary alveolar epithelial cells (HPAEpiC). 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. Fig. 3A-3B and Supplementary Movie 3 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 (Fig. 3C). The MSD results suggested that the pseudovirions were endocytosed into the host cells through active transport (Fig. 3D).
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 (Fig. 3E-3F and Supplementary Movie 4). The different dynamic trajectories, velocities, and MSD for these two parts during the separation behavior in these cells are shown in Fig. 3G-3I, demonstrating the dynamic release of the viral core into the cytoplasm.
The control virus, DiO/Vpr-mCherry dual-labeled VSV-G pseudovirions were used to infect HPAEpiC cells and showed similar entry and release processes (Supplementary Fig. 3 and Supplementary Movie 11-12). The results suggested that 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 cells (BEP-2D), a type of lower respiratory cells, and human oral epithelial cells (HOEC), 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. 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 (Fig. 4-5 and Supplementary Movie 5-8). The control group captured similar phenomena through real-time imaging of dual-labeled VSV-G pseudotyped virus in both BEP-2D cells and HOECs (Supplementary Fig. 4-5 and Supplementary Movie 13-16). The results suggested that 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.
The 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 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 HNEpC, HPAEpiC, BEP-2D cells, and HOEC (Fig. 6A). However, similar levels in productive infection were observed among the four cells types infected with VSV-G pseudotyped virus (control) (Fig. 6B). The results suggested that the differences between 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.
ACE2, the receptor for SARS-CoV-2, in these respiratory epithelial cells was tested using western blot. In Fig. 6C, 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 were used to infect respiratory epithelial cells to further determine the efficiency of virus entry (Fig. 6D). At 2 h post-infection, total RNA was extracted from the host cells, and 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.