Ultrastructural insight into SARS-CoV-2 entry and budding in human airway epithelium

The SARS-CoV-2 pandemic underscored the need to understand viral entry and budding mechanisms. SARS-CoV-2's high affinity for the ACE2 receptor, expressed apically on airway epithelial cells, contributes to rapid spread. TMPRSS2, a serine protease in the respiratory epithelium, facilitates entry by cleaving the S glycoprotein, triggering membrane fusion and nucleocapsid release. Alternative entry pathways exist in TMPRSS2-deficient cells, involving endocytosis, endosomal trafficking, and cathepsin-mediated S glycoprotein cleavage. Viral replication occurs at perinuclear sites, involving double-membrane vesicles (DMVs) and convoluted membranes. Budding likely occurs at ER-Golgi intermediate compartments (ERGIC) or via lysosomes. Emerging variants, like B.1.1.7, raise concerns about transmissibility and immune escape. This study investigated the ultrastructure of HAE cells infected with three SARS-CoV-2 isolates (B.1.1.7, B.1.258, and B.1.117.19) to gain insight into viral entry, replication, and release within the human airway epithelium.

Existing literature established the crucial role of ACE2 and TMPRSS2 in SARS-CoV-2 entry. Studies highlighted the importance of the S glycoprotein's receptor-binding domain (RBD) and the S2 subunit's role in membrane fusion. Alternative entry pathways utilizing endocytosis and cathepsins were also documented. Research described the formation of replication organelles like DMVs and the potential involvement of ERGIC and lysosomes in viral budding and egress. Studies on SARS-CoV-2 variants, including B.1.1.7, highlighted concerns about increased transmissibility and immune evasion. However, a comprehensive ultrastructural analysis of viral entry and budding in primary human airway epithelial cells was lacking.

Primary human airway epithelial (HAE) cells, differentiated at an air-liquid interface (ALI), were infected with three SARS-CoV-2 isolates (B.1.1.7, B.1.258, B.1.117.19) at a low MOI (0.01 pfu/cell). Cells were fixed at 72 h post-infection. Conventional transmission electron microscopy (TEM) was used to visualize viral structures and cellular morphology. Immunofluorescence (IF) was performed to localize ACE2 and TMPRSS2. Electron tomography (ET) was employed for 3D reconstruction of viral fusion and budding events. Immunoelectron microscopy (iEM) was used to label specific viral proteins. Nasal brushing biopsies from healthy and infected individuals were also examined. HeLa cells expressing ACE2, TMPRSS2, or S glycoprotein were used for antibody validation. Statistical analysis used paired, two-tailed Student's t-tests.

The study revealed that SARS-CoV-2 preferentially infected ciliated cells, with viral particles primarily associated with microvilli, not cilia. ACE2 and TMPRSS2 were localized to microvilli-rich regions of the plasma membrane, explaining the observed viral attachment pattern. Goblet cells showed minimal signs of infection. Electron tomography provided visual evidence of viral fusion at the plasma membrane, showing viral membrane continuity with the host cell membrane and diluted nucleocapsid content. The study observed virion budding from the membranes of viral-containing compartments (VCs), suggesting this is the primary mechanism for virion envelopment with S glycoprotein. Subtomographic averaging of plasma membrane protrusions and VC protrusions showed similarities to viral S glycoprotein structure. No significant ultrastructural differences were found between the three SARS-CoV-2 isolates examined.

This study's findings clarify SARS-CoV-2 entry and budding mechanisms in primary human airway cells. The preferential targeting of microvilli on ciliated cells, supported by the localization of ACE2 and TMPRSS2, provides valuable insights into viral tropism. The scarcity of infection in goblet cells suggests a potential protective role of mucus. The visualization of viral fusion at the plasma membrane and budding within VCs contributes to a more comprehensive understanding of the viral life cycle. The observed S glycoprotein structures on the plasma membrane and VCs, confirmed by iEM, provide a reliable marker of infected cells. The lack of discernible ultrastructural differences between the three SARS-CoV-2 variants points to a conserved entry and budding process.

This ultrastructural analysis provides novel insights into SARS-CoV-2 infection in primary human airway epithelium, demonstrating microvilli as the primary site of entry and VCs as the likely site of budding. The findings offer potential therapeutic targets. Further research should focus on characterizing VCs and elucidating the precise mechanisms of viral release and exploring alternative entry routes.

The study used a limited number of SARS-CoV-2 isolates and focused on a single time point (72 h post-infection). The generalizability of the findings to other viral variants or stages of infection requires further investigation. The techniques used, while powerful, may have limitations in fully resolving certain structural details. Additionally, the study focused primarily on ultrastructural observations, with further molecular characterization needed to confirm some conclusions.