The protein OCLN was found to play an important role in the cell-to-cell transmission of SARS-CoV-2

A recent study published in the journal PNASresearchers found that the tight junction (TJ) protein occludin (OCLN) is critical for direct cell-to-cell transmission in the host via the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike(S). proved to be

study: The tight-junction protein occludin is an internalizing factor for SARS-CoV-2 infection and mediates viral cell-to-cell transmissionImage Credit: Naeblys/Shutterstock

Background

Viral infection through close cell-to-cell contact weakens antiviral drugs and helps evade virus Neutralizing antibody (nAbs). Many human viruses, such as SARS-CoV-2, influenza A, and respiratory syncytial virus (RSV), use this mechanism, cell-free particles, or both to establish infection and survive within the host. This is proven in research.

Previous studies have also associated the formation of syncytia in human lung tissue with SARS-CoV-2. SARS-CoV-2, etc. coronavirus In (CoV), for example, Middle East respiratory syndrome (MERS-CoV), syncytial formation contributes to enhanced direct cell-to-cell transmission.However, the host factors involved in this process and the underlying mechanisms of S glycoprotein–It remains unclear whether cell-to-cell spread of SARS-CoV-2 is induced.

Human airway epithelial cells have intercellular structures called TJs that regulate the passage of ions and small solutes. It also acts as the first barrier against pathogens. TJs contain the protein OCLN, which is recognized as a key host factor for the entry of several viruses into the human host, including rotavirus and hepatitis C virus.

Since virus-induced fusion of virus-infected cells with neighboring cells and creation of syncytia requires breaking the intercellular barrier, researchers suspect that TJ proteins are involved in the spread of SARS-CoV-2 via intercellular infection. I assumed there might be.

About research

In the present study, researchers investigated the effects of SARS-CoV-2 infection on TJ proteins, SARS-CoV-2 entry and subsequent cell-to-host cell transfer. They tested Vero-E6 cells infected with replication-competent vesicular stomatitis virus (rVSV) expressing enhanced green fluorescent protein (eGFP) and SARS-CoV-2 S (rVSV-eGFP-S), or SARS-E6 cells. We used CoV-2 recombinants. Express the mNeonGreen gene to determine the distribution of OCLN in infected Vero-E6 cells.

Western blot confirmed that infection with both recombinants altered OCLN expression. Accordingly, OCLN levels became undetectable in infected Her Vero-E6 cells at 48 hours post-infection (pi). The team also confirmed these results in human-derived A549-hACE2 cells and a hamster SARS-CoV-2 infection model.

Next, the team determined whether permissive cells, such as Vero-E6 and A549-hACE2 cells, also required OCLN to establish SARS-CoV-2 infection. Therefore, Vero-E6 cells were transfected with two He-OCLN-targeting small interfering RNAs (siRNAs) and immunofluorescent assays (IFA) and quantitative reverse transcription-polymerase chain reaction (RT-qPCR) were used. He confirmed OCLN expression. Additionally, the researchers performed a co-immunoprecipitation (co-IP) assay to visualize the interaction of OCLN with the SARS-CoV-2 S protein.

OCLN is a transmembrane protein with four helical domains: an intracellular N-terminal domain, a long cytoplasmic tail also known as the C-terminus, two extracellular loops, and EL1 and EL2 linked via a short intracellular loop. . So, researchers have finally identified which OCLN domains are important for SARS-CoV-2 internalization and cell-to-cell transmission.

To this end, the team constructed four OCLN deletion constructs, namely hOCLN/ΔC, hOCLN/ΔE1, hOCLN/ΔE2, and hOCLN/ΔN, and transfected them into the OCLN KO Vero-E6 cell line. . Next, Western blot and IFA were used to confirm each expression.

result

This study pointed out that the host factor, OCLN, mediates SARS-CoV-2 internalization and subsequent cell-to-cell infection. Thus, in experiments with rVSV expressing the S protein of multiple SARS-CoV-2 variants, its knockdown reduced SARS-CoV-2 spread, whereas its overexpression promoted it. . in vitro and live SARS-CoV-2 infection decreased significantly and eventually completely destroyed the OCLN.

All rVSVs expressing the S proteins of SARS-CoV-2 Delta, Beta and Kappa variants showed enhanced cell-to-cell communication over the WA-1 strain. The gamma and alpha variants showed similar ability to promote syncytia formation as the WA-1 strain. Interestingly, Omicron has a significantly reduced ability to form syncytia in A549-hACE2 cells compared to other SARS-CoV-2 mutants and WA-1, suggesting that Omicron has a significantly reduced endocytic pathway. It shows limited ability to diffuse between cells using This observation also justifies why the Omicron BA.1/BA.2 sublineage causes less clinical symptoms than his WA-1 and other SARS-CoV-2 variants.

Furthermore, research experiments suggested that SARS-CoV-2 infection only degrades OCLN, with minimal effects on other TJ proteins such as claudin-1. Future studies should investigate the mechanisms underlying the SARS-CoV-2-induced downregulation of OCLN protein synthesis and stability in TJs. In SARS-CoV-2-permissive cells, knockdown of OCLN did not affect the expression of angiotensin-converting enzyme 2 (ACE2), but significantly reduced viral infection, especially during the internalization step and subsequent viral replication. I was. Conversely, its overexpression significantly enhanced both internalization and replication of SARS-CoV-2, but not its binding. Therefore, OCLN was not considered as a receptor or co-receptor for SARS-CoV-2 entry.

The results of this study also revealed that the endosomal entry pathway is involved in OCLN-mediated SARS-CoV-2 cell-to-cell transmission. Finally, treatment with trypsin significantly enhanced cell-to-cell transmission of SARS-CoV-2 and increased virus yield in host cells.

Conclusion

The current study provides mechanistic insight into SARS-CoV-2 entry and subsequent spread within the human host. The researchers showed robust demonstration of her OCLN-driven cell-to-cell spread of his WA-1 and all other SARS-CoV-2 variants examined in this study. In fact, OCLN is a key host factor involved in SARS-CoV-2 internalization and subsequent cell-to-cell viral infection.