The role of ARF6 in endocytosis and SARS-CoV-2 infection

In a recent study published in the bioRxiv * prepress server, researchers evaluated the role of adenosine diphosphate ribosylation factor 6 (ADP) (ARF6) in coronavirus 2 (SARS-CoV-2) infection of respiratory syndrome. acute severe.

Study: ARF6 is an important host factor for in vitro SARS-CoV-2 infection. Image credit: Corona Borealis Studio / Shutterstock


SARS-CoV-2 is a beta-coronavirus that enters human cells through two pathways: direct fusion to the plasma membrane or endocytosis, and subsequent fusion with late lysosome or endosome. Several researchers have thoroughly studied the SARS-CoV-2 receptor, various input factors, the angiotensin 2 converting enzyme (ACE2), as well as the mechanism by which the virus fuses with the plasma membrane. However, more research is needed to understand the mechanism involved in the entry of the virus through the endocytic pathway.

About the study

In the present study, researchers determined the function of ARF6 in SARS-CoV-2 endocytosis and subsequent infection.

The team examined several cell lines to evaluate the antiviral activity of a specific transmembrane serine protease 2 inhibitor (TMPRSS2) called camostat mesylate. The infection was read through an image-based channel that used SARS-CoV-2 nucleocapsid (N) antibodies to detect infected cells. The team also determined whether dynamin affected viral entry into Huh-7 cells by a specific inhibitor of dynamos guanosine triphosphatase (GTPase) called dinasora. A cholesterol-lowering agent called beta-methyl cyclodextrin was used as a positive control, as it blocked the formation of lipid ponds, which are essential for endocytosis.

The team also developed two polyclonal populations of cells targeting short palindromic repeats (CRISPR) grouped regularly between spaced with guide ribonucleic acids (gRNAs) specific for ARF6. ARF6 depletion was verified by reverse transcription polymerase chain reaction (RT-PCR). Cells were infected with SARS-CoV-2 and viral infection and replication were quantified by a 50% tissue culture infectious dose assay (TCID50) and quantitative RT PCR (RT-qPCR), respectively. .

In addition, the team tested the effect of inhibitor NAV-2729 and agonist AA147 on SARS-CoV-2 infection. The mechanism by which ARF6 supports SARS-CoV-2 replication in Calu-3 cells was also evaluated by infecting ARF6 and NTg-eliminating cells with the Omicron SARS-CoV-2 variant.


The results of the study showed that the Huh-7 human hepatocellular carcinoma cell line showed substantial resistance to the antiviral activity of the camostat. On the other hand, treatment of the cell line with the irreversible cysteine ​​protease inhibitor Z-FA-FMK resulted in strong antiviral activity. This suggested that the entry of SARS-CoV-2 into Huh-7 cells was possibly mediated by endocytosis and not by fusion of the plasma membrane. Therefore, the team used Huh-7 cells as a representation to mechanically study the endocytic entry of SARS-CoV-2.

Beta-methyl cyclodextrin treatment blocked viral infection, while dinasora treatment increased the proportion of infected cells. This suggested that the endocytic uptake mechanism used by SARS-CoV-2 depended on cholesterol. Analysis with CRISPR cells showed no difference between non-targeting (NTg) and ARF6, while viral replication was below the detection limit of ACE-2 Huh-7 cells. The team also observed a substantial reduction in the proportion of infected cells to ARF6 cells, suggesting that ARF6 played an important role in viral entry.

The NAV-2729 inhibitor showed a dose-response inhibition of the infection while the AA147 agonist increased the proportion of infected cells. This indicated that ARF6 was a crucial factor in the SARS-CoV-2 infection of Huh-7 cells. In addition, treatment of dinasora ARF-6 knockout cells did not affect infection, thus confirming that dynamin was not involved in SARS-CoV-2 endocytosis in Huh-7 cells. The team also noted that treatment with NAV-2729 substantially reduced viral replication.

In addition, the team observed that SARS-CoV-2 infection was less efficient in Calu-3 cells that were transduced with an ARF6 targeting guide compared to Calu-3 cells that were transduced. were transduced with NTg. In addition, the reduction in SARS-CoV-2 infection in Calu-3 ARF6 elimination cells was greater than in Huh-7 ARF6 elimination cells, suggesting that ARF6 had more papers in Calu-3 cells than in Huh-7 cells.

Infection of ARF6 elimination cells with the Wuhan SARS-CoV-2 strain and the Omicron variant showed a significant decrease compared to that of NTg cells. This indicated that ARF6 could be involved in the post-entry steps in Calu-3 cells.


Overall, the results of the study highlighted the potential of ARF6 as a target for the development of therapeutic agents against SARS-CoV-2 infection. Researchers believe that future studies focusing on research into ARF-6-targeted drugs could lead to the development of SARS-CoV-2 therapeutics.

* Important news

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, guided by clinical practice / health-related behavior, or treated as established information.

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