tetano
Editor, Senior Moderator
Cells
. 2021 Jul 17;10(7):1814.
doi: 10.3390/cells10071814.
SARS-CoV-2 Cellular Entry Is Independent of the ACE2 Cytoplasmic Domain Signaling
Thankamani Karthika[SUP] 1 [/SUP], Jeswin Joseph[SUP] 1 [/SUP], V R Akshay Das[SUP] 1 [/SUP], Niranjana Nair[SUP] 1 [/SUP], Packirisamy Charulekha[SUP] 1 [/SUP], Melvin Daniel Roji[SUP] 1 [/SUP], V Stalin Raj[SUP] 1 [/SUP]
Affiliations
Abstract
Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH[SUB]4[/SUB]Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s.
Keywords: ACE2; ACE2 internalization; SARS-CoV-1; SARS-CoV-2; coronavirus entry.
. 2021 Jul 17;10(7):1814.
doi: 10.3390/cells10071814.
SARS-CoV-2 Cellular Entry Is Independent of the ACE2 Cytoplasmic Domain Signaling
Thankamani Karthika[SUP] 1 [/SUP], Jeswin Joseph[SUP] 1 [/SUP], V R Akshay Das[SUP] 1 [/SUP], Niranjana Nair[SUP] 1 [/SUP], Packirisamy Charulekha[SUP] 1 [/SUP], Melvin Daniel Roji[SUP] 1 [/SUP], V Stalin Raj[SUP] 1 [/SUP]
Affiliations
- PMID: 34359983
- DOI: 10.3390/cells10071814
Abstract
Recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV)-1 and -2 initiate virus infection by binding of their spike glycoprotein with the cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and enter into the host cells mainly via the clathrin-mediated endocytosis pathway. However, the internalization process post attachment with the receptor is not clear for both SARS-CoV-1 and -2. Understanding the cellular factor/s or pathways used by these CoVs for internalization might provide insights into viral pathogenesis, transmission, and development of novel therapeutics. Here, we demonstrated that the cytoplasmic tail of ACE2 is not essential for the entry of SARS-CoV-1 and -2 by using bioinformatics, mutational, confocal imaging, and pseudotyped SARS-CoVs infection studies. ACE2 cytoplasmic domain (cytACE2) contains a conserved internalization motif and eight putative phosphorylation sites. Complete cytoplasmic domain deleted ACE2 (∆cytACE2) was properly synthesized and presented on the surface of HEK293T and BHK21 cells like wtACE2. The SARS-CoVs S1 or RBD of spike protein binds and colocalizes with the receptors followed by internalization into the host cells. Moreover, pseudotyped SARS-CoVs entered into wtACE2- and ∆cytACE2-transfected cells but not into dipeptidyl peptidase 4 (DPP4)-expressing cells. Their entry was significantly inhibited by treatment with dynasore, a dynamin inhibitor, and NH[SUB]4[/SUB]Cl, an endosomal acidification inhibitor. Furthermore, SARS-CoV antibodies and the soluble form of ACE2-treated pseudotyped SARS-CoVs were unable to enter the wtACE2 and ∆cytACE2-expressing cells. Altogether, our data show that ACE2 cytoplasmic domain signaling is not essential for the entry of SARS-CoV-1 and -2 and that SARS-CoVs entry might be mediated via known/unknown host factor/s.
Keywords: ACE2; ACE2 internalization; SARS-CoV-1; SARS-CoV-2; coronavirus entry.