The interactions of ZDHHC5/GOLGA7 with SARS-CoV-2 spike (S) protein and their effects on S protein's subcellular localization, palmitoylation and pseudovirus entry.

Abstract

BACKGROUND:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. An acyltransferase complex ZDHHC5/GOGAL7 that interacts with S protein was detected by affinity purification mass spectrometry (AP-MS). However, the palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein's subcellular localization, palmitoylation, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear.

METHODS:

The palmitoylated cysteine residues of S protein were identified by acyl-biotin exchange (ABE) assays. The interactions between S protein and host proteins were analyzed by co-immunoprecipitation (co-IP) assays. Subcellular localizations of S protein and host proteins were analyzed by fluorescence microscopy. ZDHHC5 or GOGAL7 gene was edited by CRISPR-Cas9. The entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells were analyzed by measuring the activity of Renilla luciferase.

RESULTS:

In this investigation, all ten cysteine residues in the endodomain of S protein were palmitoylated. The interaction of S protein with ZDHHC5 or GOLGA7 was confirmed. The interaction and colocalization of S protein with ZDHHC5 or GOLGA7 were independent of the ten cysteine residues in the endodomain of S protein. The interaction between S protein and ZDHHC5 was independent of the enzymatic activity and the PDZ-binding domain of ZDHHC5. Three cell lines HEK293T, A549 and Hela lacking ZDHHC5 or GOLGA7 were constructed. Furthermore, S proteins still interacted with one host protein in HEK293T cells lacking the other. ZDHHC5 or GOLGA7 knockout had no significant effect on S protein's subcellular localization or palmitoylation, but significantly decreased the entry efficiencies of SARS-CoV-2 pseudovirus into A549 and Hela cells, while varying degrees of entry efficiencies may be linked to the cell types. Additionally, the S protein interacted with the depalmitoylase APT2.

CONCLUSIONS:

ZDHHC5 and GOLGA7 played important roles in SARS-CoV-2 pseudovirus entry, but the reason why the two host proteins affected pseudovirus entry remains to be further explored. This study extends the knowledge about the interactions between SARS-CoV-2 S protein and host proteins and probably provides a reference for the corresponding antiviral methods.

• Authors: Xiao-Tao Zeng, Xiao-Xi Yu, Wei Cheng
• Journal: Virology journal (Virol J) Vol. 18 Issue 1 Pg. 257 (12 27 2021) ISSN: 1743-422X [Electronic] England
• PMID: 34961524 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2021. The Author(s).

Chemical References

• GOLGA7 protein, human
• Golgi Matrix Proteins
• Spike Glycoprotein, Coronavirus
• spike protein, SARS-CoV-2
• Acyltransferases
• ZDHHC5 protein, human
• Cysteine

Topics

• Acyltransferases
• COVID-19
• Cysteine
• Golgi Matrix Proteins (genetics, metabolism)
• HEK293 Cells
• HeLa Cells
• Humans
• Lipoylation
• SARS-CoV-2
• Spike Glycoprotein, Coronavirus (genetics, metabolism)
• Virus Internalization