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.
【저자키워드】 SARS-CoV-2, Spike protein, ZDHHC5/GOLGA7, APT2, Virus-host interaction, 【초록키워드】 severe acute respiratory syndrome coronavirus 2, coronavirus, mass spectrometry, Antiviral, S protein, knowledge, severe acute respiratory syndrome Coronavirus, Protein, CRISPR, fluorescence microscopy, cells, virus entry, SARS-CoV-2 pseudovirus, virus infection, respiratory, CRISPR-Cas9, Colocalization, SARS-CoV-2 S protein, Cas9, A549, pseudovirus entry, host proteins, Interaction, Biotin, cell lines, palmitoylation, Efficiency, the cell, independent of, cell types, cysteine, acute respiratory syndrome, acute respiratory syndrome coronavirus, no significant effect, significant effect, enzymatic activity, enzyme, complex, domain, S proteins, host protein, reason, HeLa cells, HEK293T, cell line, subcellular localization, co-immunoprecipitation, Affinity purification, co-IP, endodomain, GOLGA7, HEK293T cells, Hela, Renilla, Renilla luciferase, ZDHHC5, Effect, Affect, Cell, independent, Result, analyzed, affected, significantly, assays, provide, determine, interact, the S protein, had no, cysteine residue, HEK293T cell, 【제목키워드】 Protein, SARS-CoV-2 spike, Interaction, Effect,