Abstract
BackgroundSevere 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.MethodsThe 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.ResultsIn 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.ConclusionsZDHHC5 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.
Highlights
SARS-CoV-2 is a highly transmissible and pathogenic beta-coronavirus and causes the coronavirus disease 19 (COVID-19) pandemic, threatening human health and public safety
Confirmation of interaction between SARS‐CoV‐2 S protein and ZDHHC5 or GOLGA7 by co‐immunoprecipitated protein complexes (IP) The interaction between SARS-CoV-2 S protein and ZDHHC5 or GOLGA7 was analyzed by co-IP assays
S protein and ZDHHC5 or GOLGA7 were coexpressed in HEK293T cells by cotransfection with indicated plasmids (S/ZDHHC5 and S/GOLGA7)
Summary
SARS-CoV-2 is a highly transmissible and pathogenic beta-coronavirus and causes the coronavirus disease 19 (COVID-19) pandemic, threatening human health and public safety. The pathogenic mechanism of SARS-CoV-2 including the interaction between virus and host remains to be further explored. S protein undergoes palmitoylation and alteration of the ten cysteine residues in the endodomain (cytoplasmic tail) of S protein decreased the efficiency of syncytium formation, cell–cell fusion and pseudotyped SARS-CoV-2 infectivity [6]. Palmitoylation of the cysteine-rich endodomain of S protein from SARSCoV (severe acute respiratory syndrome coronavirus) is important for spike-mediated cell fusion [7]. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein determines virus entry and the palmitoylation of S protein affects virus infection. The palmitoylated cysteine residues of S protein, the effects of ZDHHC5 or GOLGA7 knockout on S protein’s subcellular localization, palmitoyla‐ tion, pseudovirus entry and the enzyme for depalmitoylation of S protein are not clear
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