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Glycosylation and S-palmitoylation regulate SARS-CoV-2 spike protein intracellular trafficking.

ABSTRACT: Post-translational modifications (PTMs), such as glycosylation and palmitoylation, are critical to protein folding, stability, intracellular trafficking, and function. Understanding regulation of PTMs of SARS-CoV-2 spike (S) protein could help the therapeutic drug design. Herein, the VSV vector was used to produce SARS-CoV-2 S pseudoviruses to examine the roles of the ⁶¹¹LYQD⁶¹⁴ and cysteine-rich motifs in S protein maturation and virus infectivity. Our results show that ⁶¹¹LY⁶¹² mutation alters S protein intracellular trafficking and reduces cell surface expression level. It also changes S protein glycosylation pattern and decreases pseudovirus infectivity. The S protein contains four cysteine-rich clusters with clusters I and II as the main palmitoylation sites. Mutations of clusters I and II disrupt S protein trafficking from ER-to-Golgi, suppress pseudovirus production, and reduce spike-mediated membrane fusion activity. Taken together, glycosylation and palmitoylation orchestrate the S protein maturation processing and are critical for S protein-mediated membrane fusion and infection.

SUBMITTER: Tien CF

PROVIDER: S-EPMC9250814 | biostudies-literature | 2022 Aug

REPOSITORIES: biostudies-literature

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Glycosylation and S-palmitoylation regulate SARS-CoV-2 spike protein intracellular trafficking.

Tien Chih-Feng CF Tsai Wan-Ting WT Chen Chun Hwa CH Chou Hui-Ju HJ Zhang Mingzi M MM Lin Jhe-Jhih JJ Lin En-Ju EJ Dai Shih-Syong SS Ping Yueh-Hsin YH Yu Chia-Yi CY Kuo Yi-Ping YP Tsai Wei-Hsiang WH Chen Hsin-Wei HW Yu Guann-Yi GY

iScience 20220703 8

Post-translational modifications (PTMs), such as glycosylation and palmitoylation, are critical to protein folding, stability, intracellular trafficking, and function. Understanding regulation of PTMs of SARS-CoV-2 spike (S) protein could help the therapeutic drug design. Herein, the VSV vector was used to produce SARS-CoV-2 S pseudoviruses to examine the roles of the <sup>611</sup>LYQD<sup>614</sup> and cysteine-rich motifs in S protein maturation and virus infectivity. Our results show that <s ...[more]

PMID: 35813875

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Project description:The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic caused the biggest public health crises in recent history. Many expect future coronavirus introductions into the human population. Hence, it is essential to understand the basic biology of these viruses. In natural infection, the SARS-CoV-2 Spike (S) glycoprotein is co-expressed with all other viral proteins, which modify cellular compartments to maximize virion assembly. By comparison, most of S is degraded when the protein is expressed in isolation, as in current molecular vaccines. To probe the maturation pathway of S, we redirected its maturation by fusing S to the tetraspanin protein CD81. CD81 is a defining constituent of extracellular vesicles (EVs) or exosomes. EVs are generated in large numbers by all cells, extruded into blood and lymph, and transfer cargo between cells and systemically (estimated 1012 EVs per mL plasma). EVs, like platelets, can be transfused between unrelated donors. When fusing the proline-stabilized form of strain Delta S into the flexible, large extracellular loop of CD81 rather than being degraded in the lysosome, S was extruded into EVs. CD81-S fusion containing EVs were produced in large numbers and could be isolated to high purity. Purified CD81::S EVs bound ACE2, and S displayed on individual EV was observed by cryogenic electron microscopy (EM). The CD81::S-fusion EVs were non-toxic and elicited an anti-S trimer and anti-RBD antibody response in mice. This report shows a design path to maximize viral glycoprotein assembly and release without relying on the co-expression of potentially pathogenic nonstructural viral proteins.ImportanceThe severe acute respiratory syndrome coronavirus 2 pandemic caused the biggest public health crises in recent history. To understand the maturation pathway of S, we fused S to the tetraspanin protein CD81. The resulting molecule is secreted in extracellular vesicles and induces antibodies in mice. This may be a general design path for viral glycoprotein vaccines.

Project description:MCOLN3/TRPML3 is a Ca2+-permeable cation channel that is expressed in multiple subcellular compartments with dynamic localization. Our previous studies suggest that upon macroautophagy/autophagy induction MCOLN3/TRPML3 is recruited and provides Ca2+ for the fusion process in autophagosome biogenesis. However, how intracellular trafficking and the Ca2+ channel function of MCOLN3/TRPML3 are related to autophagy are not known. Here we report that MCOLN3/TRPML3 undergoes palmitoylation at its C-terminal region, which is required for dynamic trafficking and cellular function of MCOLN3/TRPML3 in autophagy. Palmitoylation regulated MCOLN3/TRPML3 surface expression and trafficking, but not channel properties or localization and function of intracellular MCOLN3/TRPML3. Activation of intracellular MCOLN3/TRPML3 induced robust Ca2+ release, which solely increased autophagy in Ca2+- and palmitoylation-dependent manners. Palmitoylation regulated not only intracellular MCOLN3/TRPML3 trafficking to autophagic structures but also autophagic flux in induced autophagy. Importantly, nutrient starvation activated MCOLN3/TRPML3 to release Ca2+ and increased the level of MCOLN3/TRPML3 palmitoylation. Disruption of MCOLN3/TRPML3 palmitoylation, however, abolished the starvation-induced MCOLN3/TRPML3 activation without affecting channel activity. These results suggest that trafficking and channel function of MCOLN3/TRPML3 are regulated in the context of autophagy, and palmitoylation is a prerequisite for the function of MCOLN3/TRPML3 as a Ca2+ channel in autophagosome formation by controlling its trafficking between subcellular compartments. Abbreviations: 17-ODYA, 17-octadecynoic acid; 2-BP, 2-bromopalmitate; BFA, brefeldin A; DN, dominant-negative; GPN, glycyl-L-phenylalanine-beta-naphthylamide; HN, hydroxylamine; KD, knockdown; MCOLN3/TRPML3, mucolipin 3; MS, mass spectrometry; PAT, palmitoyl acyltransferase; PM, plasma membrane; WT, wild type; ZDHHC, a zinc-finger motif and an Asp-His-His-Cys sequence.

Dataset Information

Glycosylation and S-palmitoylation regulate SARS-CoV-2 spike protein intracellular trafficking.

Publications

Glycosylation and S-palmitoylation regulate SARS-CoV-2 spike protein intracellular trafficking.

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