Fig 1.
Infectivity (transducing activity) of authentic SARS-CoV-2 virus-like particles (VLPs) requires the acidic cluster in the CD of S.
(A) Organization of SARS-CoV2 virion major structural proteins and (B) the Sequence of the cytoplasmic domain of S. The cysteine-rich domain, the acidic cluster motif, and the ER-retention signal are highlighted. (C) Schematic of the authentic SARS-CoV-2 VLP luciferase transduction system [20]. We modified the published system by using HEK293T cells that express ACE2 and TMPRSS2 as both the VLP producer cells and as the targets of infection (transduction) by the VLPs. S: Spike. M-E: Membrane-IRES-Envelope. N: Nucleocapsid. Luc-RNA: RNA encoding luciferase and containing a packaging determinant from the N coding sequence. (D) Electron micrographs of negatively-stained preparations of SARS-CoV2 VLPs; particles of approximately 100nm size were identified in crude preparations, which appear decorated with Spike glycoprotein. (E) Relative infectivity of SARS-CoV-2 VLPs measured as expression of transduced luciferase activity (relative light units; RLU). Data are from three independent experiments and are normalized to wild type (“WT”) S as 100%. “(D/E)5>A” denotes substitution of the 5 acidic residues for alanines, “DE/AA” is a mutant of the N-terminal acidic residues, “DD/AA” is a mutant of the central residues, and “E/A” is a mutant of the C-terminal E of the sequence. P-values are derived from one-way ANOVA analysis with Dunnett’s multiple comparisons test; comparing mutants to WT control.
Fig 2.
Mutation of the acidic residues does not impair formation of syncytia.
Formation of syncytia was observed in VLP producer 293T cells (ACE2 + , TMPRSS2+) co-transfected with plasmids encoding the SARS-CoV2 structural proteins S, M, N, E, the luciferase reporter RNA, and GFP. Images of live cells were obtained 48 hours post-transfection, prior to VLP harvest. Single GFP-positive cells were observed in the no Spike control transfections, while Spike induced formation of large, multinuclear, GFP-positive syncytium, which were present in WT and mutant conditions. Insets are magnified regions in white boxes, scale bar = 200 µm.
Fig 3.
Virion incorporation of S requires the acidic cluster and depends largely on the N-terminal DE residues. SARS-CoV2 VLPs and VLP producer cell lysates were analyzed by western blot (see supplemental data), one example is shown in A.
VLPs were partially purified from the cultures by pelleting out cellular debris followed by filtration through 0.2 micron membranes, and then pelleting at 23,500 x g for 2 hours. (A) Top: Pelleted material from the VLP preparations (“VLP”) were analyzed by immunoblots probed with antibody to Spike as well as Membrane protein, a key structural component of virions. Bottom: aliquots of the VLP producer cells (“Cell Lysate”) were similarly probed for S, M, and the cellular protein GAPDH. VLP-associated S included a minor amount of high molecular weight unprocessed S but was prominently a doublet around 100 kDa, which is likely S processed by Furin and TMPRSS2 [21,22]. VLPs made with the S DEDDSE/AAAASA ((D/E)5 > A) and DE/AA mutants were nearly devoid of S signal, whereas VLPs made with S DD/AA have diminished signal. VLP producer cells show similar amounts of WT and mutant S proteins without obvious differences in band-patterns. (B) Mutant Spike defects in VLP incorporation were quantified; the Spike signal in VLP blots was measured and normalized to Membrane signal, with normalized Spike signal expressed as percentage of WT condition. Data from two independent experiments are shown; all mutants were significantly defective for VLP incorporation, with S DEDDSE/AAAASA ((D/E)5 > A) and DE/AA mutants consistently most impaired. P values are derived from one-way ANOVA with Dunnett’s multiple comparison test of n = 2 independent experiments.
Fig 4.
Yeast two-hybrid and Bi-molecular Fluorescence Complementation (BiFC) protein-protein interaction assays show acidic cluster-dependent interactions of the S CD with various cellular proteins.
(A) Top panel: yeast growth in the absence of tryptophan, leucine, and histidine indicates interaction between the “prey” proteins along the top and the “bait” proteins at the left. “S” indicates the wild type S CD; “S AC mut” indicates the (D/E)5 > A S mutant; “empty” indicates empty bait-vector control. Bottom panel: yeast growth in the absence of tryptophan and leucine indicates the presence of the bait and prey plasmids. (B) BiFC constructs. The Venus protein (residues 1 to 238) is divided into N-terminal (VN: residues 2 to 173) and C-terminal portions (VC: 154 to 238). VN was fused to the C-terminus of SARS-CoV-2 Spike and VC to the C-terminus of Moesin, along with epitope tags (HA or V5) as shown. (C) Cells (HEK293T) were transfected with plasmids expressing the indicated proteins, either Spike-WT-VN or Spike-(D/E)5 > A -VN, with or without co-expression of Moesin (MSN)-VC. Twenty-four hours later, the mean fluorescence intensities (MFI) of the cells were determined by flow cytometry. Results are the mean fold difference in fluorescence relative to untransfected cells. Error bars represent the SD from three biological experiments performed in technical duplicate. P-value (unpaired t test) between Spike-WT-VN or Spike-(D/E)5 > A -VN co-expressed with MSN-VC is indicated.
Fig 5.
Acidic cluster-dependent interactions of the S CD with the medium (µ) subunits of the clathrin adaptors AP1 and AP2.
(A) Pulldowns using GST-S-CD and µ1 (the medium subunit of the AP1 adaptor protein complex). (B) Same experimental design but using µ2 of the AP2 complex. All proteins were recombinant and produced in E.coli. The GST-S-CD proteins were expressed either with or without Casein Kinase II (CK-II) to phosphorylate serine residues. Pulldowns were assessed by SDS-PAGE and staining with Coomassie Blue. (C) Quantitation of replicate µ2 pull-down experiments by densitometry. P-values are derived from one-way ANOVA analysis with Dunnett multiple comparisons correction, comparing values to Spike-CD WT (+ CK-II) control.
Fig 6.
Basic domains on the surfaces of potential protein partners of the S acidic cluster.
Surfaces of the C-terminal two-thirds of the µ subunit of AP2 (PDB:1BW8), the FERM domain of Radixin (PDB:1GC6), and the M protein of SARS-CoV-2 (PDB:8CTK). Positively charged regions are colored blue, negatively charged regions are red. The positively charged regions could bind the acidic cluster in the S-CD. Spheres on the FERM domain indicate bound PIP2.