Fig 1: ApoD and SARS-CoV-2 replication or infection. A, the replicative ability of SARS-CoV-2 (multiplicity of infection: MOI 0.05) was determined in HEK293T-ACE2 cells overexpressing ApoD or control empty vector, viral titres were determined 48 hours post infection (n=4). B, the replicative ability of SARS-CoV-2 was further determined in Vero E6 cells overexpressing ApoD or empty vector at increasing concentrations of SARS-CoV-2 (MOI 0.005 to 0.1, n=2 per MOI), viral titres were measured 48 hours post infection (plaque forming units, PFU/mL). C, D, SARS-CoV-2 infectivity was determined in Vero E6 cells overexpressing ApoD or empty vector. Cells were infected with SARS-CoV-2 at the indicated MOIs, and N protein immunofluorescence was measured 24 and 48 hours post infection.
Fig 2: XL-MS upon isolated HDL and SARS-CoV-2 spike protein. A, a schematic representation of the XL-MS workflow conducted upon mixtures of ultracentrifuge-isolated HDL and spike protein. B, inter-protein crosslinks identified within isolated HDL supplemented with spike protein are represented as an interaction network, 404 crosslinks across 60 proteins. Crosslinks had a minimum of 2 associated crosslink spectral matches (CSMs) and an XlinkX score of >10. C, protein-protein interactions identified by XL-MS between ApoD and spike are separately visualised. Spike structural domains are coloured; NTD: blue, RBD: red and S2: green. XL-MS data visualisation was conducted in xiview.org. All protein abbreviations are gene names as determined by Uniprot.
Fig 3: XLMS of recombinant proteins combined with structural modelling. A, an equimolar ratio of full-length SARS-CoV-2 spike and ApoD was mixed prior to crosslinking with DSSO. Identified crosslinks are shown, highlighting a dense crosslinked region between the RBD of spike and the C-terminus of ApoD. B, the same experiment was conducted but this time using recombinant spike-RBD, again implicating the C-terminal region of ApoD in this interaction. Spike structural domains are coloured; NTD: blue, RBD: red and S2: green. Publicly available structures of ApoD (pdb.2HZR, cyan) and the spike-RBD (pdb.6M0J, green) were used to model the interaction between the two proteins. C, HADDOCK based structural modelling was then conducted using identified crosslinks and the likely core amino acid residues (ApoD: pink, spike-RBD: orange) required for the interaction as identified by DisVis. The top scoring model conforming to all crosslink distance constraints was visualised in PyMOL and crosslink distances are given in Å. The interaction model was also visualised using surface representation highlighting contacts between key amino acid residues. XL-MS data visualisation was conducted in xiview.org. D, AlphaFold2 was also used to determine a model between ApoD and spike-RBD. The resulting top scoring modelling was visualised as described above. Mapped DSSO obtained crosslinks: RBD K417–K155 ApoD, RBD K458–K156 ApoD, RBD K462–K156 ApoD. Mapped DMTMM obtained crosslinks: RBD K458–D161 ApoD, RBD D420–K155 ApoD, RBD E465–K165 ApoD, RBD K462–D161 ApoD.
Fig 4: XL-MS upon plasma derived from patients with COVID-19. A, Plasma isolated from patients in intensive care with confirmed COVID-19 (n=6) was pooled and crosslinked using DSSO. The subsequent inter-protein crosslinks identified by XL-MS are represented as an interaction network, 125 crosslinks across 68 proteins. Crosslinks had a minimum of 2 associated crosslink spectral matches (CSMs) and an XlinkX score of >40. The amino acid sequence of ApoD has been expanded, highlighting differing regions of protein interaction. Apolipoproteins are in orange, serum amyloid proteins (SAA) are in red and complement factors are in yellow. B, HDL interactions in healthy controls were determined following the same XL-MS workflow and the resulting inter-protein crosslinks identified are visualised as an interaction network. XL-MS data visualisation was conducted in xiview.org. All protein abbreviations are gene names as determined by Uniprot.
Fig 5: Specificity of ApoD and SARS-CoV-2 spike protein interaction. A, HEK293T cells were transfected with plasmids encoding either GFP, ApoD-myc or spike-V5 prior to Western blot analysis of cell lysates and releasates confirming expression. B, co-immunoprecipitations of both ApoD-myc and spike-V5 were conducted upon HEK293T cell lysates co-overexpressing both ApoD-myc and spike-V5. Flow-throughs represent depleted lysates, and bound-fractions are proteins eluted from protein G beads. Myc Ctrl represents an anti-myc immunoprecipitation conducted upon cell lysates singularly expressing spike-V5. V5 Ctrl represents an anti-V5 immunoprecipitation conducted upon cell lysates singularly expressing ApoD-myc. C, HEK293T cells were co-transfected in triplicate with plasmids encoding the SARS-CoV-1 spike protein and ApoD-myc prior to an immunoprecipitation using anti-myc antibodies and pull downs were confirmed by Western blot. D, XL-MS with SARS-CoV-1 spike protein and the identified peptide coverage for spike and ApoD is schematically visualised. E, protein-protein interactions identified by XL-MS with SARS-CoV-1 spike with only 1 CSM are separately visualised. Spike structural domains are coloured; NTD: blue, RBD: red and S2: green. XL-MS data visualisation was conducted in xiview.org. All protein abbreviations are gene names as determined by Uniprot.
Supplier Page from Abcam for Recombinant Human Apo-D protein