Fig 2: Interaction sites of NS1:ApoA1 complex identification by crosslinking mass spectrometry.(a) SDS-PAGE analysis of isNS1wt with or without the addition of disuccinimidyl sulfoxide (DSSO) crosslinker. (b) The identified crosslinks are visualized on the NS1 and bovine ApoA1 constructs. The intramolecular (NS1:NS1 and ApoA1:ApoA1) crosslinks are in magenta. The intermolecular NS1:ApoA1 crosslinks are in green. (c) The isNS1 residues that are involved in NS1:NS1 interactions are visualized on the NS1 dimer model. The NS1 cartoon model is colored by its three domains, namely the β-roll (orange), wing (blue), and β-ladder (cyan) with the intramolecular (magenta) and intermolecular (green) crosslinking sites depicted as spheres. (d) The overall model interpretation of NS1:ApoA1 complex within the crosslinker theoretical distance cutoff at <30 Å as depicted. ApoA1 dimer cartoon model with its conserved helices as labeled colored in intervals of gray and light purple. (e) The NS1:ApoA1 dimer model with validated crosslinks was fitted into the cryoEM envelope. (f) SDS-PAGE analysis of crosslinked rsNS1 alone or with human high-density lipoprotein (HDL) (lanes 3–5). Non-crosslinked rsNS1 and human HDL are the control (lanes 1–2). The crosslinked rsNS1 can be seen in higher oligomers (lane 3). (g) Identified crosslinks are mapped on the NS1 and ApoA1 constructs, colored as per panel (b). Figure 4—source data 1.Raw and annotated image for the PAGE gel visualized using silver stain. Figure 4—source data 2.Raw and annotated image for the PAGE gel visualized using silver stain.

Fig 3: sNS1 associates with ApoA1 in dengue virus (DENV)-infected mouse and human serum.(a) AG129 mice (n = 10) were infected with DENV2 NS1 T164S mutant virus and the pooled infected sera collected on day 4 post-infection was subjected to sNS1 immunoaffinity purification using anti-NS1 56.2 coupled resin as in Figure 1—figure supplement 1. 2 mg of the purified eluate was then subjected to western blot analysis after separation and transfer from Native-PAGE for detection of ApoA1 and NS1. ApoA1 was detected using the mouse monoclonal anti-ApoA1 clone 513 (Invitrogen, MIA1404) (left panel) and the oligomeric NS1 was detected using anti-NS1 56.2 IgG clone (right panel). (b) Protein AG resin (Pierce) pre-cleared DENV1-infected patient serum (n = 1) from the CELADEN trial (Low et al., 2014) was immunoprecipitated with 10 or 50 mg of rabbit polyclonal anti-ApoA1 antibody (Biorbyt, orb10643) to detect association between ApoA1 and NS1 by ELISA. The amount of ApoA1 and NS1 in the immunoprecipitated sample was determined by human ApoA1 (Abcam, ab189576) and Platelia NS1 Ag (Bio-Rad) ELISAs. Figure 7—source data 1.Raw and annotated image for the western blot analysis (anti-NS1 and anti-apoA1) on a Native gel.

Fig 4: Composition of the secreted NS1 from dengue virus (DENV)-infected Vero cells.DENV 2 WT cell culture supernatant was filtered, supplemented with protease inhibitor cocktail and 0.05% sodium azide, concentrated using a 100 kDa MWCO Vivaflow cassette and purified using 56.2 anti-NS1 antibody immunoaffinity chromatography. The eluted isNS1wt was dialyzed against PBS, concentrated, and stored at –80°C until further use. (a) Schematic of isNS1 purification to illustrate the samples used for gel analyses. % NS1 is measured by the total amount of NS1 (quantified using the anti-NS1 ELISA kit [Bio-Rad] as a percentage of total protein [quantified using the Bradford assay]) found in each sample. Details of the % enrichment in NS1 along the purification process are as shown in Figure 1—figure supplement 1b. (b) Coomassie blue detection of proteins from crude, wash, and elute immunoaffinity fractions for isNS1wt, with the recombinant sNS1 (rsNS1) obtained from Shu et al., 2022 as a positive control, after separation on a 10% Native-PAGE gel (left). The crude and elute fractions contain 1 µg of total protein. The wash fraction contains approximately 100 ng of total protein in maximum well volume of the gel. The same set of samples were also subjected to a western blot detection of NS1 using 56.2 anti-NS1 antibody after separation on a 10% Native-PAGE (right). The crude and elute fractions contain 500 ng of total protein. The wash fraction contains approximately 100 ng of total protein in maximum well volume of the gel. (c) Coomassie blue detection of proteins from crude, wash, and elute immunoaffinity fractions for isNS1wt and rsNS1 (Shu et al., 2022), after separation on a 4–20% reducing SDS-PAGE gel. The crude and elute fractions contain 1 µg of total protein. The wash fraction contains approximately 100 ng of total protein in maximum well volume of the gel. Similarly, the same set of samples were also subjected to a western blot detection of NS1 and ApoA1 using 56.2 anti-NS1 antibody or ApoA1 antibody (Biorbyt, orb10643), respectively, after separation on a 4–20% reducing SDS-PAGE (right). (d) In-gel protein identification of the purified isNS1wt by liquid chromatography mass spectrometry (LC-MS). Proportion of NS1, ApoA1 and other unidentified proteins quantified in total ion intensity, obtained from the following samples: elute in solution (boxed in blue), 250 kDa gel band (boxed in purple), 50 kDa gel band (boxed in red), and 25 kDa gel band (green). The boxed gel bands are from representative gels showing the different protein species found while the actual gel bands used for protein identification by LC-MS are as shown in Figure 1—figure supplement 2a and b. Figure 1—source data 1.Raw and annotated image for the PAGE gel stained in Coomassie blue. Figure 1—source data 2.Raw and annotated image for the western blot analysis (anti-NS1). Figure 1—source data 3.Raw and annotated image for the PAGE gel stained in Coomassie blue in Figure 1c. Figure 1—source data 4.Raw and annotated image for the western blot analysis (anti-NS1) in Figure 1c. Figure 1—source data 5.Raw and annotated image for the western blot analysis (anti-ApoA1) in Figure 1c.

Fig 5: Serum ApoA1 Concentration and eGFR. Scatter plot showing the Pearson correlation between serum ApoA1 concentration and estimated glomerular filtration rate (eGFR), an indicator of kidney function. 40 patients from our cohort were grouped into the following categories based on diagnosis: NL (normal control individual, n = 5); HPS‐3&5 (n = 10), HPS‐1 (n = 10), HPS‐1PF (HPS‐1 with pulmonary fibrosis, n = 10), and IPF&FPF (idiopathic pulmonary fibrosis/familial pulmonary fibrosis, n = 5). A moderate positive Pearson correlation (r = 0.4053, P‐value = 0.00948) was observed between serum ApoA1 levels and eGFR, as measured by ELISA. eGFR ≥ 90 is considered normal.