Secreted dengue virus NS1 from infection is predominantly dimeric and in complex with high-density lipoprotein

Reviewer #1 (Public Review):

The authors of this study seek to visualize NS1 purified from dengue virus infected cells. They infect vero cells with DV2-WT and DV2 NS1-T164S (a mutant virus previously characterized by the authors). The authors utilize an anti-NS1 antibody to immunoprecipitate NS1 from cell supernatants and then elute the antibody/NS1 complex with acid. The authors evaluate the eluted NS1 by SDS-PAGE, Native Page, mass spec, negative-stain EM, and eventually Cryo-EM. SDS-PAGE, mas spec, and native page reveal a >250 Kd species containing both NS1 and the proteinaceous component of HDL (ApoA1). The authors produce evidence to suggest that this population is predominantly NS1 in complex with ApoA1. This contrasts with recombinantly produced NS1 (obtained from a collaborator) which did not appear to be in complex with or contain ApoA1 (Figure 1C). The authors then visualize their NS1 stock in complex with their monoclonal antibody by CryoEM. For NS1-WT, the major species visualized by the authors was a ternary complex of an HDL particle in complex with an NS1 dimer bound to their mAB. For their mutant NS1-T164S, they find similar structures, but in contrast to NS1-WT, they visualize free NS1 dimers in complex with 2 Fabs (similar to what's been reported previously) as one of the major species. This highlights that different NS1 species have markedly divergent structural dynamics. It's important to note that the electron density maps for their structures do appear to be a bit overfitted since there are many regions with electron density that do not have a predicted fit and their HDL structure does not appear to have any predicted secondary structure for ApoA1. The authors then map the interaction between NS1 and ApoA1 using cross-linking mass spectrometry revealing numerous NS1-ApoA1 contact sites in the beta-roll and wing domain. The authors find that NS1 isolated from DENV infected mice is also present as a >250 kD species containing ApoA1. They further determine that immunoprecipitation of ApoA1 out of the sera from a single dengue patient correlates with levels of NS1 (presumably COIPed by ApoA1) in a dose-dependent manner.

In the end, the authors make some useful observations for the NS1 field (mostly confirmatory) providing additional insight into the propensity of NS1 to interact with HDL and ApoA1. The study does not provide any functional assays to demonstrate activity of their proteins or conduct mutagenesis (or any other assays) to support their interaction predications. The authors assertion that higher-order NS1 exists primarily as a NS1 dimer in complex with HDL is not well supported as their purification methodology of NS1 likely introduces bias as to what NS1 complexes are isolated. While their results clearly reveal NS1 in complex with ApoA1, the lack of other NS1 homo-oligomers may be explained by how they purify NS1 from virally infected supernatant. Because NS1 produced during viral infection is not tagged, the authors use an anti-NS1 monoclonal antibody to purify NS1. This introduces a source of bias since only NS1 oligomers with their mAb epitope exposed will be purified. Further, the use of acid to elute NS1 may denature or alter NS1 structure and the authors do not include controls to test functionality of their NS1 stocks (capacity to trigger endothelial dysfunction or immune cell activation). The acid elution may force NS1 homo-oligomers into dimers which then reassociate with ApoA1 in a manner that is not reflective of native conditions. Conducting CryoEM of NS1 stocks only in the presence of full-length mAbs or Fabs also severely biases what species of NS1 is visualized since any NS1 oligomers without the B-ladder domain exposed will not be visualized. If the residues obscured by their mAb are involved in formation of higher-order oligomers then this antibody would functionally inhibit these species from forming. The absence of critical controls, use of one mAb, and acid elution for protein purification severely limits the interpretation of these data and do not paint a clear picture of if NS1 produced during infection is structurally distinct from recombinant NS1. Certainly there is novelty in purifying NS1 from virally infected cells, but without using a few different NS1 antibodies to purify NS1 stocks (or better yet a polyclonal population of antibodies) it's unclear if the results of the authors are simply a consequence of the mAb they selected.

Data produced from numerous labs studying structure and function of flavivirus NS1 proteins provide diverse lines of evidence that the oligomeric state of NS1 is dynamic and can shift depending on context and environment. This means that the methodology used for NS1 production and purification will strongly impact the results of a study. The data in this manuscript certainly capture one of these dynamic states and overall support the general model of a dynamic NS1 oligomer that can associate with both host proteins as well as itself but the assertions of this manuscript are overall too strong given their data, as there is little evidence in this manuscript, and none available in the large body of existing literature, to support that NS1 exists only as a dimer associated with ApoA1. More likely the results of this paper are a result of their NS1 purification methodology.

Suggestions for the Authors:

Major:

1. Because of the methodology used for NS1 purification, it is not clear from the data provided if NS1 from viral infection differs from recombinant NS1. Isolating NS1 from viral infection using a polyclonal antibody population would be better to answer their questions. On this point, Vero cells are also not the best candidate for their NS1 production given these cells do not come from a human. A more relevant cell line like U937-DC-SIGN would be preferable.

2. The authors need to support their interaction predictions and models via orthogonal assays like mutagenesis followed by HDL/ApoA1 complexing and even NS1 functional assays. The authors should be able to mutate NS1 at regions predicted to be critical for ApoA1/HDL interaction. This is critical to support the central conclusions of this manuscript.

3. The authors need to show that the NS1 stocks produced using acid elution are functional compared to standard recombinantly produced NS1. Do acidic conditions impact structure/function of NS1?

4. Overall, the data obtained from the mutant NS1 (contrasted to WT NS1) reveals how dynamic the oligomeric state of NS1 proteins are but the authors do not provide any insight into how/why this is, some additional lines of evidence using either structural studies or mutagenesis to compare WT and their mutant and even NS1 from a different serotype of DENV would help the field to understand the dynamic nature of NS1.

Reviewer #2 (Public Review):

Summary:

Chew et al describe interaction of the flavivirus protein NS1 with HDL using primarily cryoEM and mass spec. The NS1 was secreted from dengue virus infected Vero cells, and the HDL were derived from the 3% FBS in the culture media. NS1 is a virulence factor/toxin and is a biomarker for dengue infection in patients. The mechanisms of its various activities in the host are incompletely understood. NS1 has been seen in dimer, tetramer and hexamer forms. It is well established to interact with membrane surfaces, presumably through a hydrophobic surface of the dimer form, and the recombinant protein has been shown to bind HDL. In this study, cryoEM and crosslinking-mass spec are used to examine NS1 secreted from virus-infected cells, with the conclusion that the sNS1 is predominantly/exclusively HDL-associated through specific contacts with the ApoA1 protein.

Strengths:

The experimental results are consistent with previously published data.

Weaknesses:

CryoEM:

Some of the neg-stain 2D class averages for sNS1 in Fig S1 clearly show 1 or 2 NS1 dimers on the surface of a spherical object, presumably HDL, and indicate the possibility of high-quality cryoEM results. However, the cryoEM results are disappointing. The cryo 2D class averages and refined EM map in Fig S4 are of poor quality, indicating sub-optimal grid preparation or some other sample problem. Some of the FSC curves (2 in Fig S7 and 1 in Fig S6) have extremely peculiar shapes, suggesting something amiss in the map refinement. The sharp drop in the "corrected" FSC curves in Figs S5c and S6c (upper) indicate severe problems. The stated resolutions (3.42 & 3.82 Å) for the sNS1ts-Fab56.2 are wildly incompatible with the images of the refined maps in Figs 3 & S7. At those resolutions, clear secondary structural elements should be visible throughout the map. From the 2D averages and 3D maps shown in the figures this does not seem to be the case. Local resolution maps should be shown for each structure.

The samples were clearly challenging for cryoEM, leading to poor quality maps that were difficult to interpret. None of the figures are convincing that NS1, Ab56.2 or Fab56.2 are correctly fit into EM maps. There is no indication of ApoA1 helices. Details of the fit of models to density for key regions of the higher-resolution EM maps should be shown and the models should be deposited in the PDB. An example of modeling difficulty is clear in the sNS1ts dimer with bound Fab56.2 (figs 3c & S7e). For this complex, the orientation of the Fab56.2 relative to the sNS1ts dimer in this submission (Fig 3c) is substantially different than in the bioRxiv preprint (Fig 3c). Regions of empty density in Fig 3c also illustrate the challenge of building a model into this map.

Mass spec:

Crosslinking-mass spec was used to detect contacts between NS1 and ApoA1, providing strong validation of the sNS1-HDL association. As the crosslinks were detected in a bulk sample, they show that NS1 is near ApoA1 in many/most HDL particles, but they do not indicate a specific protein-protein complex. Thus, the data do not support the model of an NS1-ApoA1 complex in Fig 4d. Further, a specific NS1-ApoA1 interaction should have evidence in the EM maps (helical density for ApoA1), but none is shown or mentioned. If such exists, it could perhaps be visualized after focused refinement of the map for sNS1ts-HDL with Fab56.2 (Fig S7d). The finding that sNS1-ApoA1 crosslinks involved residues on the hydrophobic surface of the NS1 dimer confirms previous data that this NS1 surface engages with membranes and lipids.

Sample quality:

The paper lacks any validation that the purified sNS1 retains established functions, for example the ability to enhance virus infectivity or to promote endothelial dysfunction. Peculiarities include the gel filtration profiles (Fig 2a), which indicate identical elution volumes (apparent MWs) for sNS1wt-HDL bound to Ab562 (~150 kDa) and to the ~3X smaller Fab56.2 (~50 kDa). There should also be some indication of sNS1wt-HDL pairs crosslinked by the full-length Ab, as can be seen in the raw cryoEM micrograph (Fig S5b).

Obtaining high quality structures is often more demanding of sample integrity than are activity assays. Given the low quality of the cryoEM maps, it's possible that the acidification step in immunoaffinity purification damaged the HDL complex. No validation of HDL integrity, for example with acid-treated HDL, is reported. Acid treatment is perhaps discounted by a statement (line 464) that another group also used immunoaffinity purification in a recent study (ref 20) reporting sNS1 bound to HDL. However the statement is incorrect; the cited study used affinity purification via a strep-tag on recombinant sNS1.

Discussion:

The Discussion reflects a view that the NS1 secreted from virus-infected cells is a 1:1 sNS1dimer:HDL complex with the specific NS1-ApoA1 contacts detected by crosslinking mass spec. This is inconsistent with both the neg-stain 2D class average with 2 sNS1 dimers on an HDL (Fig S1c) and with the recent study of Flamand & co-workers showing 1-3 NS1 dimers per HDL (ref 20). It is also ignores the propensity of NS1 to associate with membranes and lipids. It is far more likely that NS1 association with HDL is driven by these hydrophobic interactions than by specific protein-protein contacts. A lengthy Discussion section (lines 461-522) includes several chemically dubious or inconsistent statements, all based on the assumption that specific ApoA1 contacts are essential to NS1 association with HDL and that sNS1 oligomers higher than the dimer necessarily involve ApoA1 interaction, conclusions that are not established by the data in this paper.