A morphological transformation in respiratory syncytial virus leads to enhanced complement activation

The complement system is a critical host defense against infection, playing a protective role that can also enhance disease if misregulated. Although many consequences of complement activation during viral infection are well-established, specific mechanisms that contribute to activation by different human viruses remain elusive. Here, we investigate complement activation by human respiratory syncytial virus (RSV), a respiratory pathogen that causes severe disease in infants, the immunocompromised, and the elderly. Using a strain of RSV harboring tags on the surface glycoproteins F and G, we were able to monitor opsonization of single RSV particles with monoclonal antibodies and complement components using fluorescence microscopy. These experiments revealed an antigenic hierarchy in complement activation, where antibodies that bind towards the apex of F in either the pre- or postfusion conformation are able to activate complement whereas other antibodies are not. Additionally, among antibodies that were able to activate complement, we observed preferential targeting of a subset of particles with globular morphology, in contrast to the more prevalent viral filaments. We found that enhanced complement activation on these particles arises from changes in surface curvature that occur when the viral matrix detaches from the surrounding membrane. This transformation occurs naturally over time under mild conditions, and correlates with the accumulation of postfusion F on the viral surface. Collectively, these results identify antigenic and biophysical characteristics of virus particles that contribute to the formation of immune complexes, and suggest models for how these factors may shape disease severity and adaptive immune responses to RSV.


Introduction
The complement system is a network of proteins that play a vital role in the innate and adaptive immune responses to pathogens including viruses 1-3 , bacteria 4,5 , and parasites [6][7][8] . Activation of the complement system proceeds through three principal routes: the classical, lectin, and alternative pathways (reviewed 30 in ref. 9). These pathways differ in their mechanisms of activation: by opsonizing antibodies (classical pathway), by pathogen-specific carbohydrates (lectin pathway), or through continual low levels of attachment to surfaces (alternative pathway). Following activation, each pathway converges on C3, the central component of the complement cascade. C3 that has been cleaved by proteases or that has spontaneously hydrolyzed can covalently attach to activating surfaces via a reactive thioester. C3 35 attachment to surfaces is self-amplifying, producing new C3 convertases that further drive opsonization. C3 attachment also contributes to the terminal arm of the complement cascade, eventually leading to the assembly of a membrane attack complex that can neutralize membrane-bound targets through the formation of lytic pores.
In addition to its role in the neutralization of pathogens and infected cells, C3 also plays a central role in 40 immune signaling. Activation of complement results in the cleavage of C3 into two fragments, C3a and C3b. C3b attaches to pathogen surfaces, where its multiple degradation products interact with a variety of immune receptors. These interactions contribute to antigen transport to and within lymphoid organs 10,11 ; antigen presentation by follicular dendritic cells 12 ; activation of B cells 13,14 ; T cell priming 2 ; and the clearance of immune complexes by phagocytic cells 15 or erythrocytes 16 . Additionally, C3a that is 45 produced during complement activation is a potent anaphylatoxin, increasing inflammation and recruiting immune cells to sites of infection 17,18 . The diversity of interactions between immune cells and C3 highlights its central protective role bridging innate and adaptive immunity, as well as the potential dangers associated with misregulation of complement 19,20 . Although the disparate contributions complement makes to health and disease are well-established, the mechanisms by which different 50 human viruses activate or evade complement are less well understood. Understanding the factors that contribute to this process could help aid in the development of more effective vaccines and improve understanding of disease pathogenesis.
Here, we set out to investigate complement activation by the human pathogen respiratory syncytial virus (RSV). Activation of complement during RSV infection has been linked to both protection 21, 22 and 55 pathogenesis 17,23 , but the mechanisms that drive complement activation by RSV remain unclear. RSV is an enveloped, negative-sense single-stranded RNA virus of the family Pneumoviridae that causes severe infection among infants, the immunocompromised, and the elderly. The RSV genome encodes three membrane proteins -the fusion protein (F), the attachment glycoprotein (G), and the short hydrophobic protein (SH) -which are expressed on the surface of infected cells and packaged to varying degrees into 60 shed virus particles. Among these surface proteins, F and G serve as the primary targets of the adaptive immune response, as well as the leading candidates for vaccine development and prophylaxis [24][25][26][27] . The fusion protein, F, mediates the merger between viral and cellular membranes during RSV entry 28 , and has recently been reported to induce outside-in signaling via IGF-1R to facilitate this process 29 . The glycoprotein, G, mediates attachment through the chemokine receptor CX3CR1 [30][31][32] and, when 65 expressed in soluble form, helps antagonize immune responses 22 . While antibodies against F can provide potent protection both in vitro and in vivo, the conformational rearrangements this protein undergoes have historically presented challenges in vaccine design 33 . Although antibodies against the prefusion conformation of F (pre-F) are frequently capable of blocking viral entry, antibodies that bind to the postfusion conformation of F (post-F) often fail to do so 34 . Numerous recent breakthroughs in protein 70 design are helping to overcome this challenge through the development of stabilized pre-F antigens as vaccine candidates [35][36][37] . However, in the context of natural infection, both pre-and post-F conformations occur, and high antibody titers against post-F have been associated with enhanced disease severity and increased activation of complement in the lungs 23,38 .
Understanding how antibodies and RSV antigens contribute to complement activation could provide new 75 insights into vaccine development and mechanisms of RSV pathogenesis. To investigate how RSVspecific antibodies contribute to activation of complement, we developed a fluorescence imaging-based approach to simultaneously quantify antibody binding, the abundance of viral antigens, and the deposition of complement proteins on RSV at the single-virus level. These experiments identify an antigenic hierarchy for complement activation that is dictated by accessibility of the antibody Fc for binding by C1, 80 the initiator of the classical pathway. We also identify a role for the complement defense protein CD55 (DAF), which is packaged into virus particles and increases complement activation thresholds and decreases C3 deposition. Finally, we identify biophysical features of individual RSV particles within heterogeneous populations that enhance their tendency to activate complement. In particular, we find that the detachment of the RSV matrix from the viral envelope -a transition that can be induced by 85 physical perturbations but also occurs under normal physiological conditions in vitro -significantly enhances complement activation by a range of antibodies targeting either pre-F, post-F, or both conformations of F. Collectively, these results identify a constellation of mechanisms that contribute to complement activation and immune complex formation by RSV, and inform models for how complement may shape disease severity and the adaptive immune response to infection. 90

Results
Complement activation by F-specific antibodies varies by antigenic site. To identify determinants of complement activation by RSV, we developed a fluorescence assay to quantify antibody binding and opsonization with complement C3 across populations of individual virus particles. Following our previous work using influenza A virus 39 , we engineered a strain of RSV A that was amenable to fluorescence 95 imaging of infected cells and shed virus particles (Supporting information, Figure S1A). To track viral infection, we inserted a fluorescent reporter (mTagBFP2) downstream of the NS1 stop codon but upstream of the gene-end sequence using an internal ribosome entry site. Separately, we inserted a ybbR-tag at the C-terminus of G and a pentaglycine tag immediately following the signal sequence in F. Sfp synthase 40 can be used to conjugate CoA-based fluorescent probes to the tag on G, while the tag on 100 F becomes exposed following cleavage of the signal sequence, creating a suitable substrate for the enzyme Sortase A (SrtA) to conjugate small peptide-based fluorophores 41 . These modifications allow us to visualize RSV particles and measure densities of antigen (i.e. F or G) on the viral surface independent of conformational changes in F (i.e. prefusion vs. postfusion) and while preserving antigenic sites. Viruses labeled in this way retain levels of infectivity matching unlabeled controls ( Figure S1B), demonstrating 105 that the attachment of fluorophores is non-disruptive.
Using this system, we sought to characterize activation of the classical pathway by RSV particles. We enzymatically labeled F on the surface of RSV particles collected from A549 cells and immobilized these viruses onto pegylated coverslips functionalized with the anti-G antibody 3D3 25 . This allowed us to quantify opsonization following incubation of fluorescent viruses with normal human serum supplemented 110 with defined fluorescent mAbs and labeled C3 ( Figure 1A). Initial tests using normal human serum resulted in robust C3 deposition in the absence of supplemental mAbs ( Figure S1C, left column). Incubating virus with this serum for 30 minutes at 4 o C also inhibited binding of a high-affinity pre-F-specific mAb (5C4) ~2-fold and a post-F-specific mAb (ADI-14359) by ~10-fold, suggesting the presence of competing polyclonal IgG/IgM within the serum ( Figure S1D). In contrast, IgG/IgM-depleted normal 115 human serum showed little C3 deposition in the absence of supplemental mAbs, but robust opsonization with C3 when F-specific mAbs were added ( Figure S1C, right column). IgG/IgM-depleted serum therefore provides a means of determining the ability of individual mAbs to activate complement, allowing us to identify antibody features that are predictive of potency.
We expressed and purified a panel of human IgG1 mAbs targeting each of the known antigenic sites of 120 RSV F 26 ( Figure 1B). Each mAb was modified to contain a ybbR-tag 40 at the C-terminus of the heavy chain, to permit quantitative site-specific labeling and the ability to measure the amounts of antibody bound to individual virus particles. By using mAbs at concentrations that saturate binding, we were able to decouple binding affinity from the intrinsic capacity of a given mAb to activate complement once bound. Under conditions that saturate binding, these mAbs vary markedly in their ability to activate complement.

125
Among the pre-F-binding mAbs tested, 5C4 24 (site 0), CR9501 42 (site V), and Motavizumab 43 (site II) showed the greatest potency. In contrast, ADI-19425 34 (site III) and 101F 44 (site IV) showed only background levels of C3 deposition ( Figure 1C & D). Thus, although each pre-F-binding mAb achieved similar levels of binding to RSV F ( Figure 1D, lower plot), only three out of five activated complement above background levels. 130 Several F-specific antibodies bind to the postfusion conformation of F, either exclusively or in addition to the prefusion conformation. This includes several antibodies in our panel: 101F and Motavizumab (which bind to both pre-and post-F) as well as ADI-14359 and ADI-14353 (which bind specifically to post-F) 34 . To compare C3 deposition driven by these antibodies upon binding to post-F, we first incubated viruses bound to coverslips for 24h in buffer with low ionic strength (300mM mannitol, 10mM HEPES pH 7.2), 135 based on the previous finding that pre-F spontaneously triggers to post-F in buffers with low salt concentrations 45,46 . This treatment increased binding of the post-F specific antibodies ADI-14359 and ADI-14353 approximately 300-fold without obvious changes in the morphology of virus particles ( Figure  1E). Conversion of RSV particles to a predominantly post-F form increased C3 deposition from post-F specific mAbs ADI-14359 and ADI-14353 to levels comparable to or greater than the most potent pre-F 140 specific antibodies in the context of pre-F antigens ( Figure 1F & G). For the conformation-independent mAbs 101F and Motavizumab, C3 deposition following pre-to-post-F conversion followed different trends, increasing approximately six-fold for 101F but decreased by a similar ratio for Motavizumab, despite similar levels of antibody binding ( Figure 1F & G).
The classical pathway is activated when the C1 initiation complex binds to IgM or IgG that has assembled 145 on activating surfaces. While activation via IgG requires the assembly of a hexameric complex of antibody Fc regions 47-49 , IgM is pre-assembled as an activating platform, with C1 binding sites exposed only upon engagement with surface antigen 50 . As a comparison with our IgG antibodies, we tested activation of the classical pathway by recombinant IgM with VH and VL domains from 5C4. As expected, this high-affinity IgM was considerably more potent than its IgG1 counterpart, leading to >100-fold more C3 deposition 150 per bound heavy chain than the corresponding IgG1 mAb ( Figure 1H). Given the importance of establishing a platform for C1 binding in activation of the classical pathway, we sought to identify how the IgG antibodies in our panel may differ in this regard.
A consistent trend among the IgG1 antibodies that most efficiently activate complement deposition is the angle with which they bind to either pre-or post-F. 101F and Motavizumab, antibodies that bind to both 155 conformations but with alternating Fc orientations, illustrate this effect. For both antibodies, C3 deposition increases approximately six-fold when the Fc is oriented away from the viral membrane as opposed to towards it. Projection of the Fc region further above the viral membrane is common to all of the activating antibodies we tested ( Figure S2A), and potentially increases accessibility for binding by C1. Moreover, positioning the Fc regions on a plane above the surrounding canopy of F could also facilitate Fc hexamer 160 formation 47 by avoiding steric hindrance from neighboring proteins in the viral membrane. Consistent with this model, we observe more binding by C1 (as detected by an anti-C1q antibody) to mAbs that bind to pre-F with their Fcs projected outward (CR9501, 5C4, Motavizumab) compared to those where the Fc lies within or below the plane of F trimers in the viral membrane (ADI-19425, 101F) ( Figure S2B & C). Of note, we still observe C1 binding for mAbs that do not drive C3 deposition (e.g. ADI-19425), suggesting 165 that attachment of C1 to these mAbs may be less likely to produce an active C1 complex. This could occur if a high density of antibodies permitted C1 to attach, but assembly of the activating hexamer were occluded by F, G, or other proteins present at high densities on the viral surface.

CD55 is packaged into RSV particles and modulates sensitivity to complement deposition.
Complement defense proteins anchored in the membranes of host cells can be packaged into enveloped 170 viruses during assembly [51][52][53] , where they may function to restrict different stages of the complement cascade. To determine if host complement defense proteins restrict opsonization of RSV particles with C3, we focused on the roles of CD46 and CD55. Both proteins are abundantly expressed on A549 cells and function to limit the amplification step of the complement cascade by restricting the formation or stability of new C3 convertases 54 . Using fluorescent Fab fragments or antibodies against CD46 and 175 CD55, we were able to detect both on the surface of RSV particles collected from A549 cells. We proceeded to construct two polyclonal A549 knockout cell lines where one gene or the other was deleted via CRISPR/Cas9. Following knockout and cell sorting, we were no longer able to detect the targeted proteins in cells or shed viruses, verifying the specificity of the antibodies and confirming successful knockout ( Figure 2A&B). Using these cells lines, we proceeded to compare C3 deposition in the presence 180 or absence of CD55 or CD46. Virus released from CD55 KO cells showed increased sensitivity to C3 deposition across antibodies specific to site 0, II, and V, with percentages of opsonized particles increasing ~2-3 fold relative to virus produced by wildtype cells ( Figure 2C). Conversely, virus released from CD46 KO cells did not show significant differences in C3 deposition relative to wildtype cells using the site-V-specific mAb CR9501 ( Figure 2D). Comparing activation by CR9501 across a range of 185 antibody dilutions suggests that deletion of CD55 increases opsonization to an extent comparable to a 4-fold increase in antibody concentration ( Figure 2E&F). While additional complement defense proteins may play critical roles in other aspects of RSV infection, these results show that CD55 plays an outsized role in modulating sensitivity to opsonization with C3.

Globular particles containing postfusion F serve as dominant targets of complement activation 190
by both pre-F and post-F specific antibodies. Although the efficiency of opsonization with C3 varies depending on the activating antibody and on the presence or absence of host complement defense proteins, we observed a consistent pattern across experimental conditions, where particles opsonized with C3 had more globular morphology than those with low or undetectable levels of C3, which tended to be more filamentous ( Figure 3A & B). To investigate this further, we characterized particle morphology 195 and F conformation by simultaneously labeling RSV particles with the prefusion-specific antibody 5C4 and the postfusion-specific antibody ADI-14359. This revealed that filamentous particles contained almost exclusively pre-F while globular particles were frequently enriched in post-F ( Figure 3C), consistent with prior characterization using electron microscopy 55 . Comparing newly-released virus with virus incubated in cell culture media at 37 o C for 24 hours revealed that the proportion of post-F-containing 200 particles increased following the 24h incubation, while the proportion of filamentous particles (defined as those >1um in size with an aspect ratio >2) decreased ( Figure 3D-F). Direct labeling of RSV-infected cells with 5C4 and ADI-14359 revealed mixtures of pre-and post-F particles on the surfaces of both infected A549 cells as well as on neighboring uninfected cells, confirming that the occurrence of both particle types is not limited to circumstances where the virus is being collected or handled ( Figure S3). 205 Collectively, these results suggest that RSV particles transform spontaneously over time under mild conditions into a globular state enriched in post-F.
The bias in C3 opsonization towards globular particles could reflect a higher intrinsic sensitivity to complement activation in these particles as compared to viral filaments, or it could indicate that particle morphology is altered upon deposition of C3 or other complement proteins. To distinguish between these 210 two possibilities, we compared C3 deposition on newly-shed virus (collected over a 2-hour window) to "aged" virus (incubated at 37 o C an additional 22 hours following release from cells). If globular morphology predisposes particles to C3 deposition, we would expect to see more C3 deposition in the particles aged an additional 22 hours. Consistent with this prediction, we observed that virus collected at 2h (retaining a high proportion of filaments) was less sensitive to opsonization than virus aged an 215 additional 22h at 37 o C (retaining a lower proportion of filaments) ( Figure 4A & B), suggesting that particles with globular morphology and/or higher levels of post-F have lower thresholds for complement activation. This trend is conserved even for the pre-F-specific mAb CR9501, an unexpected result given that samples incubated at 37 o C show ~20% loss in pre-F as it converts to post-F ( Figure 3F, Figure 4B).
To further confirm that differences in particle morphology precede differences in opsonization, we 220 performed time-resolved experiments to determine the kinetics of complement deposition. For these experiments, we used CR9501 mAb as the activating antibody and tracked deposition of C4 on virus particles rather than C3. The lower concentrations of C4 in serum as compared to C3 allowed us to visualize deposition on particles without the high background signal from protein in solution that arises when using C3. Additionally, deposition of C4b is immediately downstream of activation of the C1 225 complex in the classical pathway, providing rapid detection of complement activation. Consistent with endpoint measurements of C3 deposition, C4 accumulates first on globular particles, appearing within ~10-15 minutes of incubation with complement components ( Figure 4C). Analysis of C4 deposition across n = 742 globular and n = 607 filamentous particles revealed substantial differences in patterns of opsonization; ~50% of globular particles showed detectable accumulation of C4 within 30 minutes, 230 compared to <10% of filamentous particles ( Figure 4D-F). Moreover, opsonization with C4 proceeds with different kinetics, occurring ~5-fold faster in globular particles than in filamentous ones ( Figure 4E&F). These results demonstrate that sensitivity to antibody-dependent complement deposition correlates with RSV particle morphology.

Detachment of the viral matrix increases complement activation by decreasing membrane 235
curvature. The globular RSV particles we observe resemble prior observations from electron microscopy, where the matrix protein appears to have dissociated from the membrane, resulting in more rounded morphology and less ordered distributions of surface proteins as compared to viral filaments budding from infected cells 55,56 . Although this effect has been attributed to damage during sample preparation, we reasoned that aging may result in the same morphological transformation, and that a 240 similar effect could be achieved in a controlled fashion through osmotic swelling. Treatment of RSV particles with a low osmolarity buffer (10mM HEPES pH 7.2, 2mM CaCl 2 ) transformed viral filaments into spherical particles over the course of ~1 min, with no loss of infectivity ( Figure 5A, Figure S4A). To corroborate that this morphological transformation coincides with detachment of the viral matrix, we performed photobleaching experiments on RSV particles with enzymatically labeled F. In the absence of 245 an intact matrix layer, we reasoned that F (which may interact with the matrix protein via its cytoplasmic tail 57,58 ) could freely diffuse laterally within the plane of the viral membrane and should therefore show increased recovery after partial photobleaching ( Figure S4 B&C). Consistent with this prediction, we observed significantly more recovery in bleached F on post-F enriched globular viruses and on osmotically-swollen viruses compared to viral filaments. The increased recovery on osmotically swollen 250 viruses could be reversed by treatment with 0.5% PFA while preserving the particles' spherical geometry ( Figure S4C). These results suggest that the morphological transformation we observe as RSV particles age or are subjected to physical perturbations is driven by the detachment of the matrix from the viral membrane.
We next sought to determine the effects of this controlled transformation on complement activation. We 255 found that osmotic swelling led to increased C1 binding in the presence of F-specific mAbs (CR9501, 5C4, ADI-19425), but not in their absence ( Figure 5B, left), indicating that the effect is not a non-specific consequence of swelling. Furthermore, increased C1 binding did not result from increased mAb binding, which remains constant or decreases slightly upon swelling ( Figure 5B, right). Similar to the effects of aging on virus particles (Figure 4), C3 opsonization also increases upon osmotic swelling, an effect that 260 is conserved across mAbs targeting a range of antigenic sites on pre-F and/or post-F ( Figure 5C). Recombinant IgM antibody with VH and VL domains from 5C4 shows a similar effect, with C3 deposition increasing by ~50% following osmotic swelling across a range of IgM dilutions ( Figure 5D). Collectively, these results demonstrate that detachment of the viral matrix increases sensitivity to complement activation by both IgG and IgM antibodies, and that increased sensitivity is likely due in part to increased 265 attachment of C1 to the viral surface.
The morphological transformation in RSV particles is accompanied by two notable biophysical changes: increased mobility of F in the membrane and decreased membrane curvature. While either could potentially contribute to enhanced complement activation, we observed a similar fold-increase in C3 deposition on both fixed (0.5% PFA) and unfixed viruses in the spherical state vs. the filamentous state 270 ( Figure S4D), suggesting the antigen mobility alone does not account for preferential opsonization of globular RSV particles. We therefore sought to determine if membrane curvature could be a determining factor. Curvature has previously been implicated in complement activation on antigen/antibody-coated beads and peptidoglycan nanoparticles [59][60][61] . However, these studies have reached different conclusions regarding the effects of curvature, and it remains unclear how these results would generalize to 275 enveloped viruses, where antigens are oriented and may be spaced semi-regularly 62-64 .
The transformation from filament to sphere results in decreased membrane curvature to an extent that varies depending on the size of the initial filament. While the change in curvature is negligible for particles whose length and diameter are similar (~100nm), curvature will decrease ~2-fold for a particle 1μm in length, and ~5-fold for particles 10μm in length ( Figure S5A & B). If complement activation by IgG is 280 sensitive to curvature, the effects of osmotic swelling on C3 deposition should therefore vary depending on the initial size of the particle. To test this prediction, we compared C3:F ratios between osmoticallyswollen (spherical) particles and non-treated (filamentous) particles from the same initial population of RSV. While C3:F ratios are similar for smaller viruses regardless of swelling, C3:F is increased by up to nine-fold upon swelling in larger viruses, indicating a preference for lower curvature in IgG1 activation of 285 complement ( Figure S5C). Thus, large viruses are poised to evade complement activation when they emerge from cells as highly-curved filaments, but become substantially more susceptible as they age or their morphology is physically disrupted.

Discussion
Activation of complement during RSV infection has been linked to both protective and pathogenic effects, but the mechanisms that drive complement activation by RSV remain unclear. We find that a number of factors contribute to activation of the classical pathway by shed RSV particles, including characteristics of the targeting antibody, the packaging of complement defense proteins into the viral membrane, and 295 biophysical characteristics of RSV particles. Among these factors, the dominant contribution comes from the activating antibody. We find an antigenic hierarchy in the ability of different mAbs to activate complement: although all of the IgG1 antibodies we have tested contain the same Fc and are capable of binding to pre-or post-F at similar antibody:F ratios, only a subset are able to activate complement with appreciable efficiency under the conditions of our experiments. A common feature of these activating 300 antibodies is that they are all predicted to project their Fc above the surrounding canopy of F (>15nm above the viral membrane), leading to more efficient binding by C1 (Figure 1, Figure S2). The typical distance between adjacent F trimers in the RSV membrane appears to be ~10-20nm 56 , a value that is roughly consistent with other filamentous viruses 62, 64,65 . Since this spacing is too small to accommodate an activating IgG or IgM platform between F trimers and since F appears to be largely stationary within 305 the membrane of viral filaments ( Figure S4B & C), it may be critical to position antibody Fcs in this way so as to avoid steric clashes with adjacent F trimers. A consequence of this scenario is that activation of complement would occur at comparatively large distances from the membrane. While this could potentially limit the proper assembly of a membrane attack complex (where proximity to the membrane may be beneficial 66 ), it may be inconsequential to other aspects of activation including the production of 310 C3a, which has been implicated in RSV disease severity 17 .
Perhaps the most unexpected result presented here is the observation that treatments which increase the proportion of post-F in the viral membrane can enhance complement activation by both pre-F and post-F specific antibodies (Figures 4 & 5). Given the importance of C3 opsonization in antigen presentation and B cell activation 14 , we speculate that this may contribute to bias in adaptive immune 315 responses to infection. Previous work has established that infants mount an adaptive immune response focused largely on prefusion F, and that this response shifts over time to recognize post-F as the infants age 34 . Antibodies that bind to pre-F may activate complement disproportionately on post-F containing particles, lowering thresholds for the activation of B cells that engage with these particles. Such an effect could be further enhanced by epitope masking by the pre-F antibodies, which would further reduce the 320 availability of pre-F epitopes for B cell engagement. Although this model is speculative, our results suggest that further investigation of whether or how bias in complement activation contributes to establishing immunodominance hierarchies may be warranted.
Antibody responses specific to postfusion F have been linked to increased disease severity associated with enhanced complement activation 23,38 . Post-F antibodies are poorly neutralizing and thus ineffective 325 at controlling infection, potentially leading to increased viral load. In addition, our results suggest that the high intrinsic capacity of post-F containing particles to activate complement may further enhance the damage caused by post-F specific antibodies. Mechanistically, the increased tendency of post-F enriched particles to activate complement appears to originate from the detachment of the viral matrix, which in turn decreases the mean curvature of the viral surface. Although more work is needed to understand the 330 factors that contribute to matrix detachment and the extent to which this occurs during in vivo infection, our observations that these particles accumulate over time under normal cell culture conditions suggest that their presence in vivo is feasible, where the physical and chemical environment would be considerably harsher and more complex. Other enveloped viruses, including phylogenetic neighbors of RSV from Paramyxoviridae such as measles virus and Newcastle disease virus, have been shown to 335 exhibit a similar pleomorphism arising from matrix disassembly 64,67,68 . More generally, our results suggest that any factor that alters virus morphology -whether genetic or non-genetic -could have biophysical effects on immune signaling.
The methodology presented here -combining site-specific labeling and fluorescence characterization to dissect mechanisms of complement activation by RSV -provides a model that could be generalized to 340 other pathogens. Although investigating complement activation in vitro limits our ability to asses direct immunological consequences of activation, it provides the ability to isolate the contributions that different antibodies and distinct particle subsets make towards complement activation. This may be particularly useful for viruses that vary widely in the biophysical characteristics of released progeny, including RSV, HMPV, influenza, and Ebola. Extending this methodology to investigate other viruses and their 345 interactions with immune receptors could help determine if particle heterogeneity contributes to disparate outcomes during immune signaling in other contexts as well.

Methods
Creating recombinant RSV for site-specific labeling. We introduced modifications into the RSV genome following the approach of Hotard et al. 69 . Briefly, we electroporated a BAC containing the 350 antigenomic cDNA of a chimeric RSV strain A2 with the F protein from Line 19 (obtained through BEI Resources) into the E coli strain SW102 70 . To avoid virus attenuation and fluorescence spectral overlap caused by the mKate2 reporter in front of NS1 in the initial construct, we replaced it with an mTagBFP2 reporter, expressed from an IRES following NS1. From this modified BAC, we proceeded to use a galK cassette with a 5' homology arm targeting the C-terminal region of G and a 3' homology arm targeting 355 the N-terminal region of F to insert tags on G and F simultaneously following an initial round of selection on galactose plates. We verified successfully modified BACs by sequencing a PCR-amplified region around the modified site, and we purified the BACs from 250ml cultures using a Nucleobond BAC100 kit. The genomic sequence for the final virus is given in Supporting information.
To rescue recombinant viruses, we Preparing viruses. We used viral stocks snap-frozen and stored at -80 o C to infect ~90% confluent A549 cells in 8-chambered coverglass or 96-well plates at MOI ~1. To enhance the efficiency of infection, virus diluted into virus growth media (final volume of 100ul) was centrifuged onto cells at 1200xg for 10 minutes and returned to the incubator for an additional 50 minutes before washing off the virus-containing media 375 and replacing with fresh virus growth media. Samples used for independent biological replicates were conducted using viruses from independent infections.
Enzymes and probes for site-specific labeling were generated as previously described 39,71 . At 48-60 hours post infection, F on the surface of infected cells was labeled in situ using 50μM Sortase A and 100μM fluorescent CLPMTGG substrate. With the exception of photobleaching experiments (where 380 sulfo-Cy3 was used), sulfo-Cy5 maleimide (Lumiprobe, 13380) was conjugated to the N-terminal cysteine of the SrtA peptide to label F. Labeling reactions were prepared in virus growth media supplemented with 5mM CaCl 2 . For experiments with labeled G, the labeling reaction also included 5μM Sfp, 10μM CoAprobe, and 5mM MgCl 2 . After labeling cell surface viral proteins for two hours at room temperature, the labeling reaction was washed four times with fresh media and the cells were returned to the incubator for 385 an additional two hours to allow labeled viruses to detach. Viruses prepared in this way exhibited predominantly filamentous morphology (~70%) and fewer than 5% contained detectable post-F.

Coverslip functionalization and virus immobilization. Coverslips for virus immobilization and imaging
were prepared following a pegylation protocol modified from Piehler et al. 72 . Briefly, glass coverslips were cleaned with sonication using a 50% ethanol / 50% 3M NaOH solution for 30 minutes, followed by two 390 rinses in 1L beakers of milliQ water. Coverslips were then cleaned using piranha solution (60% sulfuric acid, 40% H 2 O 2 ) and sonication for 45 minutes, rinsed, dried, and functionalized with (3glycidyloxypropyl)trimethoxysilane (GOPTS) for 1h at 75 o C. Excess GOPTS was rinsed from coverslips using anhydrous acetone, and a mixture of biotin-PEG-amine / methoxy-PEG-amine (Rapp Polymere) was prepared in anhydrous acetone at a ratio of 10 mol% biotinylated PEG. The PEG solution was 395 coupled to coverslips overnight at 75 o C, rinsed twice in 1L beakers of milliQ water, and stored in milliQ water at 4 o C until use.
For virus immobilization, pegylated coverslips were rinsed in ethanol, dried, and sealed with custom chambers made of polydimethylsiloxane with wells shaped using a 4mm biopsy punch. Wells were filled with PBS and incubated successively with streptavidin (5ug/ml in PBS) and anti-G antibody 3D3 27 with a 400 biotin site-specifically conjugated to the C-terminus of the heavy chain (see Antibody cloning, expression, purification, and labeling). After washing wells ten times with PBS to remove excess antibody, coverslips were stored at 4 o C in a humidified enclosure for <2 days, until ready for use.
Antibody cloning, expression, purification, and labeling. Antibody sequences used in this work are listed in Supporting information. VH and VL sequences were cloned into a human IgG1 backbone with a 405 C-terminal ybbR tag using Gibson assembly. Verified clones were used to transfect T75 flasks of HEK293s at ~85% confluency. At ~12h post transfection, cells were washed twice with PBS to remove any residual IgG from the serum-containing culture media and grown for an addition 6 days in serumfree OptiMEM. Media containing secreted mAbs was collected and centrifuged at 1000xg to remove detached cells before purification with Protein G resin. Fab fragments (ADI-14359) and 5C4 IgM were 410 expressed and purified analogously, with the exception that a C-terminal His(6)-tag on the heavy chains were used for affinity purification by Ni-NTA agarose in place of Protein G resin.
Eluted antibodies were quantified, diluted into a new buffer for enzymatic labeling (150mM NaCl, 25mM HEPES, 5mM MgCl 2 ), and concentrated using centrifugal concentrators (VIVAspin 100K). Antibodies concentrated to ~1mg/ml were then labeled overnight on ice using Sfp synthase and CoA-conjugated 415 dyes, prepared as previously described 39 . Following removal of excess dye using PD-10 desalting columns, labeling efficiencies were determined spectrophotometrically to be >90% for all antibodies based on the number of heavy chains. For virus immobilization, we cloned and purified the anti-G antibody 3D3 27,73 using the same protocol, but substituting CoA-biotin for the fluorescent dyes.
C3/C4 deposition assay. C3 deposition assays were performed using IgG/IgM-depleted Normal Human 420 Serum (NHS; Pelfreeze 34014). Fluorescent C3 and C4 were produced by labeling purified C3 or C4 (Complement Technologies, A113 and A105) using AF-488 dye functionalized with Nhydroxysuccinimide ester (Lumiprobe 11820). Labeling reactions were calibrated to prevent over-labeling of proteins and resulted in ~0.6-1.0 dye molecules / protein, as determined via spectrophotometry. Complement reactions to monitor C3 deposition were prepared using complement buffer (150mM NaCl, 425 25mM HEPES, 0.5mM MgCl 2 , 0.15mM CaCl 2 ) supplemented with 10mg/ml BSA, 5% IgG/IgM-depleted NHS, 50μg/ml 488-C3, and 10-20μg/ml fluorescent mAb (to assure rapid saturation of binding). Assuming a C3 concentration of 1mg/ml in NHS, approximately 30-50% of C3 in the experiment will carry a fluorophore. Prior to starting the reaction, all samples were washed thoroughly with complement buffer. Complement reactions were prepared on ice and added to virus samples before incubation at 37 o C / 5% 430 CO 2 / 100% humidity for one hour. Following incubation, samples were washed three times with PBS to terminate the reaction and imaged immediately. A similar procedure was followed for timelapse experiments using C4, except NHS was used at a final concentration of 2.5% and 488-labeled C4 was supplemented at 10μg/ml. To synchronize recording with the start of the reaction, samples containing immobilized RSV were mounted in an incubated enclosure on the microscope prior to adding the 435 complement components.
C1 binding assay. C1 binding assays were performed using purified C1 (Complement Technologies A098) in the absence of other serum proteins and the presence of defined mAbs. C1 was diluted into complement buffer to a final concentration of 10μg/ml. The reaction also contained 10mg/ml BSA and saturating concentrations of defined mAbs (10-20μg/ml, as in C3 deposition assays). This mixture was 440 incubated with enzymatically labeled RSV particles for 90 minutes at 4 o C, washed three times in complement buffer, and labeled with an Alexa Fluor 488-conjugated anti-C1qA antibody (1A4; Santa Cruz sc-53544 AF488) at a 1:500 dilution in complement buffer for 30 minutes at room temperature before imaging.
Fluorescence microscopy and image analysis. Following incubation with antibodies and complement 445 components, we imaged opsonized RSV particles using a 60X 1.40 NA objective on a Nikon T2i microscope body equipped with a Yokogawa CSU-X spinning disk and ORCA-Flash4.0 V3 camera. For each sample condition per biological replicate, we collected images of ~15 randomized fields of view, each containing ~500-1000 RSV particles. The resulting image datasets were analyzed and plotted using custom Matlab scripts. Images were segmented using the F channel to identify pixels associated with 450 each virus in the image. Following background subtraction, fluorescence intensities were integrated across all channels to obtain an integrated intensity for F, mAb, and C3/C4/C1 for each segmented particle. Integrated intensities were then plotted directly to determine population distributions (e.g. Figure  1C), or simplified further by determining the percentage of positive particles (e.g. Figure 1D).

Virus photobleaching.
Viruses labeled with Sulfo-Cy3 CLPMTGG via SrtA were immobilized on 455 coverslips using antibodies against G (3D3) and imaged on an Olympus FluoView FV1200 laser scanning confocal microscope using a 60X 1.35 NA objective. A circular region ~1μm in diameter that overlapped with a portion of the virus particle was selected and bleached using a 561nm laser, and fluorescence recovery was monitored by imaging at 5s intervals for one minute, including one image pre-bleach and one image immediately post-bleach. To identify globular particles enriched in post-F, a 488-labeled Fab 460 fragment with VH and VL domains from ADI-14359 was added as a marker. Use of a Fab fragment for these experiments prevented antibody-mediated crosslinking of F that could alter fluorescence recovery. Time series of bleaching and recovery were used determine differences in F mobility. The percentage recovery was determined by generating an image mask from the difference between the first frame postbleach and the last frame pre-bleach, to identify the bleached pixels. Intensities within the masked region 465 were then integrated to quantify signal before bleaching, immediately after bleaching, and after a 20s recovery.

Infectivity comparisons.
Comparisons of virus infectivity following various treatments ( Figure S1B, Figure S4A) used expression of the mTagBFP2 reporter to quantify infected cells. For comparisons of infectivity with or without fluorophores conjugated to F, viruses were labeled at 60hpi as described under 470 Collecting and labeling viruses. Control samples were incubated at room temperature in parallel with labeled samples, and washed in the same way, to assure that collected viruses in all cases were shed exclusively over a two-hour period. 5μl of these samples were used to infect confluent A549 cells in 96well plates, as described in Preparing viruses. Infection was quantified by counting BFP-positive cells across ~10 fields of view at 10x magnification at 12hpi. Virus quantified in this way reflects only the 475 particles shed from cells in a 2h period, and does not reflect the potentially substantial fraction of virus that remain cell-associated, or that were removed during wash steps.
For comparisons of untreated and osmotically-swollen viruses, 96-well plates containing infected A549 cells at 60hpi were washed with fresh media to remove older virus and returned to the incubator for 2h to allow new virus to shed. Collected samples were then split into experimental and control groups. For 480 control groups, 5μl of shed virus was diluted into 95ul of 1x MEM with sodium bicarbonate and 7.5mM HEPES. For experimental groups, 5μl of shed virus was diluted into 75μl of 10mM HEPES (the low osmolarity buffer used for osmotic swelling), incubated for ~1 minute, and added to 10μl of 10x sodium bicarbonate and 10x MEM, so that the final composition of control and experimental groups is matching. Samples were then used to infect confluent A549 cells in 96-well plates seeded the previous day. 485 Infection was quantified by counting BFP-positive cells across ~10 fields of view at 10x magnification at 12hpi.
Creating polyclonal A549 CD55 and CD46 knock-out lines. A549 knockout cells were generated through transduction with lentivirus generated from the lentiCRISPR v2 packaging plasmid 74 . Three sgRNA sequences were selected using CRISPR KO and the design rules described by Doench et al. 75 . 490 These were tested in small scale via transient transfection in HEK293s and the sgRNAs that yielded the highest efficiency (determined via immunofluorescence) were selected for lentivirus preparation and infection into A549s. The spacer sequences used for CD55 and CD46 sgRNAs are 5'-GCACCACCACAAATTGACAA-3' (for CD55) and 5'-GTTTGTGATCGGAATCATACA-3' (for CD46; underline indicates a nucleotide added for efficient transcription initiation). Polyclonal knockout cells were 495 further enriched at the Washington University Flow Cytometry core, using a FACS Aria II to isolate cells negative for surface staining with fluorescent antibodies against CD46 (clone TRA-2-10) or CD55 (clone JS11).
Modeling virus curvature. RSV particles are modeled in two simplified morphological states: filamentous particles -consisting of a cylindrical region of length L and radius a f and two hemispherical 500 caps -and globular particles, which we approximate as a sphere of radius a s . During the transition from a filament to a sphere, the surface area of the virus remains constant; this is constrained by the number of lipids packaged during assembly and the inability of lipid membranes to withstand area strains above ~5% 76 . Conversely, the volume of the virus may change due to a flux of water into or out of the particle. The relationships in Figure S5A were obtained by applying a constant area constraint and equating the 505 two surface areas (filament and sphere) and solving for the mean curvature in both cases.

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(D) C4 accumulation on RSV particles over time, categorized by particle morphology. Data to the left shows C4 accumulation on 742 globular particles; data on the right show C4 accumulation for 607 filamentous particles. In both plots, rows show C4 data for individual particles from 0 to 30 minutes, sorted from top to bottom according to final C4 accumulation.

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C4 accumulation in each group.
(F) Sample images of globular (top images) and filamentous (bottom images) RSV particles. Displayed images are sampled at 4 minute intervals beginning from 2 minutes after the addition of serum and complement components (scale bar = 1μm).

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(A) Comparison of infectivity (quantified as single-round infectious units per ml) of virus shed from A549 cells during a two-hour period starting at 60hpi. RSV samples were divided into control (untreated) and experimental groups (osmotic shock), and used to infect confluent monolayers of A549 cells. Infected cells were quantified at 12hpi by counting cells expressing the BFP reporter. The experiment does not account for RSV particles that remain cellassociated.

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(B) Images of photobleached / recovered RSV filaments (top) and spheres (bottom), where spheres are obtained through osmotic swelling. Magenta images show virus particles immediately after bleaching; green images show viruses after 20 seconds of recovery.
(C) Quantification of fluorescence recovery of different RSV particle subsets. Points represent individual viruses and black lines represent median values. P-values are determined using a two-sample KS test.

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(D) C3 deposition on RSV particles with (filled circles) and without (open circles) osmotic swelling. Gray lines connecting points indicate paired biological replicates. Fixation with 0.5% PFA was used to restrict antigen mobility, as shown in C. Inset: fold-change in percent C3-positive particles following osmotic swelling, with and without treatment with 0.5% PFA.