INTRODUCTIONChikungunya virus (CHIKV) is the most common alphavirus infecting humans worldwide. A series of epidemics since 2004, of nearly pandemic proportions, have affected millions of people with severe, incapacitating, and often chronic arthralgia (Weaver et al., 2018). CHIKV, like other alphaviruses, is an enveloped, positive-stranded RNA virus. Its structural proteins [capsid (CP) and envelope (E1 and E2) glycoproteins (GPs)] assemble into mature virions that have T=4 quasi-icosahedral symmetry. Two icosahedral layers protect the positive-stranded RNA genome; namely, an outer GP shell of 240 E1-E2 heterodimers arranged into 80 trimeric GP spikes that are embedded in a lipid bilayer, and an internal nucleocapsid of 240 CPs encapsulating the genome (Sun et al., 2013). CHIKV virions enter target cells via endocytosis and the acidic environment in endosomes induces conformational changes in GPs allowing for fusion between the viral membrane and the host cell membrane (Solignat et al., 2009). Consequently, nucleocapsids (NCs) are released into the cytosol and quickly disassemble. Viral nonstructural proteins are then translated from the incoming viral genomic RNA in the cytosol and form replication complexes or “spherules” to synthesize viral RNA (Jose et al., 2009). Spherules are flask-shaped membrane compartments connecting to the cytosol through a narrow “neck”. Newly synthesized viral RNAs are exported through the neck into the cytosol. Viral structural proteins are synthesized from the subgenomic RNA in the cytosol where multiple copies of CP interact with genomic RNA to nucleate the assembly of NCs, which can occur independently of GPs in situ and in vitro (Mukhopadhyay et al., 2002). GPs assemble into trimeric spikes and are delivered to the plasma membrane through intracellular membrane trafficking. At the plasma membrane, horizontal interactions between GPs and vertical interactions between the cytosolic domain of E2 GPs with CPs are thought to drive the enveloping of assembled NCs and therefore the budding of nascent virions with outer icosahedral GP shells enclosing icosahedral NCs (Forsell et al., 1996; Forsell et al., 2000).
Compared with their well-studied entry process, the temporally and spatially orchestrated assembly/budding of alphaviruses is less well understood. Antiviral antibodies are traditionally screened for their ability to inhibit virus entry into target cells, from inhibition of receptor binding to prevention of conformational changes within the viral fusion proteins. Recently, we noticed an interesting antiviral effect for a panel of human and mouse anti-CHIKV neutralizing antibodies (NAbs) targeting at different epitopes; namely, NAbs inhibited the release of CHIKV particles from infected cells in addition to their expected canonical inhibition of virus entry into cells (Fox et al., 2015; Jin et al., 2015). However, the mechanism underlying this interesting phenotype and its functional consequences remained unresolved. In principle, NAbs could inhibit virus release by disrupting one or more of the following steps during virus production: 1) the intracellular assembly of NCs, 2) the recruitment of NCs to the plasma membrane, 3) the orchestrated assembly/budding of NCs into virions with NCs enveloped in the GP layer, 4) the pinching off of virions from the cell membrane, among others. Here, we visualized CHIKV-infected human cells using a combination of various microscopy techniques to determine the mechanism underlying the inhibition of CHIKV release by two ultra-potent human NAbs (C9 and IM-CKV063) targeting the CHIKV E2 glycoprotein.
Cryo-electron tomography (cryoET) and subtomogram averaging (STA) can visualize three-dimensional (3D) macromolecular structures at a resolution of a few nanometers in a near-native context (Galaz-Montoya and Ludtke, 2017). Indeed, cryoET is optimally suited to study conformationally and compositionally heterogeneous macromolecular complexes in vitro (Shahmoradian et al., 2013) and in situ (Asano et al., 2015; Baker et al., 2018; Dai et al., 2013; Pfeffer et al., 2015). Using cellular cryoET STA complemented with super-resolution stimulated emission depletion (STED) microscopy, transmission electron microscopy (TEM) and immuno-electron microscopy (IEM), we found that upon NAbs binding and crosslinking coalesced viral GPs failed to organize icosahedrally to drive virus budding, causing structurally heterogeneous nucleocapsid-like particles (NCLPs) to remain arrested in the cytosol.
Our study describes the mechanism by which NAbs inhibit virus budding from infected cell surface via binding to viral GPs and disrupting GPs driven envelopment of NCs. NAbs bound to coalesced GPs on the surface of infected cells activate Fcγ receptors via their Fc regions, inducing a strong, antibody-dependent, cell-mediated cytotoxicity (ADCC) response from immune effector cells. Therefore, our data suggest that these NAbs might exert a multi-fold, “triply functional” protective effect in vivo by blocking the assembly/budding of virions and eliciting the clearance of infected cells in addition to preventing the infection of new cells.
RESULTSNeutralizing anti-CHIKV antibodies induced coalescence of viral GPs at the plasma membrane of mammalian cells.We used sub-diffraction resolution STED microscopy to visualize U2OS cells that were infected with CHIKV vaccine strain 181/clone 25 (CHIKV 181) and treated with NAbs or with a non-neutralizing antibody (non-NAb) (Figure S1) (we observed n > 20 cells and analyzed three for each case). For all antibody treatment experiments, we administered the antibodies at 3 h post infection to allow for virus entry into the cells and initiation of viral genome replication, as described previously (Jin et al., 2015).
In cells treated with the non-NAb IM-CKV066, we observed that the antibody co-localized with viral structural proteins in distinct puncta that were distributed evenly over the plasma membrane, with some perinuclear accumulation assumed to correspond to endocytosed GP-antibody complexes (Figure S1A). Line profile analysis of the fluorescent puncta revealed sharp fluorescence intensity peaks of varying width (84-389 nm, n=418), with IM-CKV066 and CHIKV structural protein signals overlapping (Figure 1A). The diameter of virions is ~70 nm and the distance between antigen and fluorophore can range from 15 to 20 nm in a typical primary-secondary antibody complex binding to an antigen (Mennella et al., 2015). Therefore, we estimated that the size range of the puncta in the absence of NAbs (84-389 nm) corresponded to individual virions and small clusters of 2-5 virions. Importantly, the absence of a homogeneous fluorescent signal over the entire surface of the plasma membrane suggested that GPs were not distributed evenly on the plasma membrane; instead, they concentrated at distinct, relatively sparse sites of active virus budding.
In marked contrast, treatment with the NAb IM-CKV063 yielded some large patches of intense fluorescence (Figure S1B) of varying width (187 nm-12.7 μm, n=107) (Figure 1B) with overlapping CHIKV structural protein and IM-CKV063 signals. The NAb C9 induced a similar effect (not shown). The majority of patches were over 389 nm, the maximum width of the puncta seen in the non-NAb controls. Because the NAbs and non-NAb bind to CHIKV E2 GPs, our results suggested that NAbs induced the coalescence of GPs into large patches at the plasma membrane.
NAbs crosslinked GPs at the outer leaflet of the plasma membrane of CHIKV-infected cells, preventing the envelopment of NCs with plasma membrane.To visualize virus budding in greater detail, we used thin-section electron microscopy to image CHIKV-infected RD cells that had been treated with anti-CHIKV monoclonal antibodies (Figures 2A, 2B & S2). In our controls [i.e., in the absence of antibodies (Figure S2A) or in the presence one of two non-NAbs targeting different epitopes (Fong et al., 2014), IM-CKV066 (Figures 2A & S2F) and IM-CKV062 (Figure S2G)], nascent budding particles and released virions were readily detected, yet relatively sparse, and sometimes found in small clusters of 2-5 particles, in agreement with the results obtained by STED microscopy (Figure 1A). In contrast, in the presence of human NAb C9 (Figures 2B & S2B) or IM-CKV063 (Figure S2C), NCLPs accumulated in the cytosol under the plasma membrane, which failed to curve around them; i.e., NAbs caused a phenotype that we henceforth will refer to as “NCLP arrest”. Two mouse NAbs CHK-9 (Figure S2D) and CHK-152 (Figure S2E) that we previously reported to inhibit CHIKV release caused the same “NCLP arrest” phenotype (Jin et al., 2015; Pal et al., 2013). In addition, our images captured flask-shaped viral spherules bulging from the cell surface of both control and NAb-treated cells (Figure S2). Viral spherules form early in the replication cycle, before GPs are translated, and their bulging out from the plasma membrane is thought to be GP-independent, driven by non-structural viral proteins and the synthesis of double-stranded RNA replication intermediates (Frolova et al., 2010). As expected, NAbs did not abrogate spherule formation in our experiments. Rather, NAbs thwarted virus budding by specifically inhibiting the envelopment of NCs, which normally requires for the plasma membrane incorporated with a GP lattice to curve around NCs (Garoff et al., 2004).
To better understand how NAbs induce NCLP arrest, we used IEM with anti-human antibodies conjugated to 6-nm colloidal gold to determine the localization of NAbs administered to infected cells. We observed gold particles lining the surface of infected cells and noticed a plethora of arrested NCLPs under the inner leaflet of the plasma membrane (Figures 2D, S3A & B). In contrast, in the presence of non-NAb, we readily detected released and budding virus particles labeled with gold-conjugated secondary antibodies (Figures 2C & S3C).
We previously reported that full IgG molecules but not Fab fragments of NAbs inhibit CHIKV release from cells (Jin et al., 2015). Therefore, considering such previous reports and our STED analyses above (Figure 1B), our IEM results here suggested that NAbs likely crosslinked viral spikes on the outer leaflet of the plasma membrane via bivalent binding of one IgG molecule to two E2 GPs in separate spikes. Consequently, GPs coalesced into large patches on the plasma membrane, which then failed to curve around NCs to enclose them, as required for virion assembly and budding.
In addition, our IEM images revealed the presence of GPs on the surface of viral replication spherules bound with monoclonal antibodies against E2 GP labeled with gold-conjugated anti-human antibodies (Figure S3B & C). The spherules bulging out from the cell surface often seemed to cluster at the sites of budding inhibition (Figure S3B), suggesting that NAbs might crosslink GPs on neighboring spherules. Released virions, budding particles, cytosolic NCLPs, and spherules, all of which are structural hallmarks of virus infection, were absent from control cells expressing CHIKV GPs only (Figure S3E).
To better understand the topology of CHIKV budding inhibition by NAbs, we collected TEM tiltseries of the same samples examined in Figure 2D and Figure S3A, B & D and reconstructed them into tomograms. The tomograms in the absence of NAbs showed normal virus budding and release (Movie S1 & Figure S5A). In contrast, the tomograms in the presence of NAbs (Movie S2) confirmed that NAbs bound to GPs at the outer surface of cells, leaving a host of NCLPs arrested in the cytosol (Figure S6). Furthermore, these tomograms also confirmed that spherules contained GPs on their surface (Figure S5Bi), which sometimes appeared to be crosslinked by NAbs (Figure S5Bii).
CryoET of CHIKV-infected cells suggested that treatment with NAbs might disrupt the normal organization of GPs at the plasma membrane.Alphavirus GP spikes are proposed to be delivered to the plasma membrane as sheets of hexagonal arrays (Soonsawad et al., 2010). However, the native arrangement of GPs has never been directly observed on the plasma membrane of infected cells. To avoid artifacts from chemical fixation while visualizing CHIKV budding inhibition by NAbs in human cells, we collected 20 cryoET tiltseries from regions at the periphery of ice-embedded CHIKV-infected cells (i.e., the regions thin enough for cryoET imaging) without stains or fixatives, and reconstructed them into tomograms after motion and CTF correction (Figure 2E, 2F & S4). Consistent with our TEM and IEM results (Figures 2A-D, S2, 3, 5 & 6), our cryoET data revealed budding/released viral particles in the absence of NAbs (Figure 2E & Movie S3).
In our cryoET tomograms, the plasma membrane of CHIKV-infected cells in the absence of NAbs appeared smooth (Figures 2E & 3Ai, & Movie S3). STA of plasma membrane subtomograms extracted from CHIKV-infected cells in the absence of NAbs converged to an average that matched the expected thickness of mammalian cell membranes (Figure 3Aii). Low-density masses were present on the outer leaflet, which might be attributable to cell-surface proteins and/or sparse viral GPs, suggesting that GPs were not continuously distributed in high density on the cell surface, but instead concentrated at relatively dispersed and small active budding spots, readily enwrapping budding NCs (Figure 4A), in good agreement with results from our STED experiments (Figure 1A). The average of plasma membrane subtomograms in the absence of NAbs also showed strong, filamentous internal density beneath the plasma membrane that might correspond to cytoskeletal components (Figure 3Aii), which have been hypothesized to aid virus egress.
In marked contrast, the membrane of CHIKV-infected cells treated with NAbs was coated with extra density on the outer leaflet at sites of NCLP arrest (Figures 2F & 3Bi, & Movie S4). Given our IEM observation that NAbs bound to GPs on the outer leaflet of the plasma membrane (Figures 2D & S3A), we attributed the extra density to coalesced NAb-GP complexes. The organization of GP spikes into an icosahedron during virion assembly at the plasma membrane has been hypothesized to drive virus budding (Forsell et al., 1996; Forsell et al., 2000). Therefore, we investigated whether GPs would exhibit any symmetric organization in the presence of NAbs at the sites of NCLP arrest where there was clear extra density coating the plasma membrane. We extracted subtomograms containing thickened membrane patches and subjected them to STA. Our results showed extra density beyond the cell membrane, on both the inner and the outer leaflets (Figure 3Bii). The extra density in the cytosol might be attributable to a combination of intracellular GP tails, components of the cellular milieu, and arrested NCLPs. Of note, these intracellular densities lacked the cytoskeleton-like order seen in the absence of NAbs (Figure 3Aii). On the outer leaflet of the plasma membrane, the ~8–9 nm thick layer of extra density matched the protruding height of GPs above the lipid bilayer in mature virions (Sun et al., 2013), and was thus attributable to coalesced viral spikes whereas the lighter layer extending further out might be attributable to floppy NAbs on top (Figure 3Bii). Of note, coalesced GPs did not appear to be arranged symmetrically, suggesting that they were conformationally and/or compositionally heterogeneous. However, if GPs were indeed natively hexagonally ordered in flat sheets on the plasma membrane as previously hypothesized (Soonsawad et al., 2010), our results here suggested that NAb binding disrupted such organization, thereby inhibiting CHIKV budding.
Arrested NCLPs inside CHIKV-infected cells in the presence of NAb were pleomorphic and heterogeneous.The tomograms of fixed, CHIKV-infected cells treated with NAbs allowed to measure the size of the NCLPs in 3D, which can be challenging or impractical in two dimensions because of overlapping densities that might come from different Z planes in the specimen. In our IEM tomograms, the NCLPs (n=98) were structurally heterogeneous, with spherically averaged radii (or “radius of gyration”) ranging from 5.3 to 16.0 nm (μ=12.8 nm, σ=1.78 nm) (Figure S6B), in good agreement with manual measurements from the mid-Z slice of representative NCLPs taken directly from the tomograms. The known radius of the CHIKV nucleocapsid core inside mature virions is ~19.3 nm, while the radius of a subpopulation of icosahedrally symmetric particles among purified Venezuelan equine encephalitis virus (VEEV, another alphavirus) NCs was ~21 nm in two independent studies (Lamb et al., 2010; Paredes et al., 2003). In addition to the icosahedrally symmetric map derived from a small set of 90 projection images, Lamb et al., 2010 also reported a wide size range for purified NCs (15.5-27 nm in radius, μ=21.5 nm, σ=1.75 nm). This suggested that budding-arrested NCLPs in our IEM tomograms were either incomplete (either not fully assembled or starting to disassemble) and/or shrunk as an artifact due to chemical fixation during specimen preparation.
Our cryoET tomograms of CHIKV-infected cells in the absence or presence of NAbs allowed us to visualize normal virus budding and budding-arrested NCLPs without artifacts from chemical fixation. We first used self-symmetry alignment (SSA) (Dai et al., 2013; Galaz-Montoya et al., 2015) to create a reference-free initial model for released virions (n=47) in the absence of NAbs. Iterative-refinement STA of virions using this initial model yielded an icosahedrally symmetric map (Figure 4B) resolved to ~3.4 nm resolution (Figure S7A), in good agreement with the known structure of CHIKV (Sun et al., 2013). Next, from our cryoET tomograms of CHIKV-infected cells treated with NAbs, we extracted 1,163 budding-arrested NCLPs (Figure 5), which appeared structurally heterogeneous and pleomorphic upon visual inspection (Figure 5A). We could not rule out the possibility that a subset of NCLPs might be icosahedrally symmetric; however, after extensive analyses (see STAR Methods), including the use of a symmetric initial model, we could not find any evidence for strict icosahedral symmetry among this set of in situ NCLPs. Since refinement (iterative alignment and averaging) of structurally heterogeneous particles together can prevent them from converging to a robust average, we classified the NCLPs by size and carried out STA among particles with the same radius (the classes were 7.9-27.9 nm in radius, μ=18.3 nm, σ=3.0 nm) (Figure 5B). The size distribution for NCLPs was shifted towards larger radii compared to that for particles from our IEM tomograms and was much closer to the size range reported for purified NCs (15.5-27 nm in radius) (Lamb et al., 2010). In our cryoET tomograms, most NCLPs had sizes near the mean, within a small (~5%) difference from the radius of NCs inside virions. The size classes at either end of the spectrum (representing particles with radii ~8-15 nm and ~23-28 nm) were sparsely populated and failed to yield good subtomogram averages. On the other hand, five classes within the 15.8-22.2 nm radius range (Figure 5C, 49 < n < 107 per class) converged to structures at ~3.6-4 nm resolutions (Figure S7B), comparable to the resolution obtained for the subtomogram average of released virions (~3.4 nm) (Figure S7A), considering that NCLPs were not icosahedrally averaged.
NAbs induced clustering of viral spherules at the cell surface.Consistent with the results from TEM and IEM experiments (Figures S3B & S6), cryoET revealed clusters of viral spherules in the presence of NAbs, often close to the sites of NCLP arrest (Figures 2F & 6A, & Movie S4). The spherules (n=99) spanned a wide range of sizes (~400,000 nm3-5,000,000 nm3 in volume)(Figure 6B). Furthermore, their shapes often deviated from spherical, being ellipsoidal or pleomorphic. Spherules that were not clustered by NAbs appeared smaller and more uniform, closer to a spheroid (with minor and major axes ranging between 47-58 nm and 56-68 nm, respectively), in good agreement with the reported diameter of 58 nm for Semliki Forest virus (SFV) spherules (Kallio et al., 2013). In contrast, spherules clustered by NAbs were larger and more strongly ellipsoidal on average (with minor and major axes ranging between 45-103 nm and 69-132 nm, respectively) (Figures 6B). In our cryoET tomograms, the spherules seemed to contain wrapped fibrillar densities (Figure 6A) resembling those reported for dsRNA inside spherules of Flock House Nodavirus (Ertel et al., 2017). On the other hand, in our TEM and IEM experiments, chemical fixation condensed the dsRNA into a small clump in the middle of the spherules with a few fibrils connected to the center (Figures S2, S3 & S5).
NAbs that crosslinked CHIKV GPs at the cell surface induced ADCC from immune effector cells against CHIKV-infected human cellsResults from our microscopy experiments above suggested that NAbs crosslinked GPs at the surface of CHIKV-infected cells, inducing the formation of large, dense patches of NAb-GP complexes. We therefore hypothesized that this dense layer of coalesced NAb-GPs should be able to bind and crosslink Fc receptors on the surface of immune effector cells, leading to the induction of ADCC. To test this hypothesis, we incubated reporter cells expressing FcγRIIIa with antibody-treated, CHIKV-infected cells. We quantified the induction of ADCC using the luciferase reporter that was produced as the result of activating the nuclear factor of activated T-cells (NFAT) pathway. We found that NAbs C9 and IM-CKV063 were indeed able to specifically induce strong ADCC from effector cells bearing FcγRIIIa in a concentration-dependent manner, while their N297Q mutants failed to activate ADCC response due to a lack of the glycosylation required for interaction with FcγRIIIa (Figure 7A). Two non-NAbs IM-CKV066 and IM-CKV062 (which did not crosslink GPs into large, dense patches) only induced a weak ADCC response at very high concentrations. A control NAb against dengue virus failed to induce ADCC against CHIKV-infected cells (Figure 7A). These functional results support our hypothesis that IgGs crosslink GPs at the surface of CHIKV-infected cells via the Fab regions to block virus budding, while the Fc regions crosslink Fcγ receptors at the surface of effector cells to induce cytotoxicity against infected cells (Figure 7B).
DISCUSSIONCHIKV is transmitted to humans by Aedes mosquitoes and causes an acute febrile illness that is typically accompanied by severe arthralgia, with relapses that last weeks to years (Weaver et al., 2018), resulting in great economic losses due to loss of productivity. In the past decade, CHIKV has spread from endemic areas of Africa and Asia to Europe and the Americas, with millions of individuals infected during the 2000s, resulting in thousands of deaths (Johansson, 2015). Although we, and others, have isolated CHIKV NAbs and demonstrated their ability to protect against infection (Fong et al., 2014; Jin et al., 2015; Pal et al., 2013; Smith et al., 2015), there is currently no approved vaccine or treatment against CHIKV infection. Previous studies have evaluated antibodies for their ability to block virus entry in vitro, following the traditional paradigm in immunology. Although we previously reported a dual antiviral effect in which CHIKV NAbs block virus entry and virus release (Jin et al., 2015), the mechanism for blocking virus release was not resolved, and its functional implications were not explored. Here, we report the structural basis underlying the mechanism by which NAbs inhibit CHIKV release. Importantly, the model we described here for NAb function targeting infected cells kills two birds with one stone: inhibition of nascent virus budding at the surface of infected cells and activation of immune effector cells to clear infected cells. Considering that these NAbs also block virus entry into cells, their protection seems to be triply-functional, providing a mechanistic explanation to their known antiviral super-potency.
Inhibition of viral release by antibodies has been reported for viruses from a number of different families (Corboba et al., 2000; Kajihara et al., 2012; Masrinoul et al., 2014; Vanderplasschen et al., 1997); however, the underlying mechanisms remain largely unknown. Previous thin-section TEM studies with two non-neutralizing antibodies against Marburg virus (Kajihara et al., 2012) and a broadly reactive antibody against influenza A virus (Yamayoshi et al., 2017) have suggested a tethering model for the inhibition of virus release. It was proposed that those antibodies might allow for normal virus budding while tethering released virions to viral GPs on the cell surface, but the underlying mechanisms for this proposed tethering model are yet to be elucidated. On the other hand, here we showed NAbs crosslinking viral GPs to inhibit virus budding, leaving a myriad of NCs arrested inside cells. Therefore, we propose that a lower concentration of NAbs might be required to block virion budding than to tether every budded virion to the cell surface, or to completely neutralize virus entry. Furthermore, the inhibition of virus budding through the mechanism we describe here elicited a strong ADCC response from immune effector cells, a response not yet demonstrated for antibodies that tether virions to the surface of cells. Of note, the anti-budding mechanism we describe here might apply to other viruses whose budding is driven by GPs at the cell surface. Furthermore, the activation of ADCC suggests that these antibodies might promote the clearance of infected cells in vivo, warranting further studies to test such hypothesis.
Although CPs can independently assemble into NCs both in cells and in vitro, nucleocapsid preassembly in the cytosol is not a prerequisite for alphavirus budding. Indeed, the budding of assembly-deficient NCs can be rescued by horizontal spike-spike interactions and vertical spike-capsid interactions (Forsell et al., 2000), suggesting that those interactions drive virus budding. In situ electron tomography of chemically fixed SFV-infected cells in a previous study suggested that trimeric GP spikes might already be assembled and organized into hexagonal lattices in cellular membrane compartments prior to being delivered to the plasma membrane, where they are required for virus budding (Soonsawad et al., 2010). Mutations in the D-loop of the SFV E2 protein that disrupt the contact between the viral E1 and E2 proteins inhibit SFV budding, further suggesting that perturbations to the native organization of the GP lattice can result in budding arrest (Byrd and Kielian, 2017). Here, we demonstrated that NAbs can crosslink viral GPs on the extracellular side of the plasma membrane to induce their coalescence. STA of dense NAb-GP patches from cryoET tomograms yielded averages with no hexagonal organization or any regular pattern, suggesting that NAb crosslinking disrupts the normal organization of GPs, which are hypothesized to drive alphavirus budding (Forsell et al., 1996; Forsell et al., 2000).
Alphavirus nucleocapsid cores purified from cells vary in size and density and are less structurally stable than mature virions (Lamb et al., 2010). In two previous studies, a subpopulation of VEEV NCs isolated from virions stripped from their envelope (Paredes et al., 2003) or purified from infected cells (Lamb et al., 2010) was icosahedrally symmetric when visualized in vitro by cryoEM single particle analysis (SPA). Yet, in both studies the organization of CPs in the isolated NCs differed from that inside mature virions, suggesting that NCs are highly plastic and undergo GP-driven and/or lipid-driven structural changes upon envelopment. Importantly, the previous studies did not address structural heterogeneity among NCs. Indeed, Lamb et al. (2010) discarded many particles due to poor correlation against the average refined with icosahedral symmetry imposed. The discarded particles included those that differed in size or were otherwise distorted/damaged, as is commonly done in SPA aiming to computationally purify specific conformations of macromolecular complexes in order to determine their structure at the highest possible resolution. However, a more recent study reported that even NCs inside mature Ross River virus (another alphavirus) virions presented structural heterogeneity deviating from icosahedral symmetry (Wang et al., 2015). For in vitro studies, there is always the concern that harsh chemical manipulation to purify macromolecular complexes can result in structural damages, giving rise to artificial, non-native heterogeneity. On the other hand, the heterogeneity that we observed here across budding-arrested NCLPs in situ in the presence of NAbs might be attributable to a combination of assembly/disassembly intermediates, natural structural variations among fully assembled NCs, and both of these types of NCLPs interacting with host factors inside the cytosol, where molecular crowding can have dramatic effects on the composition, conformation, and function of macromolecular complexes (Grunewald et al., 2003).
Resolving CHIKV assembly/disassembly intermediates in 4D (a time course study combined with 3D cryoET) inside cells in the absence of NAbs might reveal mechanistic aspects of virus production in great detail, such as the interactions of NCs with cellular host factors, and how the organization of GPs on the plasma membrane drives the envelopment of NCs. However, CHIKV budding appears to be a relatively sparse and quick process that is challenging to capture by high-magnification cryoET restricted to the thin cell periphery. Indeed, intracellular NCs were incredibly scant in our TEM and cryoET data in the absence of NAbs. Therefore, the budding arrest induced in our system by NAb treatment, which leads to the accumulation of arrested NCLPs inside the cytosol, provides a unique opportunity to study alphavirus nucleocapsid assembly/disassembly intermediates in situ, warranting future infection time-course studies (Murata et al., 2017), with much larger datasets, and enhancing the contrast and resolution, possibly with the aid of phase plates (Asano et al., 2015; Dai et al., 2013). The advent of structural biology in situ (Galaz-Montoya and Ludtke, 2017) brought about by the recent emergence of revolutionary technology in cryo electron microscopy and tomography such as direct electron detection cameras (McMullan et al., 2014), phase plates (Danev and Baumeister, 2017), correlative light and electron microscopy, and focused ion beam milling (Wang et al., 2012), will increasingly yield insights into viral infection and other biological processes in a native cellular context.
The synthesis of viral dsRNA is required for the formation of spherules at the plasma membrane very early during alphavirus infection, before the synthesis of viral structural proteins (Frolova et al., 2010). Unlike SFV and Sindbis virus spherules, which are internalized at later time points during infection, the majority of CHIKV spherules remain on the cell surface throughout the course of infection (Thaa et al., 2015). In our TEM images, spherules seemed to be distributed dispersedly on the plasma membrane in the absence of NAbs. Unexpectedly, spherules in tomograms of NAb-treated cells agglomerated in clusters. Since our 2D and 3D TEM/IEM images and tomograms revealed the presence of GPs bound with NAbs on the surface of spherules, spherule clustering might result from NAbs crosslinking GPs on neighboring spherules. Interestingly, the spherules in intertwined clusters were enlarged compared with SFV spherules for which the size of the viral genome is thought to be the main determinant of spherule size (Kallio et al., 2013). Since the CHIKV and SFV genomes are similar in length (11.8 kb for CHIKV vs 11.4 kb for SFV), the size discrepancy between SFV spherules and the enlarged, clustered CHIKV spherules we observed might arise from differences in experimental factors (chemical fixation vs near-native preservation by plunge freezing) and data analysis (reporting only one parameter for spherule size vs measuring the major and minor axes of the central cross section of each ellipsoidal spherule). Alternatively, the clustering of spherules through NAb crosslinking might cause them to swell by perturbing their normal function and dynamics.
Functionally, our study demonstrated that NAbs that crosslink viral GPs on the plasma membrane of CHIKV-infected cells were able to engage Fc receptors on effector cells to induce ADCC against CHIKV-infected cells. The density of IgG molecules coating the infected cells is a critical factor for the crosslinking and activation of Fcγ receptors as well as for the downstream induction of ADCC (Bournazos and Ravetch, 2015). Our results here, taken together with our previous studies (Jin et al., 2015; Fox et al., 2015), lead to a model in which bivalent binding of IgGs to viral GPs via Fab regions induces coalescence of viral spikes on the surface of CHIKV-infected human cells. In turn, a dense display of IgGs on coalesced GP patches activates Fcγ receptors on the surface of effector cells via their Fc regions. Fcγ receptor-mediated cellular activation of effector leukocytes, such as monocytes, macrophages, neutrophils, and NK cells, should result in ADCC, phagocytosis, and cytokine and chemokine release, facilitating the cytotoxic lysis and clearance of infected cells. Our results here provide an answer to one of the central questions in a recent review of super-potent antibodies regarding why in vitro neutralization does not always correlate well with in vivo efficiency (Walker and Burton, 2018). As we showed here, NAbs can inhibit pathogens at different stages of their life cycle. These observations warrant further studies in vivo to determine whether the same or similar clearance mechanisms can better protect organisms against infection and the associated pathogenesis compared to only preventing the entry of virus particles into cells.
In summary, the results of our study suggest a model that explains the mechanism by which NAbs inhibit CHIKV release from infected cells. We found that the specific step of the CHIKV production process that is inhibited by NAbs is the envelopment/budding of NCs. IgG molecules crosslink viral GP spikes on the plasma membrane of CHIKV-infected cells, likely disrupting the normal organization of GPs that drives the plasma membrane to curve around NCs. As a result, structurally heterogeneous NCLPs remain arrested under the plasma membrane, and Fc receptors are activated to induce ADCC from immune effector cells to clear virus-infected cells. Our findings provide a better understanding of super, multi-functional antibodies and shed light on alphavirus assembly in and budding from cells, highlighting the importance of evaluating antibody functions throughout the complete virus replication cycle. This paradigm shift towards a comprehensive model of virus inhibition, deviating from the traditional view that focuses heavily on strength of entry neutralization, opens avenues for improved antiviral therapy development.