Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

Domain: physics
Published: 2026-01-20
Editorial outcome: major_revision

Abstract
Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we image tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing membrane modification and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virus particles bud. Naturally occurring furin site variants determine whether virus particles mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

Introduction
Orthoflaviviruses (henceforth flaviviruses) are a large genus of arthropod-borne, positive-sense RNA viruses within the Flaviviridae family. The mosquito-borne dengue virus alone is estimated to yearly cause hundreds of millions of human infections, some progressing to the severe condition known as dengue shock syndrome1. Human infections with tick-borne flaviviruses are less frequent, but can have severe outcomes. Tick-borne encephalitis virus (TBEV) is the namesake virus of the “TBEV serocomplex” which includes other tick-borne flaviviruses such as Powassan virus and the low-pathogenic Langat virus (LGTV). Pathogenic tick-borne flaviviruses have a strong neurotropism in mammals, and can cause encephalitis with debilitating or deadly outcome in humans2.

After entering the cell through endocytosis, the flavivirus genome is translated as a single, transmembrane polyprotein, which is subsequently cleaved by host and viral proteases into ten individual proteins. Seven of these are the non-structural (NS) proteins, which serve to replicate the viral genome. Of the NS proteins, NS3 and NS5 are cytoplasmic enzymes that serve as protease and helicase (NS3), and RNA-dependent RNA polymerase and methyl transferase (NS5). The remaining NS proteins include the endoplasmic reticulum (ER) lumen-resident peripheral membrane protein NS1, and the integral membrane proteins NS2A, NS2B, NS4A, and NS4B. Viral genome replication takes place on a transformed, dilated ER containing multiple bud-like membrane invaginations3,4. These invaginations, referred to as replication organelles (ROs), are the site of viral RNA replication5–9. RO-like membrane rearrangements can be formed by a subset of NS proteins even in the absence of viral RNA replication5,10,11, but require interactions with host ER proteins5–9,12,13. Electron microscopy of resin-embedded, infected cells has shown that the RO is a 80–90 nm, near-spherical bud with a ~10 nm opening towards the cytoplasm14–17. However, due to the destruction of protein structure by resin embedding, the organization of proteins and RNA in the RO is still unknown.

Virion assembly also takes place at the ER, when a cytoplasmic complex of viral RNA and C protein interacts with the transmembrane envelope proteins prM and E, followed by budding into the ER lumen. NS2A has been suggested as a key viral protein coupling replication and assembly18–20, and resin-embedding electron microscopy has visualized putative virions in the immediate vicinity of ROs14,16,21. Newly formed, immature virus particles have a spiky surface covered with extended prM-E trimers22–24. The current view of virion maturation, primarily based on studies of mosquito-borne flaviviruses, outlines a sequence of events that leads to formation of mature virions: the exposure of the immature virus particles to the lower pH of the Golgi apparatus leads to a conformation change in the glycoproteins, which rearrange on the virion surface to produce a smooth particle. In this smooth particle, the glycoprotein prM is cleaved by the host-cell protease furin to produce pr and M, leading to the infectious, mature virion25–27. If virion assembly and maturation are directly linked to ROs is currently unknown.

To shed light on the interactions between flavivirus replication, assembly, and maturation, we performed in situ cryo-electron tomography28–32 on human cells and mouse brain tissue infected with LGTV, and a novel, chimeric LGTV carrying TBEV structural proteins. The data suggest a mechanism for RO membrane remodeling, the presence of a protein complex tethering the RO membrane to an apposed ER membrane, and a close proximity of virion assembly and maturation.

Results
Cryo-electron tomography reveal two states of replication organelles in Langat virus-infected cells
To explore the macromolecular architecture of flavivirus ROs, we grew human A549 cells on EM grids and infected them with LGTV. Throughout this study, we consistently used amounts of virus that infected 50–90% of cells on grids. Cells were plunge-frozen at 24 h post infection (h.p.i) and subjected to focused-ion-beam milling, after which lamellas containing the infected cytoplasm were imaged using cryo-electron tomography (cryo-ET) (Table 1). The tomograms revealed a dilated ER inclusive of clustered ROs (Fig. 1 and Supplementary video 1). ROs were clearly identified as near-spherical membrane invaginations into the ER lumen, of a kind not present in uninfected cells (Fig. S1A, B). The vicinity of the remodeled ER contained bona fide ribosomes as well as mitochondria immediately apposed to the ER membrane (Fig. 1A–D and Movies S1 and S2). ROs frequently appeared in clusters within the lumen of dilated ER, as in Fig. 1A–D in which a single, dilated ER cisterna contained >10 ROs within the field of view (bearing in mind that the RO cluster probably extended beyond the depth of the lamella). The same ER cisterna additionally contained a virus particle with the characteristic spiky appearance of immature flaviviruses (Fig. 1A, B, orange arrow, and Fig. 1D). The majority of ROs contained filamentous densities, presumably the replicating double-stranded form of the viral RNA, within their lumen (Fig. 1A–D). On the other hand, several ROs were devoid of internal filamentous structures (Fig. 1A–C, white arrows). These two types of ROs will henceforth be denoted as “filled” and “empty”, respectively (Fig. 1E). In 23 tomograms, 83±14% of ROs were filled (Fig. 1F). The empty ROs were significantly smaller with an average diameter of 46±8 nm (N=25), compared to the filled ROs at 85±5 nm (N=63) (Fig. 1G). In summary, we established a workflow to image flavivirus replication by cryo-ET, revealing that ROs exist in two forms: with and without luminal filamentous densities.

A combination of membrane reinforcement and RNA-induced pressure determines RO morphology
ROs of alphaviruses, which have a similar membrane bud shape, depend on the pressure from intraluminal double-stranded RNA (dsRNA) to inflate and stabilize the curved RO membrane33. The presence of empty LGTV ROs speaks against this mechanism for flaviviruses, and we thus reasoned that a coat might confer a spontaneous curvature to the RO membrane. To investigate whether a curvature-inducing coat, or some other type of locally acting membrane modification, is present on ROs, we assessed if RO membrane thickness is in line with such membrane modifications. Indeed, by visual inspection of tomograms, the membranes of both empty and filled ROs appeared thicker than the surrounding ER membrane (Fig. 2A). At the current tomogram resolution, no distinct, repeating macromolecules were visible on the RO membranes. While this does not exclude the presence of a protein coat composed of smaller or membrane-integral proteins, it makes it unlikely that subtomogram averaging of the current data sets could conclusively determine the nature of any RO membrane modifications. Instead, we extended our previously developed surface morphometrics toolbox to allow for local estimation of membrane thickness34,35. This software allows calculation of the average membrane thickness within a user-selected area, based on average density profiles normal to the membrane. Both for single ROs and their surrounding ER membrane, this yielded reliably interpretable density profiles (Fig. 2B, C). Color coding membranes by thickness indicated that RO membranes are consistently thicker than the surrounding ER membrane (Fig. 2D). Indeed, in four tomograms, we measured a significant difference in membrane thickness with surrounding ER membrane regions being 3.4±0.3 nm (N=103), and RO membranes 4.1±0.2 nm (N=133) (Figs. 2E and S2A). On the other hand, the RO membrane thickness appeared largely independent of RO size (Fig. 2F). The estimated membrane thickness was independent of tested tomogram binning parameters (Fig. S2B), and the estimated ER membrane thickness was not significantly different in infected and uninfected cells (Fig. S2C).

The observation that flavivirus ROs can form without detectable luminal dsRNA, and the indication that their membranes are locally reinforced, distinguish them from alphavirus ROs that have a near-identical membrane bud shape. Based on cryo-ET data, we recently published a mathematical model of alphavirus RO membrane budding, which showed that the pressure from intraluminal dsRNA, together with constraint of the membrane neck, is sufficient for the creation of the RO membrane bud33. We next adapted this mathematical model to explain flavivirus RO membrane remodeling. We assume that the membrane reinforcement generates a spontaneous curvature H0 of the RO membrane. Such a spontaneous curvature can be generated, e.g., by protein structure, asymmetry of lipids or other membrane components, as well as by crowding36. Furthermore, we consider that in ROs that enclose dsRNA, the RNA exerts a pressure P on the membrane. The total energy E of the RO membrane is then composed of an integral over the membrane surface A and the contribution of the pressure, which scales with the volume V,

E = \int_A dA [2\kappa (H - H_0)^2 + \sigma] - PV, (1)

where the first term describes the bending energy according to the Helfrich model, with \kappa the bending stiffness and H the mean curvature37. The second term in Eq. (1) contains the membrane tension \sigma. Motivated by the experimentally observed shapes, we describe the ROs as spheres with a radius R, as schematically depicted in Fig. 2G, simplifying Eq. (1) to

E = 8\pi\kappa(1 - H_0 R)^2 + 4\pi\sigma R^2 - \frac{4}{3} \pi P R^3 (2)

However, there are two unknown factors in Eq. (2), the spontaneous curvature H0 and the pressure P in the RO. Based on the observation that the measured membrane thickness is largely independent of RO diameter (Fig. 2F), the membrane reinforcement can be assumed to be comparable for empty and filled ROs. Thus, we can take advantage of the imaging of empty ROs to obtain H0 at vanishing pressure, P=0. Minimizing Eq. (2) with respect to R, we obtain

H_0 = \frac{\sqrt{1 + 2\sigma \kappa R^2}}{2R} (3)

From the experiments we have obtained an average diameter of the empty ROs to be 2R=46 nm. By using previously estimated parameters33 for the membrane properties, i.e., \sigma=10^{-5} N/m and \kappa=10 k_B T, we predict the spontaneous curvature to be H0 = 0.04 nm^{-1}, which corresponds to a radius of curvature 1/H0 = 25 nm. Next, we want to predict the influence of the pressure generated by the RNA on the RO size. Since the RNA does not affect the spontaneous curvature H0, we keep the above prediction and minimize Eq. (2) with respect to R, giving

P = \frac{2(2\kappa H_0^2 R - 2\kappa H_0 + \sigma R)}{R^2} (4)

Now including the predicted H0 = 0.04 nm^{-1} with the measured average diameter of RNA-filled ROs 2R=85 nm, we obtain the pressure P = 5*10^{-4} k_B T nm^{-3}. To interpret this value, we compare it with our previous study of alphavirus ROs, which shows that a dsRNA with a length of 2000–10,000 base pairs generates an internal pressure of 10^{-4} - 10^{-3} k_B T nm^{-3} (see “Materials and Methods”, section “Estimating RO intraluminal pressure”).

Taken together, flavivirus ROs have a thicker membrane than the surrounding ER, consistent with a local membrane modification that sets a baseline RO size in the absence of luminal RNA. The size increase from empty to filled ROs is consistent with a single copy of the genome in dsRNA form.

Virions form and undergo maturation in the immediate vicinity of replication organelles
In our cryo-electron tomograms, we consistently observed virions in the vicinity of ROs, underscoring the strong association between replication and virion assembly (Fig. 3A–C and Movies S3 and S4). We next wished to use the structure preservation in cryo-ET to study the spatial relation between virion budding and maturation. In the tomograms, virus particles at different stages of maturation were distinguishable. Immature virus particles may in principle be either fully mature, furin-cleaved virions, or virions that have undergone a pH-dependent conformational change but are yet to be cleaved by furin. For simplicity, virions with smooth appearance in tomograms will henceforth be referred to as mature virions. Subtomogram averaging on a small number of virions confirmed the distinct morphology of the immature (N=84) and mature (N=51) virus particles (Fig. 3D, E), and a good match between the in situ averages and structures of purified flaviviruses (Fig. S3). The tomograms also included examples of what seemed to be nearly or recently completed virion budding (Fig. 3A, B). In such events, immature virus particles could be observed right at the membrane, across from ROs (Fig. 3A, orange arrow). Both immature and mature virus particles were consistently found close to ROs, in separate but intertwined membrane compartments (Fig. 3A–C). In principle, seemingly discrete membrane compartments may be connected beyond the limited thickness of a lamella tomogram. However, the consistent lack, across many tomograms, of observable, colocalized immature and mature particles suggests that they are indeed in separate, albeit closely spaced compartments. To quantitate the relation of immature and mature particles to ROs, we measured the center-center distance of virions to ROs in 5 tomograms. Immature virus particles were 95±56 nm (N=37), and mature virions 147±41 nm (N=24) from the closest RO (Fig. 3F). The small but significant distance difference (p=0.0003, unpaired t test), together with the observation that immature and mature virus particles are present in separate compartments, indicates that virion maturation is coupled to a slight spatial separation from ROs but does not necessitate longer-range trafficking. To further investigate the spatial relationship between genome replication, virion assembly, and the Golgi apparatus, we performed immunofluorescence light microscopy on fixed, LGTV-infected cells. Furin exhibited co-localization with the Golgi marker GM130 in both uninfected and LGTV-infected cells (Fig. S4). To assess the distribution of ER and Golgi markers in infected cells in more detail, we imaged LGTV-infected cells stained for dsRNA, GM130, and the ER marker Calnexin using super-resolution structured illumination microscopy (SIM) (Fig. 3G–K). This revealed that dsRNA is consistently surrounded by clusters of Calnexin and GM130 staining (Fig. 3G). At the resolution of the SIM imaging (80–100 nm in x-y), dsRNA, GM130, and Calnexin typically showed close but distinct staining, with all three markers being present in multiple foci within areas smaller than the field of view of our tomograms, i.e., (1.2 μm)^2 (Fig. 3K). These results corroborate the cryo-ET, reinforcing the close proximity between viral RNA replication, LGTV assembly, and maturation within the infected cell. In summary, both virion assembly and maturation can occur in the immediate proximity of ROs, in distinct but intertwined compartments.

The TBEV furin site variants R86 and Q86 differ in virion maturation near replication organelles
In purified flaviviruses, a transition from immature to smooth particles can occur due to pH change alone38,39. Since our tomograms allowed us to determine the morphology of individual, intracellular virions, we wished to test if the observed, RO-proximal virion maturation was dependent on the action of furin and the furin site sequence in prM. We first established conditions that alter the prM cleavage by furin in LGTV-infected cells. The well-established treatment of cells with 20 mM NH4Cl led to a near-complete block of prM-to-M cleavage as judged by western blotting (Figs. 4A and S5). However, NH4Cl treatment reduces furin activity by raising Golgi pH, and hence does not allow disentanglement of the cause of the conformational change. Treatment of cells with Furin Inhibitor I, which instead acts by covalently modifying furin’s active site, decreased the prM-to-M conversion in a concentration-dependent manner, albeit less completely than NH4Cl (Figs. 4A and S5). To correlate the differences in prM cleavage to the conformation of single, RO-proximal virions, we proceeded to acquire cryo-ET data on cells treated the same way (Fig. S6), and calculated the percentage of mature virions in these sets of tomograms. Whereas the number of virions in individual tomograms was sometimes small, hence leading to a large spread, the average percentage of mature virions over several tomograms showed a clear trend. Inhibition of prM-to-M cleavage by furin was reflected in a reduction of mature virions in tomograms (Fig. 4B). These data suggest a role for furin cleavage in the RO-proximal conformational change in virions. We next wanted to probe this by perturbing the virions instead of the cells. To do so, we took advantage of our recently characterized chimeric LGTV, which carries the structural proteins prM and E (ectodomain) from TBEV (Fig. 4C). This virus, rLGTVT:prME, is genetically stable and has a low pathogenicity similar to that of wild type LGTV40. The rLGTVT:prME prM and E come from the human TBEV isolate 93/783 of the European subtype, whose prM has an unusual arginine (R) in position 86, where most other strains have a glutamine (Q) (Fig. 4C). This residue is located at the P8 position of the furin cleavage site in prM, and previous studies of flaviviruses have shown that modifications here might influence prM cleavage and virus export41. We thus reasoned that R86 may affect furin cleavage efficiency and provide a naturally occurring tool to study maturation. Hence, we also produced a version of rLGTVT:prME with the more common glutamine 86. These chimeric viruses, henceforth referred to as R86 and Q86, only differ in this amino acid residue (Fig. 4C). Both viruses replicated with similar kinetics in A549 cells (Fig. S7), but the R86 virus had a slightly higher lethality in immunocompromised Ips1-/- (IFN-β promoter stimulator 1) mice (5 of 5 mice died with R86, 7 of 10 died with Q86, Fig. 4D). No difference in neurovirulence was detected (Fig. 4E). We next wanted to investigate whether R86 and Q86 conferred different prM cleavage kinetics. In a cleavage assay with a peptide corresponding to residues 81-94 of prM, the R86 sequence was cleaved faster by furin than Q86 (Fig. 4F, G). We noted that the R86 sequence generates a putative second, minimal recognition site (KR) for other proprotein convertases such as PC1/3 and PC242. In a peptide cleavage assay, the R86 sequence was also cleaved faster than Q86 by PC1/3. However, the cleavage was still completely dependent on the furin recognition site (RTRR), i.e., the putative second PC1/3 cleavage site K85-R86 was not sufficient for cleavage by PC1/3 (Fig. 4G). Next, we looked for the presence of unprocessed prM protein in cell supernatant, which would indicate release of immature virus particles. For both viruses, the bulk of cell-bound M was in the form of uncleaved prM, whereas most M in the supernatant was cleaved (Fig. 4H). The Q86 chimeric virus had a higher percentage of uncleaved prM in supernatant at 48 h post-infection (Fig. 4I), but the majority of released Q86 particles still exhibited mature morphology as assessed by cryo-EM (Fig. S8). The biochemical and cellular assays thus converge on the interpretation that the R86 sequence variant confers incrementally faster and more complete furin cleavage. Having characterized the different rates of furin cleavage, we then returned to the question of individual virion conformation inside infected cells. We infected cells with R86 and Q86 chimeric viruses and recorded cryo-electron tomograms of the infected cytoplasmas for wild type LGTV. Both for R86 and Q86, the tomograms showed a similar overall appearance as for wild type LGTV, including an abundance of filled and empty ROs as well as new virions inside dilated ER compartments (Figs. 4J, K and S9 and Movies S5 and S6). In the R86 tomograms, both immature and mature particles were seen (Fig. S9), whereas immature virus particles predominated in the Q86 tomograms (Fig. 4J, K). The average fractions mature particles over several tomograms showed a clear trend (Fig. 4L). For Q86, 2.5±5.9% of virions were mature (N=7 tomograms), whereas 46±46% of R86 virus particles were mature (N=7 tomograms), a significantly higher percentage (p=0.02, unpaired t test). Taken together, a single residue in the distal part of the TBEV furin site affects prM cleavage rates by furin and PC1/3, mean survival in immunocompromised mice, and virion maturation state in areas near ROs.

A protein complex connects the replication organelle to an apposed ER membrane
A recurring feature in the cryo-electron tomograms was the close proximity of a second ER membrane to the ER membrane containing the ROs (Figs. 1 and 2). We wanted to study the spatial relation of RO membrane necks to this second ER membrane. Putative necks were hard to identify in tomograms, possibly since they are narrower than those from some other bud-type ROs14,33. The challenge in identifying RO necks may also be due to the lower resolution of some tomograms acquired on thicker lamellas and the need for necks to be in a favorable orientation in order to be identified. Nevertheless, where an RO neck was identified, it consistently carried a protein complex on its cytoplasmic side, appearing to mediate the connection of its membrane to the adjacent, second ER membrane (Fig. 5A–D). These complexes appeared at the necks of both filled and empty ROs (Fig. 5A–D). While limited occurrences of these complexes hindered structural analysis by subtomogram averaging, we performed manual segmentations of individual complexes to estimate their volumes. Assuming average protein density, the volumes of 606±183 nm^3 translated to a complex molecular mass of 500±151 kDa (Fig. 5E). Interestingly, we observed similar-looking complexes connecting the replication organelle to the site of immature particle budding (or possibly core assembly) in the neighboring ER cisterna (Fig. 5F–H and Movies S7 and S8). This observation suggests that the protein complex might act as a membrane zipper in infected cells, coordinating RNA production with the packaging of RNA into nascent immature virus particles.

Cryo-ET reveals structural signatures of LGTV replication in ex vivo mouse brain
We next wished to explore if the structural features of LGTV replication that we observed in cell lines can also be identified directly in a complex, infected tissue. To do so, we proceeded to set up a workflow for cryo-ET of LGTV-infected mouse brain tissue. We based our approach on our recent publication, in which we imaged entire, ex vivo, LGTV-infected brains from type I interferon receptor knockout (Ifnar-/-) mice using fluorescence optical projection tomography43. In the 3D volumes of infected brains, immunofluorescence staining against LGTV NS5 protein was particularly strong in the choroid plexus (ChP) regions of the brain (Fig. 6A). ChP is an anatomical substructure responsible for secreting cerebrospinal fluid (CSF) into the ventricles, and as such it interfaces both with the blood and the CSF-filled ventricles (Fig. 6A). The ventricular side of the CSF-producing ependymal cells is covered with cilia and microvilli (Fig. 6A). Based on the consistently strong infection of the ChP, we developed a cryo-ET workflow for imaging ChP that was surgically removed from unfixed, infected brains post mortem. Due to the thickness of this sample, we opted for vitrification through high-pressure freezing. The vitrified tissue was trimmed using a cryo-ultramicrotome after which lamellas were milled in place and subjected to cryo-ET (Fig. 6A). The tomograms of ChP revealed a multitude of subcellular structures such as mitochondria, nuclear pore complexes, and a centriole (Fig. S10A, B). In addition, some tomograms contained virus-related features consistent with the observations in infected cell lines. This included mature virions encapsulated in vesicles close to membranes containing several ROs (Fig. 6B–D and Movie S9). Another tomogram contained a bona fide empty RO with a seemingly thicker membrane than its limiting ER membrane, mirroring the features seen in cellular tomograms (Fig. 6E, F). Taken together, we present the first cryo-ET data on virus replication in brain tissue. The data supports our cell line-based findings of RO-proximal virion maturation, and the presence of empty ROs, and provides a proof of principle that neurotropic virus replication can be studied by cryo-ET directly in infected brain samples.

Methods
Cell line and culturing
The human A549 lung epithelial cell line was grown in DMEM medium supplemented with 10% fetal bovine serum (FBS) and Penicillin Streptomycin GlutaMAX Supplement (Gibco) at 37°C in a 5% CO2 environment.

Virus preparations
Wild type LGTV (strain TP21, a kind gift Gerhard Dobler, Bundeswehr Institute of Microbiology, Munich, Germany) for cell biology and cryo-ET was propagated in A549 mitochondrial antiviral signaling protein MAVS-/- cells66 (kind gift of Gisa Gerold, Medical University of Innsbruck) as described previously40. A detailed description of chimeric LGTV (rLGTVT:prME) generation, rescue, and characterization can be found elsewhere40. In brief, the infectious clone of LGTV, strain TP21 was used as genetic background into which the prM and ecto-E from TBEV strain 93/783 (GenBank: MT581212.1) was inserted. Point mutation resulting in the R86Q amino acid substitution in the prM protein were introduced by overlapping PCR with primers; For: 5′ GGACGCTGTGGGAAACAGGAAGGCTCACGGACA 3′, Rev: 5′ TGTCCGTGAGCCTTCCTGTTTCCCACAGCGTCC 3′ (Sigma-Aldrich). RNA was generated from linearized DNA by in vitro transcription and transfected into BHK21 cells using Lipofectamine 2000 (Invitrogen). Supernatant from transfected cells was passaged once in A549 MAVS-/- cells, confirmed by sequencing, and used for all downstream cell biology and cryo-ET experiments without further passaging. To purify rLGTVT:prME Q86 for cryo-EM, A549 MAVS-/- cells were grown to 70% confluency in T175 bottles and infected with LGTV Q86 MOI 0.1 diluted in infection media (DMEM + Penicillin/Streptomycin + 35 nM Rapamycin). Cells were incubated at 37°C in 5% CO2 for 72 h before harvesting and purified as described previously40.

Sample preparation for cryo-electron tomography of cells
Ultrafoil Au R2/2 200 mesh grids (200 mesh, Quantifoil Micro Tools GmbH) were glow discharged. Under laminar flow, the grids were dipped in ethanol before being placed in µ-Slide 8 Well Chamber (IBIDI) wells. DMEM medium with 10% FBS was added to each well and incubated while cells were being prepared. Fresh medium was added to wells and cells were seeded out at 1.5 × 10^4 cells/well. The seeded cells were then placed in a 37°C, 5% CO2 incubator for 24 h. The medium in the wells was then replaced with serum-free DMEM and either Langat virus wild type (LGTV), recombinant chimeric R86, or recombinant chimeric Q86 were added. Virus was added so that 50–90% of the cells on grids were infected, as judged by immunofluorescence staining on EM grids using staining protocols described below. Depending on virus preparation, this amount of virus typically corresponded to a nominal MOI of 10–40, bearing in mind that the viruses were titrated under conditions that are substantially different from subconfluent cells grown on EM grids. The cells were incubated for one hour before replacing the medium with 2% FBS in DMEM and left to incubate for 24 h. Where indicated, 60 µM Furin Inhibitor I (344930, Sigma-Aldrich) or 20 mM NH4Cl was added to infected cells at 15 h.p.i. The medium was replaced with fresh DMEM including 2% FBS before being taken for freezing. Plunge freezing into an ethane/propane mix was performed with a Vitrobot (Thermo Fisher Scientific) at 22°C, 100% humidity, blotting time of 5 s, and blot force of -5.

Preparation of cryo-lamellas of cells
Lamellas were milled from plunge-frozen cells using the Scios or Aquilos 2 dual beam FIB/SEM microscope (Thermo Fisher Scientific). Samples were first coated with a platinum layer using the gas injection system (GIS, Thermo Fisher Scientific) operated at 26°C and 12 mm working distance for 10 s per grid. Lamella were milled at an angle range of 16°–20°. The cells were milled stepwise using a gallium beam at 30 kV with decreasing current starting at 0.5 nA for rough milling and ending at 0.03 nA for final polishing of the lamella. Lamellas were milled to a nominal 200 nm thickness and stored in liquid nitrogen for less than a week before being loaded into a Titan Krios (Thermo Fisher Scientific) for data collection.

Cryo-ET data collection on cells
Data were collected using a Titan Krios (Thermo Fisher Scientific) at 300 kV in parallel illumination mode. Tilt series acquisition was done using SerialEM67 on a K2 Summit detector (Gatan, Pleasanton, CA) in super-resolution mode. The K2 Summit detector was fitted with a BioQuantum energy filter (Gatan, Pleasanton, CA) operated with a 20 eV width silt. Later data were acquired using SerialEM67 and PACEtomo68 at the same microscope, now fitted with a Falcon 4i direct electron detector and a Selectris energy filter. Areas to be imaged were selected from low-magnification overview images using a condenser 2 aperture, after coma-free alignment done using Sherpa (Thermo Fisher Scientific). Tilt series were collected using the dose symmetric scheme with a starting angle of -13° to account for lamella pretilt. The parameters used for acquisition were: 33,000× nominal magnification with a corresponding object pixel size of 2.145 Å in super-resolution mode using the K2 Summit detector, and 3.6 Å using the Falcon 4i detector, a tilt range of typically -50° to +50°, defocus between -3 and -5 µm, tilt increment of 2° or 3°, and a total electron dose of 110 e/Å^2.

Cryo-ET image processing
Motion correction, tilt series alignment, CTF estimation and correction, and tomogram reconstruction was performed as described previously69, using MotionCor270 with Fourier binning of 2, IMOD71,72, and CTFFIND473. For visualization and segmentation, tomograms were 3 times binned using IMOD, resulting in a pixel size of 12.87 Å. Tomogram reconstruction from the Falcon 4i data was performed using WARP74 and AreTomo75. Tomograms were denoised using cryoCARE76 or IsoNet77, occasionally combined with a non-local means filter as implemented in Amira (Thermo Fisher Scientific). Segmentation of tomograms was performed in Amira, with initial membrane tracing and segmentation done using MemBrain V278. Subtomogram averages of mature and immature virus particles were incorporated into segmentation using UCSF Chimera79. Amira was used for counting of visually recognizable features (empty and filled ROs, immature and mature particles), measurements of the distances between them, and the volume of the neck complex densities. RO diameters were only measured for ROs completely contained in the tomogram volume, and at the largest diameter (central section) through the RO. The neck complex volumes were manually segmented to give volume estimates, which were then converted to estimated molecular masses assuming 825 Da/nm^3 80. Local membrane thickness was estimated as described elsewhere, with the average thickness being calculated for single ROs and for subregions of the ER membrane as illustrated in Fig. S3A35.

Subtomogram averaging of virions
From tomograms generated with WARP74 at 10 Å/px object pixel size, immature and mature particles were manually picked based on their clearly distinguishable appearance. 84 immature and 51 mature particles were extracted from 11 and 3 tomograms, respectively, with a box size of 80×80×80 voxels. Subtomogram averaging was done in Dynamo81,82, following the same procedure for both the immature and mature datasets. Initially, all particles were translationally and rotationally aligned to a single, high-contrast particle from the respective datasets, without symmetrization. These C1 averages were manually rotated and saved in UCSF Chimera79 to approximately fit Dynamo’s icosahedral convention, after which they were used as a template for a Gold-standard alignment with imposed icosahedral symmetry, using Dynamo’s Adaptive bandpass Filtering function. Gold-standard Fourier shell correlation curves estimated the resolution at a cutoff of 0.143 to 33 Å for the immature particles, and 80 Å for the mature particles, respectively. The final averages were filtered to this resolution and masked using the spherical alignment mask.

Estimating RO intraluminal pressure
In recent work33, we proposed a model that describes the relation between the length of an RNA and the volume of the surrounding spherule. Spherules with a volume of V = 10^3 - 2*10^3 nm^3 contain 4*10^3 - 10^4 base pairs (Fig. S11A), where the number of base pairs N is well described by

N = L_bp / l_bp * (1 + \sigma R^2 / \kappa) * 4/3 * \pi R^3 / V_N (S1)

with L = 333 nm, \sigma R^2 = \kappa = 0.02, R = 9.6 nm and the length per base pair l_bp = 0.256 nm. Furthermore, a theoretical model was used to determine the relationship between the scaled pressure PR^3/\kappa and the scaled volume V/R^3 (Fig. S11B). Combining both results, we obtain a relation between the pressure P and the number of base pairs N, which shows that an RNA with a length of 2*10^3 - 10^4 base pairs corresponds to a pressure of 10^{-3} - 10^{-4} k_B T nm^{-3} (Fig. S11C).

Immunofluorescence staining
Cells were grown on cover glasses and infected with LGTV as for cryo-ET. At 24 h.p.i., cells were fixed with 4% formaldehyde for 20 min at room temperature and then rinsed with PBS. The fixed cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min at room temperature and then rinsed with PBS. The cells were then blocked with 2% BSA in PBS containing 0.05% Tween-20 (PBS-T) for 1 h at room temperature. For both confocal and SIM imaging, cells were stained with the primary antibody against GM130 (mouse monoclonal clone 35/GM130, 1:300, BD Transduction), followed by secondary fluorescent antibody (goat anti-mouse Alexa Fluor 568, 1:1000, Invitrogen) before staining against dsRNA (mouse monoclonal clone J2, 1:1000, Scicons, NordicMUbio; conjugated to allophycocyanin, Abcam). For confocal imaging, cells were further stained against furin (goat polyclonal, 1:50, Invitrogen) with secondary fluorescent antibody donkey anti-goat Alexa Fluor 488 (1:1000, Invitrogen). Cells for SIM imaging were instead stained against calnexin (rabbit polyclonal, 1:200, Sigma-Aldrich), with secondary fluorescent antibody goat anti-rabbit Alexa Fluor 488 (1:1000, Invitrogen). DAPI was added in blocking buffer for nuclear counterstaining. Each staining step was performed for 1 h at room temperature.

Fluorescence microscopy
Fluorescence images were acquired using a Leica SP8 confocal microscope with a HC PL APO 63×/1.4 oil CS2 objective (Leica). Super-resolution SIM images were acquired using a Zeiss Elyra 7 microscope in lattice SIM mode with 15 phases. SIM2 post-processing was performed using Carl Zeiss Zen Black 3.0 software. Confocal fluorescence images were analyzed using ImageJ Fiji software83.

RNA isolation and qPCR
RNA was extracted from cell supernatant using Viral RNA kit (Qiagen) according to manufacturer’s instructions. The elution volume was kept constant and cDNA was subsequently synthetized from 10 µl of eluted RNA using high-capacity cDNA Reverse Transcription kit (Thermo Fisher). LGTV RNA was quantified with qPCRBIO probe mix Hi-ROX (PCR Biosystems) and primers recognizing NS384 on a StepOnePlus real-time PCR system (Applied Biosystems).

Western blot of chimeric virus prM cleavage
At indicated time points, supernatant was collected, and A549 cells infected with rLGTVT:prMER86 or Q86 were lysed in 350 µl of lysis buffer (50 mM Tris-HCl pH 7.5 + 150 mM NaCl + 0.1% Triton X-100) complemented with 1× protease inhibitor (cOmplete™ ULTRA, Roche, Basel, Switzerland) on ice for 20 min. Following lysis, cellular debris was removed by centrifugation at 14,000×g for 10 min at 4°C. Supernatant or pre-cleared cell lysate was mixed with Laemmli buffer to final concentration 1× and boiled at 95°C for 5 min. Proteins were separated by standard SDS-PAGE and transferred to an Immobilon®-P PVDF Membrane (GE Healthcare, Chicago, IL, USA). Blots were blocked overnight at 4°C in blocking buffer (PBS + 0.05% Tween 20 + 2% Amersham ECL Prime Blocking Reagent; Cytiva), stained with primary antibodies against NS385 (chicken polyclonal, diluted 1:1500), tubulin (rabbit polyclonal, diluted 1:4000, Abcam-ab6046) or M86 (in-house rabbit polyclonal serum, diluted 1:500) overnight at 4°C followed by secondary antibodies (goat anti-chicken Alexa-555, donkey anti-rabbit Alexa-647 (dilution 1:2500, Invitrogen, Waltham, MA, USA) for 1 h at room temperature. Blots were visualized on Amersham™ Imager 680 (GE Healthcare).

Western blot of LGTV prM cleavage under furin inhibition
To examine the effect of Furin inhibitor I (344930, Sigma-Aldrich) and ammonium chloride (NH4Cl) on prM cleavage, A549 cells were infected with LGTV and treated at 15 hpi with either DMSO (vehicle control), Furin inhibitor I (20, 40, and 60 µM), or NH4Cl (20 mM). Cells were harvested at 24 hpi and lysed directly in 2× Laemmli sample buffer, followed by heating at 95°C for 5 min. Protein lysates were subjected to SDS-PAGE and transferred onto PVDF membranes for immunoblotting. Blots were probed with antibodies against β-actin (1:10,000; Sigma-Aldrich), LGTV-NS3 (rabbit polyclonal manufactured by Sino Biological, 1:1000), and M (in-house rabbit polyclonal serum, 1:500) at 4°C followed by secondary antibodies (goat anti-rabbit-HRP, Thermo Fisher, Cat# 31460, 1:10,000 and goat anti-mouse-HRP, Thermo Fisher, Cat# 31430, 1:10,000) for 1 h at room temperature. Blots were visualized using chemiluminescence (Bio-Rad) and imaged using a ChemiDoc imaging system (Bio-Rad).

Enzymatic assays
Synthetic peptides (Biomatik) corresponding to the P13 to P'1 residues of prM from TBEV strain Torö (Dabcyl-GRCGKQEGSRTRRG-E(EDANS)) and 93/783 (Dabcyl-GRCGKREGSRTRRG-E(EDANS)) or corresponding peptides with an impaired furin recognition site (Dabcyl-GRCGKREGSRTRAG-E(EDANS), Dabcyl-GRCGKQEGSRTRAG-E(EDANS)) was used as substrate. Cleavage efficiency was assayed using an adapted fluorogenic peptide assay87. For furin, 3 U furin (Thermo Fisher, Waltham, MA, USA) was mixed with 100 µM of substrate in a total volume of 100 µl reaction buffer (100 mM HEPES pH 7.5 + 1 mM CaCl2 + 0.5% Triton X-100) at 30°C for 3 h. For proprotein convertase 1/3 (PC1/3), 1 µg recombinant human PC1/3 (R&D Systems, Minneapolis, MN, USA) was mixed with 100 µM of substrate in a total volume of 100 µl reaction buffer (25 mM MES pH 6.0 + 5 mM CaCl2 + 1% (w/v) Brij-35) at 37°C for 1 h. The emission at 490 nm measured every 3 min and the average rate calculated by linear regression.

Virus infection of mice
All animal experiments were conducted at the Umeå Centre for Comparative Biology, under approval from the regional Animal Research Ethics Committee of Northern Norrland and the Swedish Board of Agriculture, ethical permit A9-2018, A41-2019, and conducted as described previously43,84. Briefly, Mavs-/- mice in C57BL/6 background (kind gift of Nelson O Gekara, Umeå University) were infected by intraperitoneal injection of 10^4 focus-forming units (FFU) or intracranial injection of 10^2 FFU of rLGTVT:prMER86 or Q86 diluted in PBS. Ifnar–/– mice were intracranially inoculated with 10^4 FFU of LGTV and sacrificed when they developed either one pre-defined severe sign or at least three milder signs. Mice were monitored for symptoms of disease and euthanized as previously described criteria for humane endpoint43.

Cryo-ET of LGTV-infected mouse brain tissue
Based on optical projection tomography that visualized the infection distribution in entire, ex vivo brains43, fourth ventricle choroid plexuses were surgically removed from brains of LGTV-infected Ifnar–/– mice post mortem. The choroid plexuses were perfused with ice-cold phosphate-buffered saline (PBS) and rapidly transferred to ice-cold artificial CSF88. Just prior to high-pressure freezing, the tissue was placed in a 3 mm copper high-pressure freezing carrier (Wohlwend), which can be clipped into an Autogrid. The sample was covered with a 20% dextran solution in PBS as cryoprotectant and covered with a sapphire disk. The assembled carrier was rapidly vitrified using a Leica EM HPM100 high-pressure freezer. The frozen carrier was trimmed at cryogenic temperatures using a Leica EM FC7 cryo-ultramicrotome with a diamond knife. The copper carrier was trimmed to leave a flat tissue sample on the carrier, measuring 100 µm in width, 20 µm in thickness, and 30 µm in depth89. Frozen carriers were clipped into Autogrids (Thermo Fisher) prior to cryo-FIB milling with a Scios dual-beam FIB/SEM microscope (Thermo Fisher Scientific).

The sample was coated with a protective platinum layer using a GIS for 15 s at a working distance of 7 mm. The cryo stage was tilted at an angle of approximately 10° for milling. A rough milling was initially performed with an ion beam accelerating voltage of 30 kV and a current ranging from 0.79 to 2.5 nA to reach a thickness of 1 µm. Additionally, the two sides of the lamella were milled above and below to allow cryo-ET data collection by preventing the thick edges of the tissue from obstructing transmission EM imaging89. After rough milling, one edge of the lamella was detached from the main platform to relieve stress. When the lamella reached a thickness of approximately 1 µm, the ion beam current was lowered to 80 to 230 pA for fine milling, resulting in a final lamella thickness of around 200 nm. SerialEM was used to collect tilt series data with tilt angles ranging from 40° to -40° in 2° increments. The total electron dose for a single tilt series was approximately 100 e-/Å^2, with defocus between -5 and -10 µm. Tomograms were generated as described above for cells.

Statistics and reproducibility
Data and statistical analysis were performed using Prism (GraphPad Software Inc., USA). Details about replicates, statistical test used, exact values of n, what n represents, and dispersion and precision measures used can be found in figures and corresponding figure legends. Values of p<0.05 were considered significant. All tomograms shown are representative of larger datasets as indicated in Table 1.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, Dahmane et al. report an in situ tomographic study of flavivirus replication organelles (ROs) using a cryo-FIB cryo-ET workflow on unfixed samples from infected cells and fixed samples from infected mouse brains. The results reveal, with unprecedented detail, the shape of ROs and their connections to surrounding membranes of viral factories and the RE. The study highlights the relationship between ROs and viral particles assembling in the RE, tracking the maturation of these virions near the ROs. The authors utilize mutants with lower maturation rates, showing how these impact virion maturation in proximity to the ROs. They also identify empty ROs and ROs filled with dsRNA, the replication intermediate that serves as a template for viral genome synthesis. (This point should be noted in the introduction.)\nThe study shows that the spherule membrane thickness exceeds that of the RE or surrounding membranes (in both empty and full ROs), suggesting the involvement of membrane-binding proteins in RO formation. Additionally, the authors identify a protein complex at the interface of the spherule neck, viral factory, and RE membrane, potentially directing newly synthesized RNA genomes from ROs to assembling viral particles in the RER. Observations in infected mouse brains corroborate findings from cell studies and set a protocol for ex vivo cryo-EM tomography. Overall, I found the manuscript very informative for understanding flavivirus RO biology and virus assembly.\nI find the biophysical calculations part a bit speculative and in the end I dont see the biological relevance. A better contextualization and some glances at the possible biological implications would be desirable.\nSuggestions for improvement:\nUnfortunately, the authors do not provide a tomographic reconstruction of this protein complex. Even at low resolution, it would be valuable to understand how the complex associates with membranes, possibly integrating through the RE membrane like the CoV pore complex or bending the spherule neck similar to the Alphavirus replication complex. Insights here could advance our understanding of flavivirus pore architecture and the evolution of +RNA virus replication complexes.\nSpecific Points to Address:\n• Fig2 B-C: Clarify what red/blue continuous and dashed lines represent, either in the legend or with an offset. For panel E, the near-flat slope suggests little correlation between membrane thickness and RO radius.\n• G: The 2000-10000 intraluminal pressure range implies a fivefold variability; this model could benefit from a clearer interpretation. Could you plot RO pressure against dsRNA size and indicate 2000 and 10000 on the graph? What if the incorporation of lipids and more proteins in the mature spherule is changing the spontaneous curvature H0 of the RO and not the pressure from the packed dsRNA? I find this part very very speculative, theoretical and not experimentally proven.\n• Ln 89: Specify the MOI used for cell infection here and in later references.\n• Ln 88-107: Comment on the absence of intermediate ROs (no \"growing\" forms) and explain implications for RO maturation. Given the high MOI (presumably) and 24-hour infection, have you tested shorter times or lower MOIs?\n• Ln 109 section: Reframe RNA as a proposed \"pushing factor\" in RO formation for Alphavirus rather than a proven mechanism. Note that Alphavirus spherules can form without RNA in cells expressing nsps with specific mutations (Hellstrom et al., J. Virol 2017, Fig 5D).\n• Ln 120: Replace “Allowes” with “Allows.”\n• Ln 178: Indicate the number of averaged events here.\n• Ln 201: Simplify: “virion maturation near replication organelles” for clarity.\n• Ln 248: Include volume estimates and provide more detailed discussion.\n• Ln 294: Consider spherule formation in Alphavirus, which does not always require RNA. This finding might suggest that Alphavirus packs more efficiently into spherules rather than requiring dsRNA to “push” into the structure. Refer to the paper above for context.\n• Ln 317: Replace “server” with “play.”\n• Ln 325: Provide references for “maturation is more closely colocalized than previously thought,” or rephrase to “than might have been expected.”\n• Ln 334: Comment on the resolution loss due to brain fixation in this workflow. Is it possible to avoid fixation, perhaps with fluorescently labeled NS5?\n• Ln 415: Replace “we demonstrated” with “we proposed a model” to clarify that this is a theoretical relationship between RNA length and spherule volume/pressure.\n• Ln 615: Specify the number of events (counted particles) for each case.\n• Figure S5: For data on rLGTVT Q86 and R86, include MOI details for each experiment to facilitate comparison across early and late infection stages. Different MOIs could affect particle maturation outputs in both mutants and wild-type; standardizing MOI will support clearer comparisons.", "reviewer_2": "Dahmane et al describe the cryo-electron tomography analyses of flavivirus replication and assembly organelles.\nThe manuscript has provided new outlook on the organization of virus infected cells. They used both a wild-type Langat virus and a chimeric Langat-TBEV virus for EM analysis using A549 cells. They also used mutants with reduced furin cleavage efficiency to analyze the maturation state of the virus inside the cell. Finally, they could identify structures similar to what they had observed in the cell culture model using infected mouse brains. Using these analyses, the authors report key observations and propose models for flavivirus assembly and maturation. The methodology is superior to previous studies using resin-embedded tomography. Therefore, authors have better preservation of native protein structures within the cell, which is evident from the results. Also, the author’s collective experience and previous observations about mouse brain imaging and tomography studies of alphavirus replication organelles and the arrangement of RNA inside spherules have been instrumental in the analyses provided here.\nDespite this positive aspect, some aspects of the manuscript of concern need to be addressed.\nThe study regarding replication organelles is mostly observational, and the assumption is based on observed structures without clear validation. The authors claim they observed different replication structures of various sizes, some with and without RNA. They also claim that virus particles within the lumen of the replication structure contain RNA spherules. Observational studies are helpful and supported through experimental validation, which this manuscript lacks. Some experimental validation for mature and immature structures in the same compartment, which they claim to be ER-derived, is paradigm-shifting and, therefore, needs extensive experimental evidence for the scientific community to accept. Providing an IFA image using confocal microscopy of double-stranded RNA and furin colocalization is insufficient to prove that the maturation process occurs in ER-derived organelles. The authors also mention that there is a neck-like structure at the spherule neck that might be similar to the structures observed in alphavirus replication organelles, and they speculate that it could be ns1.\nFlavivirus replication proteins are not similar to alphavirus replication proteins as the authors state several membrane proteins have no structural information. Super-resolution microscopy or CLEM analysis would be required to locate their organization in the replication organelles. Previous elegant studies from several research groups have independently shown that these proteins are involved in the formation and rearrangement of the ER to form replication structures. Without more experimental support, this claim cannot be substantiated in the current state of the manuscript.\nThe assumption seems to be that the furin cleavage leads to the rearrangement of structural proteins. Therefore, immature viruses become mature viruses in the same compartment containing furin. Furin is a membrane protein present in several organelles in the cell. This is inaccurate as several elegant structural studies, including X-ray crystallography and single particle cryoEM describing the flavivirus maturation pathway, have shown that the cleavage site in prM protein is inaccessible to furin in the immature spikey conformation. Flavivirus maturation requires low pH mediated rearrangement of the virus particle into smooth structures, which allows access to the cleavage site to furin, which becomes accessible only when spiky trimers of prM/E heterodimers rearrange to dimeric prM/E heterodimers. What authors have observed is possible, provided that the organelles are low pH-containing compartments that originate from Golgi compartments. However, no such validation experiments were performed in this study. They also could have used inhibitors of trafficking or maturation for validation experiments. To support the claims authors will need to establish the nature of membrane compartments with extensive analyses. No studies are reported here to confirm whether the replication organelles and virus particles are from the same membrane compartments using other validation methods. The study using cleavage mutants neither supports nor confirms their hypothesis; the mutants are not cleavage deficient, and they are infectious. Since flavivirus maturation is an incomplete process, there are mature, partially mature, and immature particles are observed in Golgi-derived compartments in the secretory pathway that are in the process of trafficking as well as in the virus supernatants. The determination of immature and mature virus structures from the infected cell is convincing; however, it is not established that such structures are present in the ER-derived compartments. Segmentation of structures reported here does not establish boundaries of ER and the various types of replication organelles that were clearly described in the past. Therefore, the conclusions must incorporate comparisons of previously described replication, assembly, and maturation-related virus structures that have been well-defined for several flaviviruses. Instead, the current manuscript focuses on snapshots of sections not clearly defined as modified ER or Golgi-derived structures, which is a significant disadvantage of this manuscript.\nSpecific points\nLine 49: should be dengue\nLines 74-77: These sentences must be rewritten for clarity and accuracy. It should include that the prM is cleaved by furin or furin-like protease, which exclusively occurs at a low pH compartment in late Golgi. Also, pH-mediated rearrangement of prM/E trimers occurs before the furin cleavage.\nFigure 1 and lines 98-103. The size of the replication organelles should be compared from the same plane as the classical structures connected to the same ER membrane with the spherule opening. Based on Fig 1, it appears that these organelles must be connected to different membrane structures and, therefore, must be on different Z dimensions, leading to the appearance of various diameters. Authors must address this possibility, provide images of spherules from the same ER membrane, and discuss whether there are different types/sizes of spherules. Also, the possibility of small and empty organelles in various stages of formation and viral RNA replication must be considered. It is not clear whether the virus particle shown in figure 1 is an authentic virus particle. Calculate the number of the observed occurrence with supporting images.\nLine 129-131: These are not substantiated and speculative based on the current data. The authors have not performed any experiments to localize nonstructural proteins on spherules and, therefore, can't confirm the organization is based on host proteins. Also, it is important to consider that the alphavirus replication proteins are soluble cytoplasmic proteins except for the membrane attached nsP1, and the organelles are found on the plasma membrane and endosomal and lysosomal membrane, not on ER. Therefore, a direct comparison of these spherule structures might need to be revised.\nLine 167-170 Explain the nature of ‘curvature stabilizing small proteins’ and describe the possible localization of flavivirus nonstructural proteins that are integral membrane proteins.\nLine 189: Authors show that immature and mature viruses are present in close but separate compartments. This indicates that depending on the selected region of modified cellular membranes, the replication, assembly, and maturation-related compartments may vary, albeit in proximity. Therefore, classifying them based on the nature or origin of the membrane may not be accurate. The confocal microscopy presented here lacks the resolution to confirm the colocalization of the ds RNA antibody with Golgi. As the conclusions the authors are making here have not been observed before, authors need to test host and viral proteins that are resident to ER, as well as viral and host protein markers that are Golgi-specific, for clarification. As described, the experiment does not conclusively show that furin-dependent virus maturation occurs in the immediate vicinity of ROs.\nLine 195-196: Define close proximity, in terms of measured distance, as it might significantly vary in confocal compared to cryoET.\nSince the furin cleavage site mutation described here does not prevent virus maturation, it is unclear how this information fits into the narrative of general flavivirus maturation. Authors may need to present the analysis of purified virions to make an informed conclusion rather than western blot analyses of the supernatant. In Figure 4E, the western blot analysis lacks controls, markers, and information on antibodies used in the figure legend. It is also unclear why the level of M stays the same in all samples, indicating the presence of varying levels of prM.\nLines 248-258 Similar observations were shown for DENV previously. However, it is unclear whether it is the core assembly or the formation of an immature virus without additional validation using antibodies to make conclusions.\nLines 299-301 Without any experimental evidence related to host or viral protein present in the ROs, the role of ns1listed here is speculative and should be removed. Also, ns1 is not an ER liminal resident protein; while it is on the ER membrane and lumen, it forms hexamers and is secreted through the secretory pathway.\n317: ‘server’\n324: Authors do not have supporting evidence to show that what they present here contradicts the current flaviviruses model. The location of RO could be close to Golgi an analysis that could have helped the interpretation. Recent publications have shown that replication organelles are dynamic and trafficked to the perinuclear region where the Golgi also resides.\nLine 329-321: It is unclear how authors can conclude this based on the data related to furin cleavage.", "reviewer_3": "This manuscript reports cryo-electron tomography of membranous sites of RNA replication and virion assembly in cells infected by tick borne Langat flavivirus (LGTV). The results build on prior non-cryo electron tomography of other flavivirus infected cells (e.g., Welsch et al. (2009) Cell Host and Microbe), which previously established the intimate association of membrane compartments involved in viral RNA replication and virion formation and varied other points. New observations in this manuscript include (a) a small (~17%) population of RNA replication vesicles characterized by smaller diameter and absence of detectable interior filaments presumed to be viral dsRNA, (b) the membranes of the invaginated RNA replication vesicles or replication organelles (ROs) are thicker than adjacent ER membranes (4.0 vs 3.4 nm), (c) a rare circular complex connects some RO necks to adjacent ER membranes, including immature virion budding sites, (d) virions mature from spiky to smooth morphology while still near ROs, and (e) cryo-ET of samples from mouse brains infected with an LGTV-TBEV chimera also showed proximity of ROs and virion compartments and one empty RO.\nIn general, most experiments appear well-performed and are presented clearly, but the following points should be addressed:\n1. Starting from line 109, the manuscript proposes that an observed additional 18% thickness of RO membranes relative to ER might be due to a membrane associated, curvature inducing protein coat. Although no further support for this proposal is provided and “no distinct, repeating macromolecules were visible on RO membranes” (lines 116-117), in the Abstract and other prominent points the manuscript presents the protein coat model as a firm conclusion. While a hypothetical protein coat is one possible explanation for thicker membranes and formation of empty vesicles apparently lacking dsRNA filaments, it cannot be accepted without direct evidence. Among other alternate explanations, membranes associated with viral ROs often have atypical lipid compositions that likely affect their thickness and curvature. Also, many cellular vesicles including exosomes and some budding virions or virus-like particles are formed without fully filled interiors or a permanent protein coat, but by often transitory action of various vesicle shaping pathways. The literature is far from complete, but formation of functional flavivirus ROs is already known to depend on host membrane shaping factors such as reticulon 3.1A (ms citation 12), TMEM41B (ms citation 13) and ESCRT factor ALIX (Tran et al. 2022 J Virol).\nFor these reasons the present references to a possible curvature inducing membrane coat as an established conclusion are overstated and should be modified to report the observation of a difference in membrane thickness without claiming a defined mechanism. Such statements include at least those of lines 40-41, line 109, use of the hypothetical coat as the founding assumption for a series of calculations from lines 129-166, and lines 296-298. At any place where reference to the possibility of a membrane coat is retained, alternate potential explanations should also be presented.\n2. Figure 2E, lines 124-126 and 576-577 - In comparing RO and ER membrane thickness, why was ER thickness distribution only represented by n=4 measurements while RO membrane thickness was calculated from n=132 measurements? Could a larger number of ER measurements be used to provide readers with more balance and certainty on this significant comparison? (Figure 2D appears to embody many more than 4 ER membrane thickness measurements.)\nWere comparable ER membrane thickness measurements conducted on uninfected A549 cells (Figure S1) as a control for general membrane thickness changes induced by viral replication?\n3. Fig. 5 illustrates circular structures, likely made of protein, joining the openings of some ROs to adjacent ER membrane or budding immature virions. However, “limited occurrences of these complexes hindered structural analysis” (lines 252-253) and “larger tomographic data sets would be required to get a decisive subtomogram average from infected cells” (lines 318-319). Since the manuscript reported useful subtomogram averages from 51 mature and 84 immature virions (lines 402-403), presumably significantly fewer than 51 such circular complexes were observed. To allow fuller consideration of these complexes and their possible roles, please comment more fully on the frequency of such complexes relative to all ROs.\nPlease also explain how, if relatively few such complexes were seen, it can be justified to say that “close proximity of a second ER membrane to the ER membrane containing the ROs … was consistently mediated by a protein complex at the membrane neck of the (RO),” and that such a complex “consistently appears at the necks of both filled and empty ROs” (lines 249-254, underlining added).\n4. It is surprising that this study identified no RO associated protein structures other than potentially the small circular complex of point 3 above, which again appears to be a rare constituent of LGTV ROs. Cryo-ET of ROs from coronavirus, alphavirus and nodavirus infected cells immediately identified at all RO necks prominent crown complexes formed of viral replication proteins, similar to the flavivirus NS proteins. Each flavivirus NS polyprotein totals nearly 300 kDa and includes individual proteins of 70 to over 100 kDa, which should be visible even if not present in 6 or 12 fold complexes as in previously studied ROs. If the authors’ proposal that the viral NS1 protein might form a hypothetical coat in RO vesicles (lines 300-303) should be correct, this should favor replication protein detection. The many NS1 copies required to induce the observed even curvature of RO vesicles would imply correspondingly large numbers of the other viral NS replication proteins, since flaviviruses synthesize their 7 NS replication proteins as a polyprotein in 1:1 ratios. The manuscript should comment on the fact that, unlike prior RO studies with other viruses, complexes of polymerase and other viral replication proteins were not visible in this study, and perhaps briefly consider possible explanations.\n5. As a technical point, the membrane thickness measurements were reported as 3.4 ± 0.2 nm for ER (N=4) and 4.0 ± 0.2 nm for ROs (N=132) (lines 124-126). This implies measurement accuracy of 0.2 nm = 2 Å. The tilt-series were collected at a pixel size of 2.145 Å (line 384), with tomograms reconstructed using Fourier binning of 2 (line 389), resulting in raw tomograms with pixel size 4.19 Å. Subsequent 3D visualization and tomogram segmentation were performed on 3x binned tomograms, yielding a final pixel size of 12.87 Å (line 391; final total binning factor of 6). While such image binning enhances contrast for visualization and segmentation, it may compromise finer structural details. Were the membrane thickness measurements conducted on bin1 tomograms rather than bin2 tomograms? If the measurements were performed on bin2 or higher tomograms, how was sub-pixel accuracy (0.2 nm) achieved?\n6. Line 324 – “Contrary to the prevailing model” - Please briefly explain this model and provide citations to show its prevalence.\n7. Minor point - line 613 – segmentation of the tomogram in (G), not (A)", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We thank the reviewer for the positive assessment of our paper, as well as for the constructive criticism below.\nI find the biophysical calculations part a bit speculative and in the end I dont see the biological relevance. A better contextualization and some glances at the possible biological implications would be desirable.\nAlthough this part of the paper may not be of equal interest to all readers, we are convinced that a rationale for the biophysical mechanisms helps advance our understanding of the process. We agree with you that the link with the theoretical description could be improved and and have added some contextualising text to the discussion (revised manuscript lines 336-339) to improve this. In addition, there are also several other changes in how we interpret the modification of the RO membrane modification. As a whole. We belive this part of the paper is placed in a better context.\nSuggestions for improvement:\nUnfortunately, the authors do not provide a tomographic reconstruction of this protein complex. Even at low resolution, it would be valuable to understand how the complex associates with membranes, possibly integrating through the RE membrane like the CoV pore complex or bending the spherule neck similar to the Alphavirus replication complex. Insights here could advance our understanding of flavivirus pore architecture and the evolution of +RNA virus replication complexes.\nWe agree that such a structure would be of great interest to the field. Unfortunately, with our current data and even including the new data sets for the revision, we have been unable to compute a meaningful subtomogram average of a neck complex. Our preliminary Alphafold modelling suggests that the neck complex may be narrow and consist of deeply embedded transmembrane proteins, which may explain the challenge in averaging this assembly. We believe our alphafold models are too premature to be included without experimental validation. Thus, we feel it it prudent to refrain from adding any material on this topic.\nSpecific Points to Address:\n• Fig2 B-C: Clarify what red/blue continuous and dashed lines represent, either in the legend or with an offset. For panel E, the near-flat slope suggests little correlation between membrane thickness and RO radius.\nWe have now clarified this further in the figrue legend (revised manuscript lines 678-9 in the version having figures). For panel F (which we think the reviewer refers to when they say E), we already mentioned in the results text that “the RO membrane thickness appeared largely independent of RO size, as estimated from their radii of curvature (Fig. 2F).” (revised manuscript lines 136-137). We now also mention this on lines 160-161.\n• G: The 2000-10000 intraluminal pressure range implies a fivefold variability; this model could benefit from a clearer interpretation. Could you plot RO pressure against dsRNA size and indicate 2000 and 10000 on the graph? What if the incorporation of lipids and more proteins in the mature spherule is changing the spontaneous curvature H0 of the RO and not the pressure from the packed dsRNA? I find this part very very speculative, theoretical and not experimentally proven.\nThe plot suggested by the reviewer is an excellent idea, and it was already present in Figure S2C of the original submission (now Figure S11C). With respect to the reviewer’s alternative interpretation regarding lipid and protein incorporation affecting spontaneous curvature rather than RNA pressure, we have to respectfully disagree. It is well established in physics that a confined polymer exerts a pressure, and well described in the scientific literature (e.g., DOI 10.1088/0305-4470/2/2/001 and DOI: https://doi.org/10.1103/PhysRevE.79.011924). As such, we still contend that our reasoning (which is of course based on a combination of experimental measures and theoretical predictions) provides a minimal and predictive model, providing a physical mechanism for the observed phenomenon. In fact, the confined RNA would need to violate fundamental physical principles if it did not exert a pressure on the membrane, and we thus see no reason to put forward such a model. We also note the close agreement of the experimental data with expectations for ROs +/- RNA, and the fact that we see an absence of correlation between RO size and membrane thickening (Fig. 2F).\nWe do recognise that the reviewer identified lacking clarity in how we phrased our findings in the original manuscript. In the revised manuscript, we have rephrased the results section so that it does not make any conclusive statement that the membrane thickening is a “coat”. See multiple changes to the text in the revised manuscript lines 42, 114, 120-121, 139-40, 145-7, 161, 180, 335, 341-346.\nAs for lipids, we are happy that the reviewer brough up this point. We have now incorporated this possibility in the discussion, lines 343-345 .\n• Ln 89: Specify the MOI used for cell infection here and in later references.\nWe have now added this. To the materials and methods, we have also added a further explanation about nominal MOI versus infections on grids.\n• Ln 88-107: Comment on the absence of intermediate ROs (no \"growing\" forms) and explain implications for RO maturation. Given the high MOI (presumably) and 24-hour infection, have you tested shorter times or lower MOIs?\nWe agree that the biogenesis aspect is very interesting, but we cannot really address it with the data we have. Early in this project, we tested one earlier time point (16 h p.i.) but found no conclusive difference except for fewer replication events. Since we don’t have any quantitation to support that statement we decided to leave it out. As for “growing forms” we do state that the empty ROs are smaller, and in the revised discussion we have now added some more text about the possibility that they represent assembly intermediates (revised manuscript lines 338-339).\n• Ln 109 section: Reframe RNA as a proposed \"pushing factor\" in RO formation for Alphavirus rather than a proven mechanism. Note that Alphavirus spherules can form without RNA in cells expressing nsps with specific mutations (Hellstrom et al., J. Virol 2017, Fig 5D).\nThanks! We have rephrased the statement to make it less absolute (line 329 of the revised manuscript). However, we have kept the general notion for the following reason: only in cryo-EM can it be conclusively seen whether luminal RNA is present in a RO. And in all cryo-EM images available to date, alphavirus and nodaviruses replication organelles contain luminal RNA. In the referenced paper, the authors in fact write that “We cannot exclude that cellular or plasmid-derived RNAs could be unspecifically recruited to the invaginations in the absence of the viral template” in a paragraph that also concludes “under replicative conditions, the RNA contributes to the size and appearance of the spherules” and hence concurs with our interpretation of their work.\n• Ln 120: Replace “Allowes” with “Allows.”\nThanks, done!\n• Ln 178: Indicate the number of averaged events here.\nThanks, done! New manuscript lines 192-193.\n• Ln 201: Simplify: “virion maturation near replication organelles” for clarity.\nThat’s a good suggestion, thanks – done!\n• Ln 248: Include volume estimates and provide more detailed discussion.\nThanks. We added some more information, revised manuscript lines 290-292, along with other changes to that section.\n• Ln 294: Consider spherule formation in Alphavirus, which does not always require RNA. This finding might suggest that Alphavirus packs more efficiently into spherules rather than requiring dsRNA to “push” into the structure. Refer to the paper above for context.\nThanks for mentioning this. To place our previous paper on alphaviruses in a clearer context, the current revised manuscript now reads “We recently proposed” (instead of “We recently showed”) in the sentence describing our published alphavirus work (revised manuscript line 329). We feel that further discussion of our past alphavirus paper would make the current discussion very hard for a reader to follow – it would then revolve around a (single) paper published by Hellstrom et al on another virus type than what we are studying here. Also, as mentioned above, Hellstrom et al do not themselves conclude that alphavirus spherules can form without luminal RNA (se quotes above from that paper). It is also worth mentioning that another study by the Ahola group (PMID: 23760239) showed a clear correlation between template RNA length and spherule size, which matches the model we put forward in our past alphavirus paper. Hence, we don’t think it would serve the readers to extend the discussion of the current paper with the kind of text we have produced in the response letter here. If the reviewer and editor disagree, we would suggest adding such a discussion about alphaviruses as a supplementary text.\n• Ln 317: Replace “server” with “play.”\nThanks, changed!\n• Ln 325: Provide references for “maturation is more closely colocalized than previously thought,” or rephrase to “than might have been expected.”\nGood point! We added a reference.\n• Ln 334: Comment on the resolution loss due to brain fixation in this workflow. Is it possible to avoid fixation, perhaps with fluorescently labeled NS5?\nThanks for pointing out that the text was perhaps not entirely clear on this. In fact, there was no fixation of the samples used for cryo-ET, only for the OPT which was done on separate brains. We have now mentioned it on line 309 of the revised manuscript. The orginal Fig. 6A already indicated that cryo-ET was done on unfixed material, and is thus kept unchanged.\n• Ln 415: Replace “we demonstrated” with “we proposed a model” to clarify that this is a theoretical relationship between RNA length and spherule volume/pressure.\nThanks! Changed on line 504 of the revised manuscript.\n• Ln 615: Specify the number of events (counted particles) for each case.\nThis is a good point. We have now added this data, which would ba hard to fit in the figure legend, as a table in the “source data file” which we have submitted along with the revised paper.\n• Figure S5: For data on rLGTVT Q86 and R86, include MOI details for each experiment to facilitate comparison across early and late infection stages. Different MOIs could affect particle maturation outputs in both mutants and wild-type; standardizing MOI will support clearer comparisons.\nThank you. This supplementary figure is now split in two: S7 and S9. For S7, the MOI data was already present in the figure legend of the original paper, and is kept in the legend of the revised paper. For all tomography including Fig. S9, the MOI is described in the Materials and Methods section.\nWe thank the reviewer for their insightful and constructive comments as well as for their appreciation of our work. We address specific comments below.\nWe thank the reviewer for their thoughtful and constructive comments, which have helped us clarify and strengthen the manuscript.\nIn our original submission, we stated that immature and mature virions were found in “separate but intertwined membrane compartments” and in another section as “distinct but intertwined compartments.” We thus did not intend to imply that they coexisted within the same compartment. To make this more clear, we have revised the relevant text (lines 198–201) to: “In principle, seemingly discrete membrane compartments may be connected beyond the limited thickness of a lamella tomogram. However, the consistent lack, across many tomograms, of observable, colocalized immature and mature particles suggests that they are indeed in discrete but closely spaced compartments.” This wording more accurately reflects our observations and removes ambiguity.\nAs for the biochemical identity of the “maturation compartments”, we agree with the reviewer that the standard IF images of the original manuscript were not sufficient. In the revised manuscript, we have remedied this in two ways: (i) we incorporated super-resolution SIM imaging to look at the distribution of dsRNA, ER and Golgi markers in infected cells, (ii) we reference a paper that we published in the meantime, where we report the role of an NS4B interaction with host protein ACBD3 for remodelling the ER-Golgi interface in infected cells. Taken together, the addition of new data and reference to our new separate study paints a clearer picture that the virion maturation occurs in membrane compartments that are biochemically Golgi-like/Golgi-derived, but recruited to the RO-containing ER membrane through a tighter, modified ER-Golgi interface that the virus creates. The new SIM data is shown in main figure 3G-K and mentioned in the results section on lines 210-216, and the new discussion text pertaining to this is found on lines 380-383 of the revised manuscript (see also other changes to that paragraph).\nAs for the final reviewer comment in the paragraph above: the original submission contained no text about NS1 being part of the neck complex, nor did we make any suggestions as to the neck complex composition. Our discussion of NS1 related to the measured “thickening” of the replication organelle membrane. The mention of NS1 in this context has been updated based on multiple reviewer comments. We no longer refer to the thickening as a membrane coat, and we discuss potential alternative explanations to an NS1 membrane coat (revised manuscript lines 42, 114, 120-121, 139-40, 145-7, 161, 180, 335, 341-350).\nWe thank the reviewer for raising this point. We would like to clarify that in our original manuscript we did not make any claims about the composition of the neck complex. As for the possible role of NS1 as a factor coating the RO membrane, we agree that the manuscript needed changes. We have tempered this suggestion as we mention above. We also agree with the reviewer that much is still to be discovered when it comes to the arrangement of flavivirus NS proteins in the replication organelles, but the complete resolution of their arrangement unfortunately goes beyond the scope of a revision of the current manuscript\nThanks for these remarks. We decided to deal with them in three ways: Firstly, the additions mentioned above (SIM imaging and discussing our recent separate manuscript on NS4B-ACBD3), and making it more clear that we interpret the “maturation compartments” that we see close to ROs as being Golgi-like, recruited to the RO-containing ER membrane through viral remodelling of the ER-Golgi interface. Secondly, we have added new experimentation specific to the furin cleavage (new Fig. 4A-B). We established furin inhibition protocols in cells infected with wildtype Langat virus using two different inhibitors (Furin Inhibitor I and NH4Cl). The mechanism of these inhibitors is different: Furin Inhibitor I is an irreversible inhibitor that covalently modifies the furin active site, whereas NH4Cl alters the pH of the Golgi which in turn reduces furin activity (that is optimal at pH=6). We reasoned that by comparing the two, the confounding effects of pH and furin activity could begin to be disentangled. Western blotting was used to quantitate the effect on the prM to M conversion in infected cell lysates, showing that the different inhibitors decreased the fraction of cleaved M to different extents (Fig. 4A). We then acquired new cryo-ET data sets on infected cells inhibited in these two ways and we could measure an increased fraction of spiky (immature) virions in cellular tomograms, in correlation with the decrease in cleaved M protein (Fig. 4B). The revised manuscript describes these new results on lines 226-238. Based on these new data and the two furin site variants Q86 and R86, we have to conclude that the switch from immature to smooth conformation that we observe in the vicinity of replication organelles, for Langat and the chimeric virus, appears to be dependent on furin cleavage. Thirdly, we performed cryo-EM on purified Q86 chimeric virus (new Fig. S8). This (together with the western blots shown in Fig. 4H) shows that the Q86 virus, although slowed in its RO-proximal maturation, is eventually largely cleaved by furin and, when released, primarily found in smooth conformation. In the revised manuscript, line 223-224, we clarified the statement describing that purified flaviviruses can undergo a switch to smooth conformation based on pH alone. The introduction is also updated, as discussed in our response to another suggestion below.\nFinally, we have also extended the discussion related to maturation and furin, also taking into account the recent Nat Commun paper (PMID: 40796726) on furin maturation of tick-borne flaviviruses (revised manuscript lines 388-390).\nThanks! Fixed (now on line 51).\nThanks, this was needed! We now rewrote this part (lines 76-81 of the revised manuscript).\nThe reviewer makes a valid point about the size of the ROs. We apologise for the incomplete wording related to this and have now added this description to the methods section, lines 482-484 of the revised manuscript. In fact, we always measured the ROs at the height in the 3D tomogram at which their diameter appears the biggest, i.e. their central section. This is, as the reviewer points out, necessary to get accurate measurements. As for the request to “provide images of spherules from the same ER membrane”, this is something we unfortunately we cannot do with reasonable certainty since we cannot know if ER membranes seen at different positions in the tomograms are connected beyond the limited thickness (~300 nm) of the tomogram. As for the identification of different “types/sizes of spherules”, the best we can do in this respect is the analysis in Fig. 1G. All statistics shown in the paper are based on several tomograms recorded on several cells, and the figure legends state what each data point refers to (tomogram, RO, virion). We have further clarified this in a few figure legends. As for the “authenticity” of the individual virion in Fig. 1: this is a fair point, which we assume is a comment on fundamental cryo-ET resolution limitations. Fundamentally, we can only gain increased confidence in the nature of individual particles by averaging several of them together to improve the resolution, which is done later in the manuscript (Fig. 3). To account for the remaining uncertainty related to the individual virion in Fig. 1, we chose to change the designation of the virion as a “bona fide” immature virion in the legend of figure 1 (lines 662,664 in the manuscript version with figures).\nThanks for this comment. We agree. Based on this and other comments, we revised several positions in the text related to Fig. 2. In the specific sentence and in other places, we now discuss this in terms of a “local membrane reinforcement” (to describe the local thickening of the membrane using a more general term than a “protein coat”) and we mention in the results and discussion sections that the local thickening of the RO membrane may have several molecular explanations. Some of these changes are on lines 42, 114, 120-121, 139-40, 145-147, 161, 180 and discussion lines 335, 341-350. As for the mathematical model, it is very general, focusses on the energetics of membrane bending, and is thus robust to the details of which molecules are involved.\nThanks for the suggestion. Based on several suggestions from reviewers, we made this sentence less speculative by replacing the phrase cited above with a more cautious one. The new version is on line 179-180 of the revised manuscript.\nThank you for these comments.\nWe agree that the resolution of confocal microscopy is too low. As described above, we therefore extended the revised manuscript with super resolution SIM microscopy with different ER and Golgi markers to better understand the distribution of the RO (dsRNA) with furin, Golgi and ER.\nWe have described the revisions of the manuscript that are relevant to this comment above, in response to the similar comments the reviewer made there.\nIn brief, these are additions of new data and changes to the manuscript in relation to (i) the possible Golgi nature of the maturation compartments, and (ii) the effect of furin inhibition on virion maturation.\nGood point! We agree that the original wording, based on the previous confocal images, was not precise. We have rewritten this part based on the SIM imaging and to strengthen the link to the cryo-ET (lines 213-216 of the revised manuscript).\nThanks for the suggestions. As requested, we have now performed a cryo-EM analysis of purified Q86 virus (the slower maturing species) and added it to the new Fig. S8. The outcome is described on lines 263-265 of the revised manuscript. As for the western blots, we think the reviewers comments arose from our insufficient figure legend, as the reviewer points out. While we keep the nitty-gritties in the Materials and Methods, the figure legend (of what is now Fig. 4H) now contains the key information that prM and M were both visualised “using an anti-M antibody”. The goal with the blots is to compare the relative strength of the prM and M bands detected in the same sample by the same antibody. For such an analysis, loading controls would strictly speaking not be necessary and we disagree that “appropriate controls” would be missing. Still, the cell lysate blots have loading controls (tubulin) and NS3. In addition to the experimental details found in the Materials and Methods, uncropped blots will be submitted as part of the source data file as per Nat commun policy.\nGood point, we have now revised the text to say that the observation may be core or immature virus assembly (line 294 of the revised manuscript).\nThanks. Based on suggestions from this and other reviewers, we have rewritten this part of the discussion. We have fixed and extended the description of NS1, as suggested. We have also more carefully discussed alternative mechanisms that may be behind the observed spontaneous curvature. We have kept a discussion of NS1 here, since previous publications (which we cite) point towards its possible role in this process.\nThanks – fixed! (line 367 now)\nWe thank the reviewer for pointing out this weakness in the original manuscript. Our additional data, described in responses above, address this. In addition, as described above, we now cite and discuss our recent publication that establishes a molecular mechanism for modification of the ER-Golgi interface in flavivirus replication (NS4B-ACBD3 interaction).\nThanks for this comment. We have now changed this statement to more accurately summarise the data (lines 383-387 of the revised manuscript). We also now discuss our findings in the light of a recent article in Nat commun on the topic of furin cleavage of tick-borne flaviviruses (lines 388-390).\nWe thank the reviewer for their appreciation of our work as well as the constructive criticism.\nWe thank the reviewer for this insightful comment. All reviewers brought up this in one way or another, and we have revised the manuscript thoroughly according to the suggestions. The main change, as suggested by the reviewer, is that we now discus the membrane modification in more general terms as a “modification” or “reinforcement” that may have several different molecular explanations. The new text pertaining to this is in the revised manuscript on lines: 42, 114, 120-121, 139-40, 145-7, 161, 180, 335, 341-350.\nThis is a good question. In the original manuscript, we had calculated one average ER thickness per tomogram. Thus, which that average was probably quite precise, it didn’t portray the possible variability across the ER, and it was also a potential problem for the statistical significance calculation. For the revised manuscript, we extended our scripts such that they now calculate one average thickness for each continuously segmented piece of ER membrane. While the size of these pieces vary, they are now closer in size to individual ROs and the outcome gives a better view (we feel) of the thickness variability across the ER. We prefer to avoid further sub-divisions because sections of continuous membrane may influence each other, limiting the ability to accurately estimate the number of independent variables in the statistical analyses; however, separated components are distinct enough to ensure independence.The new measurement is in Fig. 2E, and a visualisation of the ER pieces is in the new Fig. S2A. We have now also added the suggested control of measurements on the ER in uninfected cells (new Fig. S2C) which show that the bulk ER thickness is not altered by infection.\nThanks for this comment. We agree that the original wording seemed contradictory. In fact, the putative membrane necks of flavivirus ROs are quite hard to identify in the tomograms - we assume they are quite narrow and contain protein density that is hard to distinguish from membranes. Due to the anisotropic resolution of tomograms, it also depends on the RO orientation in the tomogram if we get lucky to see the neck, and tomogram resolution is further dependent on lamella thickness, which was variable within our data sets. Hence, it is correct that we have consistently seen the described densities when we identify RO necks, but we rarely do. The densities that were clear enough for segmentation were only 7. With viruses, which are larger and benefit from icosahedral symmetry, we could generate the reasonable averages shown from 50-80 particles. With the smaller neck complexes and only seven good events, we would unfortunately get nowhere.\nWe have now made adjustments to the text to explain the apparent conundrum of why we see so few neck complexes (revised manuscript lines 283-287).\nWe agree with the reviewer that this was surprising, and thank you for the suggestion to elaborate on it. In brief, we would guess that the reason lies in the fact that many of the NS proteins are membrane-integral. Hence, much of the mass of the neck-associated proteins may be embedded in/on the membranes of the RO, and not be detected at the resolution of our tomograms. We took the liberty of incorporating this line of thought into the discussion, revised manuscript lines 368-373.\nThis is a good point. The original measurements were indeed conducted on binned tomograms. It is possible to achieve subpixel precision in these measurements, since they are based on fitting the centres of Gaussians for data averaged over thousands of measurements – essentially, we report the localization precision of the averaged relative position of two leaflets, which is generally finer than the FSC or Nyquist resolution in the same sense that atomic positions in cryo-EM maps are generally more precise than the resolution of a cryo-EM map, and often are more precise than the pixel size of the data (generally better than 1Å C-alpha RMSD). Further, the averaging across many triangles achieves some degree of improvement in precision due to an effect akin to dithering, where individual triangles have different subpixel positioning. Despite this theoretical basis for our measurement precision, the concern raised about the impact of binning is valid and we have now added the results of a measurement conducted on unbinned tomograms to the new Fig. S2 (panel B).\nGood point. We have now elaborated on this in the introduction, lines 76-80 of the revised manuscript.\nThanks, fixed (now panels J-K in Fig. 4).\nThanks for your effort! We hope it was a good learning experience.\nThat is great to hear!\nIn response to the final comments of the reviewers, we now use the terms “immature virus particle” and “mature virion” throughout, as requested by reviewer 2. We have also updated Figure S4 to improve the image quality as requested by reviewer 2.\nWe would like to express our gratitude to the reviewers for their comments, which substantially improved the final manuscript.\nBest wishes,\nLars-Anders Carlson on behalf of all authors"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have very nicely addressed all my concerns on this beautiful article.", "reviewer_2": "The revisions presented in the current manuscript has substantially improved the overall quality and answered the comments. The authors have addressed the reviewer comments in their response and have made changes in the manuscript. They have also added experiments, such as SIM, that have significantly improved the IF imaging quality.\nHowever, there are two points that need to be addressed:\n188-191 and elsewhere.\nThe term \"virions\" is used to refer to virus particles here. Technically, virions can be used for only fully infectious virus particles. Therefore, please use mature virions and immature virus particles.\nLine 210: Figure S4: Inset images in the regions of interest are low quality and appear pixelated. These need to be improved or removed, as higher-quality SIM images are available to demonstrate colocalization.", "reviewer_3": "In the first review all three reviewers had extensive comments on certain statements that were not sufficiently confirmed and additional data and validation needed to take the manuscript to a higher level. The responses to these points in some cases added experiments but unfortunately not noteworthy new mechanistic findings. In other important directions the responses modified the text to acknowledge limitations of the results and alternate explanations raised by the reviewers. Apparently largely due to real technical challenges, multiple central issues raised in the reviews remain unresolved, including some of the most potentially valuable observations. One is the rings connecting replication organelles to an apposed membrane. It is now acknowledged in the responses (but not in the manuscript, lines 281-290) that these were only seen clearly seven times. No information was obtained on their structure, whether they contain viral proteins, or what they do. Another unresolved issue raised by multiple reviewers is the cause of slight thickening of replication organelle membranes compared to ER membranes, and particularly if it might involve a viral protein coat. The outcome is that the manuscript is improved in some ways but is still largely descriptive without notable mechanistic conclusions. While presenting some observations that could be the starting point for important further work, it has not settled the main questions of the prior review. Without such results, the manuscript seems mainly of interest to flavivirus specialists.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": ""}]

Decision letter