{"paper_id":"99d8df7e-9e61-487e-98ec-a6d237782e38","body_text":"HCV replication imaged in close-to-native conditions  \n \n1 \n \n 1 \nThe architecture of membrane structures involved in 2 \nhepatitis C virus genome replication revealed in close-to-3 \nnative conditions by cryo-electron tomography 4 \n 5 \n 6 \nUpasana M. Sykora1,2, Thomas J. O’ Sullivan1,2, Yehuda Halfon1,2, Juan Fontana3,§, 7 \nMark Harris1,2,§ 8 \n 9 \n1 School of Molecular and Cellular Biology, Faculty of Biological Sciences, University 10 \nof Leeds, Leeds, LS2 9JT, United Kingdom 11 \n2 Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 12 \n9JT, United Kingdom 13 \n3 Current address: Instituto Biofisika, CSIC-UPV/EHU, Barrio Sarriena s/n, Leioa, 14 \nBizkaia, Spain, 48940 15 \n§ Corresponding authors: 16 \n Juan Fontana: juan.fontana@csic.es 17 \n Mark Harris: m.harris@leeds.ac.uk  18 \n 19 \nShort title: HCV replication imaged in close-to-native conditions 20 \n6 figures, 5591 words  21 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n2 \n \nAbstract 22 \nHepatitis C virus (HCV) infection induces extensive rearrangements of host 23 \ncytoplasmic membranes, leading to the formation of  multiple membranous structures 24 \nthat facilitate RNA replication. Current knowledge of these membranous structures 25 \nhas largely relied on correlative light and electron microscopy (CLEM) techniques 26 \nusing chemical fixation and resin embedding. To overcome these limitations, cryo -27 \npreserved cell s were prepared using cryo -focused ion beam (cryo -FIB) milling and 28 \ncryo-ultramicrotomy. For the first time, the contents within the membranous structures 29 \nhave been observed in-situ using cryo-electron tomography (cryo -ET) performed on 30 \nlamellae (prepared via cryo -FIB) and on ultrathin sections (prepared via cryo-31 \nultramicrotomy) from HCV subgenomic replicon harbouring cells. Observations from 32 \n112 cryo-electron tomograms of cryo -FIB-derived samples revealed the presence of 33 \ndensities within the inner vesicles of a subset of single -, double-membrane vesicles 34 \n(SMVs, and DMVs respectively), as well as within multi -vesicular bodies (MVBs) , 35 \nwhich might represent the viral replication machinery . Notably, this study represents 36 \nthe first direct visualisation of the arrangement of non-structural proteins within a multi-37 \nmembrane vesicle ( MMV) observed from c ryo-electron microscopy of vitreous 38 \nsections (CEMOVIS) . The cryo -ET methodologies established here lay the 39 \ngroundwork for future investigations into the architecture of the HCV replication 40 \ncomplex, leveraging advanced computational tools for deeper structural and functional 41 \nanalysis. 42 \n 43 \n  44 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n3 \n \nIntroduction 45 \nHepatitis C virus (HCV) is a positive sense RNA virus that primarily infects hepatocytes 46 \nin the liver, leading to both acute and chronic hepatitis. The latter results in progressive 47 \nliver damage, including cirrhosis, liver failure, and hepatocellular carcinoma (Martinello 48 \net al., 2023) . In common with other p ositive-sense RNA viruses, HCV  induces 49 \ncytoplasmic membrane rearrangements in the host cell to form specialised structures 50 \nthat support genome replication (Wolff et al., 2020). Current ultrastructural insights into 51 \nhost membrane rearrangements during HCV infection  have primarily come from 52 \nfluorescence microscopy (FM), electron microscopy (EM), and soft X -ray microscopy 53 \n(Ferraris et al., 2010; Romero -Brey et al., 2012; Paul et al., 2014; Lee et al., 2019) . 54 \nFor example, these studies have shown that the membrane rearrangements arise from 55 \nthe ER and autophagy pathways (Mohl et al., 2016; Pérez-Berná et al., 2016). These 56 \nmembranes form vesicular networks, observed during both viral infection and after 57 \ntransfection with subgenomic replicons (SGR), which are often referred to as viral 58 \nreplication factories or the membranous web (MW) (Gosert et al., 2003; Moradpour et 59 \nal., 2003; Ferraris et al., 2010) . The MW comprises various virus-induced vesicles, 60 \nand includ es single, double, and multi-membrane vesicles (SMV, DMV, MMV, 61 \nrespectively), multivesicular bodies (MVBs) and lipid droplets (LDs) (Romero-Brey et 62 \nal., 2012). These structures have been proposed as potential replication organelles 63 \n(ROs), serving as platforms for the replicase machinery.  64 \nHCV RNA replication is orchestrated by the five non-structural proteins NS3, NS4A, 65 \nNS4B, NS5A and NS 5B, which are necessary and sufficient to form  MW in the 66 \ncytoplasm of infected or transfected cells (Lohmann et al., 1999; Gosert et al., 2003). 67 \nNS3/4A functions as a protease and helicase, facilitating HCV polyprotein cleavage 68 \nand the unwinding of RNA secondary structures  (Bartenschlager et al., 1995). NS4B 69 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n4 \n \ncontributes to MW formation alongside NS5A  (Moradpour et al., 2003) . NS5A is a 70 \nmultifunctional viral protein involved in viral replication and assembly (Hughes et al., 71 \n2009); notably it binds NS5B and modulates  its RNA-dependent RNA polymerase  72 \nactivity (Shirota et al., 2002) . Specifically, within the MW, DMVs have been primarily 73 \nhypothesised as the main HCV replication site through immunogold labelling of NS3 74 \nand NS5A. Thus far antibodies against NS5B have not been used  to designate a 75 \nspecific vesicle by immunogold labelling. Additionally, double-stranded RNA (dsRNA) 76 \n(a marker for RNA replication ) showed colocali sation with NS5A  by 77 \nimmunofluorescence and correlated with the production of DMVs over the course of 78 \ninfection, further supporting DMVs as active sites of HCV RNA synthesis  (Romero-79 \nBrey et al., 2012). Furthermore, another study employed an SGR containing an NS5A-80 \nmCherry fusion protein and suggested that the majority of NS5A -positive regions 81 \n(~35%) were in DMVs and MMVs (Grünvogel et al., 2018). However, the same studies 82 \nalso showed that immunogold labelling of NS3 and NS5A also localised to SMVs, LDs 83 \nand ER (Romero-Brey et al., 2012), and that a relevant proportion of NS5A -mCherry 84 \nregions corresponded to MVBs (~20%), ER (~7%), LDs (~7%) and mitochondria 85 \n(~5%) (Grünvogel et al., 2018) . Additionally, chronically infected liver tissue samples 86 \nfrom patients does not present any DMVs, and the MW appeared primarily as clusters 87 \nof SMVs closely associated with  ER and  LDs (Blanchard and Roingeard, 2018).  88 \nOverall, a lthough DMVs are suggested as the primary  ROs for HCV , the type of 89 \nmembranous structure utilised as the major HCV replication site is still inconclusive.  90 \nTo date, the EM methods employed to study cellular architecture following infection 91 \n(or in cells harbouring an SGR) have yielded insights into membrane rearrangements 92 \nand the roles of individual NS proteins in these processes  (Ferraris et al., 2010; 93 \nRomero-Brey et al., 2012). However, the sample preparation methods for visualisation 94 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n5 \n \nby EM utilised chemical fixation, high pressure freezing with freeze substitution, resin 95 \nembedding and sectioning techniques . T herefore, we explored the recent 96 \nadvancements in cryogenic sample preparation techniques and cryo -electron 97 \ntomography (cryo-ET), which facilitate imaging in close-to-native conditions, to gain 98 \ninsight into the ultrastructure of these membranous structures in cells harbouring SGR. 99 \nOverall, our results confirm the presence of all the above -mentioned vesicles within 100 \nthe HCV MW, and supports a model in which internal densities, potentially 101 \ncorresponding to viral and/or cellular components, accumulate inside inner vesicles 102 \n(InVs), present inside both DMVs and SMVs, and in which NS5A-eGFP accumulates 103 \naround MMVs within cells stably harbouring an HCV SGR.  104 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n6 \n \nMaterials and Methods  105 \nGenome sequences  106 \nThe HCV genome sequence for JFH-1 (genotype 2a) used in the analysis was sourced 107 \nfrom GenBank with accession number AB047639.1. The JFH-1 SGR (Wakita et al., 108 \n2005) was modified to replace the firefly luciferase (FF-luc) reporter with a fusion of 109 \nFF-luc and neomycin phosphotransferase (termed Feo) to create the pSGR JFH-1 110 \nFeo NS3-5B construct (Wyles et al., 2009; Ross-Thriepland et al., 2015) and a green 111 \nfluorescent protein (GFP) was inserted in NS5A-domain III (using insertion site P418) 112 \n(Moradpour et al., 2004). The final construct used for this study was SGR JFH -1 Feo 113 \nNS3-5B (NS5A-eGFP), hereafter referred to as SGR-NS5A-eGFP.  114 \nLinearisation of plasmid DNA for generating in-vitro RNA transcripts 115 \nPlasmid DNA (~5 µg) was digested with XbaI (NEB) in CutSmart buffer (NEB) at 37 116 \n°C for 1 hour and purified by phenol-chloroform extraction. The purified linearised DNA 117 \n(~1 µg) was used for RNA synthesis using  Ribomax Express T7 kit (Promega)  and 118 \npurified using an RNA clean and concentrator kit (Zymo Rese arch). The RNA 119 \ntranscript integrity was analysed on a 1% agarose gel, and concentration was 120 \nmeasured using a Nanodrop spectrophotometer. 121 \nCell culture 122 \nHuh7 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Sigma) 123 \nsupplemented with 10% foetal bovine serum (FBS, Gibco), 50 Units/ml penicillin, 50 124 \nμg/ml streptomycin, 1% nonessential amino acids (NEAA, Lonza) and 2.8% HEPES 125 \n(Lonza). Cells were maintained in a humified incubator at 37 °C with 5% CO2.  126 \n 127 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n7 \n \nTo generate stable  SGR-NS5A-eGFP harbouring Huh7 cells, 2 µg of replicon RNA 128 \nwas electroporated into  2 x 10 6 cells using a square -wave protocol (260 V, 25 ms). 129 \nElectroporated cells were mixed with non-electroporated cells to a final density of 1.8 130 \nx 10⁵ cells  and seeded in 6 -well plates. After 48 h of incubation, cells were washed 131 \nwith PBS and subjected to selection with 700 µg/ml G418. Following multiple selection 132 \nrounds, the G418 concentration was reduced to  400 µg/ml for maintenance of the 133 \npolyclonal cell population.  134 \nFluorescence microscopy 135 \nTo prepare coverslips for  confocal fluorescence microscopy, 22 mm coverslips were 136 \ncleaned with dH ₂O, treated with  1M HCl, washed, incubated in ethanol, dried, and 137 \nautoclaved. Mock or SGR -expressing cells (5 x 10⁴)  were seeded on coverslips or 138 \nsorted by FACS for fluorescence-based selection. Sorted cells were incubated for ~21 139 \nh, fixed with  4% formaldehyde, permeabilized with  0.2% Triton X -100, and blocked 140 \nwith 3% BSA-PBS. Coverslips were then stained with BODIPY dye (1:1000), washed, 141 \nand mounted with  ProLong Gold Antifade reagent with DAPI. Mounted slides were 142 \nincubated for 2.5 h at room temperature and stored at 4 °C. 143 \nZ-stack confocal microscopy images were captured using a Zeiss LSM880 upright 144 \nconfocal microscope. After acquisition, the images were processed with Fiji software  145 \n(Schindelin et al., 2012). Wide-field fluorescent images were acquired with the EVOS 146 \nmicroscope (Thermo Fisher Scientific). 147 \nResin embedding and sectioning for cellular EM 148 \nHuh7 cells (control and  stably expressing SGR -NS5A-eGFP) were trypsinised and 149 \npelleted. The resulting cell pellet was fixed in with 2.5% glutaraldehyde (EM Grade, 150 \nAgar Scientific) in 0.1M sodium phosphate buffer for at least 2.5 h at room 151 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n8 \n \ntemperature. The cell pellet was washed twice for 30 min in 0.1M sodium phosphate 152 \nbuffer, post-fixed with 1% osmium tetroxide in 0.1M phosphate buffer for 1 h  and 153 \nwashed again twice for 30 min with the same buffer. Dehydration was achieved by 154 \nincubating the pellet for 20 min in increasing concentrations of ethanol: 20%, 40%, 155 \n60%, 80%, and twice in 100%. The pellet was then treated with propylene oxide twice 156 \nfor 20 min each. A freshly prepared resin mixture of 50% propylene oxide and 50% 157 \nAraldite epoxy resin was then added to the pellet and allowed to infiltrate for several h 158 \nto overnight (Luft, 1961). This was followed by infiltration with a 25% propylene oxide 159 \nand 75% Araldite epoxy resin mixture for 3 h, and with 100% Araldite epoxy resin for 160 \n3-8 h. Polymerization was then carried overnight at 60 °C. Ultrathin sections (80 -100 161 \nnm) were collected on slotted copper EM grids with formvar support and stained with 162 \nsaturated uranyl acetate for 2 h and Reynolds lead citrate for 15 min.  163 \nImage acquisition using the FEI Tecnai T12 TEM  164 \nCellular EM images from ultrathin sections prepared through resin embedding and 165 \nsectioning were captured using the FEI Tecnai T12 TEM operated at 120 kV. 166 \nCell culture workflow on EM grids 167 \nFor cryo-ET, Quantifoil Finder 200 mesh gold grids R1.2/1.3 (Gilder G200F1) and C -168 \nflat 200 mesh gold 1.2/1.3 (Electron Microscopy Sciences) were utilised. The grids 169 \nwere glow discharged in a Quorum GloQube (Quorum Technologies) to enhance 170 \nhydrophilicity and impart a negative charge. A negative polarity cycle of 20 mA was 171 \napplied for 30-60 seconds. 172 \nNext, the grids were positioned in the centre of a glass-bottom cell culture dish (Nunc 173 \nTM, 150680). Grids were functionalised with 7 μl  of fibronectin (Sigma -Aldrich) at a 174 \nconcentration of 25 μg/ml and incubated at 37 °C for 1 h. Concurrently, fluorescence-175 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n9 \n \nactivated cell sorting (FACS) was performed to collect 5 x 10 3 and 10 x 103 cells and 176 \nwere pelleted and resuspended in 50 μl of complete DMEM before being seeded onto 177 \nthe EM grids at various cell densities. Following a 20  min incubation at room 178 \ntemperature, 2 ml of complete DMEM was added to each dish, and the cells were 179 \nincubated at 37 °C with 5% CO 2 for 20-24 h to promote optimal cell attachment and 180 \nspread. 181 \nPlunge freezing of cells on EM grids 182 \nAfter culture, the cells on the EM grids were plunge -frozen using a Leica EM GP 183 \nAutomatic plunge freezer (Leica Microsystems) in liquid ethane at -180 °C and 184 \ntransferred in liquid nitrogen. To facilitate blotting, 3 μl of 1X PBS was applied to the 185 \nside of the grid containing the cells, and 0.5 μl was applied to the back of the grid 186 \nwithin the humidifier chamber. The chamber was maintained at 90% humidity and 8 187 \n°C, with blotting times adjusted between 5 and 8 seconds for each sample. The grids 188 \nwere blotted using No.1 Whatman paper, then stored in liquid nitrogen. Subsequently, 189 \nclipped into C -rings and placed into autogrids for screening and data collection 190 \ncompatible with the FEI Titan Krios TEM (Thermo Fisher Scientific). 191 \nSample thinning by cryo-focussed ion milling-scanning EM (cryo-FIB-SEM)  192 \nThe lamellae generation was carried out at the Electron BioImaging Centre (eBIC) at 193 \nDiamond Light Source, utili sing the Scios cryo-FIB-SEM (Thermo Fisher Scientific) 194 \nand Aquilos cryo-FIB-SEM (Thermo Fisher Scientific). Lamellae were prepared using 195 \na focused gallium ion beam on a dual -beam focused ion beam -scanning electron 196 \nmicroscope (FIB-SEM) at a stage temperature of -180 °C. Cells located in the centr e 197 \nof the grid squares were selected for milling. To achieve uniform milling of the cells 198 \nduring the process, an organo-metallic platinum layer was applied prior to milling. This 199 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n10 \n \nwas followed by a thin layer, or splutter coat, of Pt to enhance SEM imaging. The rough 200 \nmilling was conducted in three gradual steps at a stage angle of 12° using a gallium 201 \nbeam, followed by a final fine milling step to achieve the desired nominal thickness of 202 \nthe lamellae. The final beam current used when milling was performed on SCIOS was 203 \n37 pA, while on Aquilos was 50 pA. Progress was monitored using SEM on the SCIOS 204 \nsystem (operating at 2.2-10 kV with ETD and T2 detectors) and on the Aquilos system 205 \n(operating at 2-5 kV with ETD detectors and Auto TEM).  206 \nSample vitrification by high-pressure freezing 207 \nSGR-NS5A-eGFP cells were vitrified using a high -pressure freezing method.  208 \nApproximately 1 x 106 cells per ml were resuspended in 100 µl of cryoprotectant (20% 209 \nw/v Dextran 40,000 in DMEM). The sample was placed into the 100 µm deep wells of 210 \nan Au type-A specimen carrier  (Leica, catalogue no.16770152) , ensuring the wells 211 \nwere slightly overfilled to prevent air bubbles, and then covered with the flat side of a 212 \nlipid-coated (L-⍺-Phosphatidylinecholine, Sigma, 61771 ) Au type -B carrier  (Leica, 213 \ncatalogue no.16770153 ). Excess liquid was absorbed with a filter paper, and the 214 \nsample was then high-pressure frozen at approximately 2100 bar and -190 °C for 300 215 \nms using a Leica EM ICE High-Pressure Freezer (Leica Microsystems). 216 \nCryogenic fluorescence microscopy 217 \nHigh-pressure frozen, cryo -preserved carriers and cryo -EM grids containing SGR -218 \nNS5A-eGFP cells were screened and imaged using a Leica THUNDER Imager EM 219 \ncryo CLEM (Leica Microsystems)  equipped with a HC PL APO 50x/0.9 NA cryo 220 \nobjective, an Orca Flash 4.0 V2 sCMOS camera (Hamamatsu Photonics), and a Solar 221 \nLight Engine (Lumencor). Z-stack images were acquired with a frame size of 2048 x 222 \n2048 pixels, at 30% intensity and an exposure time of 0.2 seconds, utilizing the LASX 223 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n11 \n \nsoftware (Leica Microsystems). The resulting images were processed using Fiji 224 \nsoftware (Schindelin et al., 2012).  225 \nSample thinning by cryo-ultramicrotomy 226 \nHigh pressure frozen samples were sectioned at cryogenic temperatures ( -160 °C) 227 \nusing a Leica UC7 equipped with an FC7 chamber , trimming (Diatome, Trim20), and 228 \ncryo-sectioning (Diatome, cryo immune, 3 mm ) Diamond knifes, and micro -229 \nmanipulators (Studer et al., 2014) . Images were captured with a stereomicroscope 230 \ninside the FC7 chamber and correlated with fluorescent images obtained from the 231 \nLeica Thunder Imager (Leica Microsystems) to precisely identify the regions for  cryo-232 \nET. A trapezoidal block of tissue measuring 150 x 100 x 40 µm was shaped  around 233 \nfluorescent targets, from which ribbons of either 70 or 40 nm thickness were produced. 234 \nThese ribbons were then adhered to Quantifoil R2/2, Cu 300 mesh grids (EMS) using 235 \nan electrostatic gun, after the grids had been glow discharged for 60 seconds at 30 236 \nmA by glow discharge (Easy Glow, Cressington).  237 \nImage correlation of cryo-EM and cryo-FM maps 238 \nMaps software (Thermo Fisher Scientific) was utilised to correlate fluorescent images 239 \non-the-fly at the TEM. Low magnification electron micrographs (atlas map: 125X, pixel 240 \nsize 819.2 Å, spot size: 7, illuminated area: 1100 µm) were aligned based on broken 241 \ngrid squares and distinctive patterns embedded in the centre of Quantifoil grids. At 242 \nmedium magnification (overview: 580X, pixel size 224.4 Å, spot size: 6, illuminated 243 \narea: 313 µm), specific grid squares were matched. At high magnifications (search 244 \nmap: 8700X, pixel size 28.8 Å, spot size: 10, illuminated area: 12 µm), holes in the 245 \ncarbon support film served as fiducial markers to facilitate correlation. The correlation 246 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n12 \n \nbetween the fluorescent images and electron micrographs after tomogram collection 247 \nwas achieved using the Matlab script Correlate.tex (Schorb and Briggs, 2014). 248 \nCryo-ET 249 \nCryo-ET was conducted using the FEI Titan Krios TEM (Thermo Fisher Scientific), 250 \noperated at 300 kV and equipped with a Falcon 4i direct electron detector and a 251 \nSelectris imaging filter (Thermo Fisher Scientific) in energy -filtered TEM (EFTEM) 252 \nnanoprobe mode.  253 \nTwo different acquisition schemes were employed for imaging lamellae. The first one, 254 \ninvolved a magnification of 42,000 X, corresponding to a pixel size of 3.0 Å at the 255 \nspecimen level, using Tomography 5.11 or 5.13 software (Thermo Fisher Scientific). 256 \nA dose-symmetric acquisition scheme  (Hagen et al., 2017)  was implemented from -257 \n60º to 60º in 2º increments, maintaining a constant electron dose of ~1.5 e−/Ų per 258 \nprojection, corresponding to a total dose of ~95 e−/Ų. The target defocus was 259 \nbetween -3 and -5 μm and an energy filter slit set to 10 eV was used. Individual 260 \nprojections were captured in counting mode through dose fractionation. Four frames 261 \nper projection were aligned and summed 'on-the-fly' using custom Python scripts that 262 \nutilized commands from the MotionCor2 (Zheng et al., 2017) and IMOD (Kremer et al., 263 \n1996) software packages. The second acquisition scheme was similar to the first one, 264 \nwith the following differences: the magnification employed was 64,000 X, with a 265 \ncorresponding pixel size of 1.9 Å. The electron dose was ~2.9 e−/Ų per projection, 266 \ncorresponding to a total dose of ~120 e−/Ų. The tilt range covered was from 71.2° to 267 \n-46.8° in 3° increments, starting at an angle of 12.14°. Eight frames per projection were 268 \ncollected. 269 \n 270 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n13 \n \nSimilarly, tilt series for the cryo-ultrathin sections were collected at a magnification of 271 \n53,000 X, with a corresponding pixel size of 2.40 Å. A dose -symmetric acquisition 272 \nmethod was employed from -60° to 60° in 3° increments, maintaining a constant 273 \nelectron dose of ~2.2 e−/Ų per projection, resulting in a total electron dose of ~91 274 \ne−/Ų. The target defocus ranged from -3 to -5 μm, and an energy filter slit of 10 eV 275 \nwas utilized. Six frames per projection were collected. 276 \nTomogram reconstruction  277 \nRaw images were motion corrected using MotionCor2 (Zheng et al., 2017) . The tilt 278 \nseries images were aligned and reconstructed via the eTOMO interface of the IMOD 279 \nsoftware package  (Kremer et al., 1996) . For lamellae dataset 1, tomograms were 280 \ngenerated using fiducial -less alignment. All tomograms were reconstructed with 281 \nweighted back-projection. For lamellae dataset 2 and the tomograms of cryo-ultrathin 282 \nsections, reconstruction was carried out using Aretomo software (Zheng et al., 2022). 283 \nAll tomograms were binned 4 times. Tomograms from datasets 1 and 2 are presented 284 \nafter filtering with 3D Gaussian blur function in Fiji. For the CEMOVIS dataset, images 285 \nare presented without filtering. 286 \nStatistical analysis on diameter of the vesicles 287 \nTo evaluate the size of each membranous structure, identifiable vesicles and cellular 288 \nfeatures were summarized for each tomogram based on the existing literature. The 289 \ndata on the lamellae were collected without specific correlative guidance for the NS5A-290 \neGFP foci, leading to the analysis of all measurable membrane structures (SMV, DMV, 291 \nMMV, MVB, LD) across 112 tomograms  (combined dataset) . The predominant 292 \nvesicles visible in the tomograms and measurable included SMVs, followed by DMVs, 293 \nLDs, MVBs, MMVs, and mitochondria. For each structure, both the minor and major 294 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n14 \n \naxes were measured using IMOD’s ‘measure’ function with the diameter calculated by 295 \naveraging these two measurements. The data w as exported to GraphPad Prism f or 296 \npresentation. 297 \n  298 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n15 \n \nResults 299 \nEstablishing a system to study HCV MW 300 \nMost ultrastructural studies examining HCV MW have utilised the JFH-1 virus or JFH-301 \n1-SGR-NS5A-eGFP, supporting its use for structural studies  (Ferraris et al., 2010; 302 \nRomero-Brey et al., 2012; Ferraris et al., 2013; Lee et al., 2019). NS5A plays a critical 303 \nrole in both replication and assembly of the virus and is distributed as puncta 304 \nthroughout the cytoplasm (Eyre et al., 2014), which makes it a key protein to track and 305 \nstudy HCV replication. 306 \nTo validate the use of SGR-NS5A-eGFP (Figure 1A) harbouring cells for subsequent 307 \ncryo-EM based analysis , the localisation of NS5A -eGFP in proximity to the LDs, as 308 \npreviously reported (Lee et al., 2019), was confirmed by confocal microscopy (Figure 309 \n1B). To confirm architectural differences between control Huh7 cells and those stably 310 \nharbouring the  SGR-NS5A-eGFP, resin -embedded cell samples were sectioned, 311 \nstained, and imaged by transmission electron microscope (TEM) prior to implementing 312 \na cryo-electron tomography (cryo-ET) workflow. Overall, noticeable differences were 313 \nobserved in the cellular architecture between control cells and cells harbouring SGR-314 \nNS5A-eGFP.  Whereas control cells lacked any evidence  of intracellular membrane 315 \nreorganisation, stably harbouring cells contained apparent MWs (Figures 1C and 1D). 316 \nTherefore, cells stably harbouring SGR-NS5A-eGFP were employed to establish in-317 \nsitu cryo-ET workflows and to gain knowledge into the organisation of the different 318 \ntypes of membranous vesicles present within the MW of HCV.  319 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n16 \n \n 320 \n  \nFigure 1: Huh7 stably harbouring SGR-NS5A-eGFP show membrane rearrangements. \nA) Schematic of the HCV SGR-NS5A-eGFP employed in this study. B) Confocal image of \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n17 \n \nHuh7 cells harbouring SGR -NS5A-eGFP. Cells were fixed with 4% formaldehyde . LDs \nstained with 1:1000 dilution of BODIPY (558/568) for 1 hour at room temperature and nuclei \nstained with ProLong gold antifade  mountant with DAPI. NS5A-eGFP (green), LDs (red) \nand nuclei of cells stained with DAPI (blue) are shown . Scale bar: 20 µm . C and D) \nRepresentative TEM of ultrathin sections of Huh7 cells (C) and Huh7 cells harbouring HCV \nSGR-NS5A-eGFP (D). MW: Membranous web, LD: Lipid droplets, Nu: Nucleus, ER: \nEndoplasmic reticulum. Scale bars: C1,5 µm; D1,4 µm; C2 and D2, 2 µm; C3 and D3, 1µm. \n 321 \nSample preparation workflow to generate lamellae by cryo-FIB for cryo-ET 322 \nNext, to gain insight into the architecture of the HCV MW in the SGR-NS5A-eGFP 323 \nharbouring cells in close-to-native conditions, two different approaches were explored: 324 \ncryo-focused ion beam m illing (cryo-FIB) and cryo-electron microscopy of vitreous 325 \nsections (CEMOVIS).  326 \nTo establish a cryo-FIB workflow, a homogenous population of cells harbouring SGR-327 \nNS5A-eGFP were fluorescently activated cell sort ed (FACS) and selected for the 328 \nbrightest eGFP intensities (Figure 2A). These cells were seeded on negatively glow-329 \ndischarged and fibronectin-functionalised gold grids (Figure 2B). Since cell adherence 330 \nwas variable on each grid , even after functionalisation , optimal cell distribution was 331 \nanalysed by widefield fluorescence microscopy before selecting grids for plunge 332 \nfreezing (Figure 2B and C). Subsequently, grids were screened by cryo -EM for ice 333 \nthickness and target cells suitable for cryo -FIB milling were selected  (Figure 2D ). 334 \nGenerally, the success rate of grids with optimum cell targets and ice thickness was ~ 335 \n50% per session. Cryo-FIB was then performed and  the presence of LDs in the 336 \nlamella, as observed through SEM imaging, confirmed that the lamella’s thickness was 337 \nsuitable for cryo-ET imaging (Figures 2E and F). 338 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n18 \n \n \n \nFigure 2: Schematic sample preparation workflow to generate lamellae by cryo -FIB \nfor cryo-ET of Huh7 cells stably harbouring SGR-NS5A-eGFP. Schematics (A, C and E) \nand images of workflow (B, D and F) to prepare samples for cryo-FIB and cryo-ET imaging \nof HCV MW. A) Schematic of sample preparation for grid preparation. Cell cultures of Huh7 \ncells stably harbouring SGR-NS5A-eGFP (left) were FACS sorted (centre) and cultured on \nglow-discharged and fibronectin-functionalised gold EM grids (right). B) The cell adherence \nand distribution on the grid was analysed by fluorescence microscopy. Scale bars: 1  mm \n(centre) and 0.5 mm (right).  C) Schematic of plunge freezing process . D) Ice thickness of \nplunge-frozen grids and identification of cell targets suitable for cryo-FIB were assessed by \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n19 \n \ncryo-EM. Scale bars: 100 µm. E) Schematic of cryo -FIB milling to generate lamella e for \ncryo-ET. F) SEM images showing the process of cryo -FIB: top, SEM ( F1) and ion ( F2) \nimages of a target cell prior to cryo -FIB; bottom, SEM image of a lamella in which LDs are \napparent (F3) and low magnification SEM image of a lamella (F4). Scale bars: C1, 30 µm; \nC2 and C3, 10 µm; and C4, 200 µm.  \n 339 \nQuantitative and qualitative analysis of HCV MW using the cryo-FIB/cryo-ET 340 \nworkflow 341 \nTo investigate the native architecture of HCV the different membranous vesicles within 342 \nthe MW , cryo-ET was applied to  the lamellae generated from SGR-NS5A-eGFP 343 \nharbouring cells. To locate regions for tilt-series acquisition, search maps were utilised 344 \n(Figure 3A), looking for areas containing MWs. A total of 112 reconstructed tomograms 345 \nfrom two datasets were analysed. Among these, 50% contained MVBs, 39% included 346 \nSMVs, 29% featured DMVs , 15% contained LDs, 12% included ER, 11% featured 347 \nMMVs, 9% exhibited extensive MW, and 5% contained mitochondria. 348 \nBased on the hypothesis that DMVs are the main sites of replication (Romero-Brey et 349 \nal., 2012; Romero-Brey and Bartenschlager, 2015), the initial investigation centred on 350 \nstudying the ultrastructure of DMVs and their surrounding environment. DMVs  were 351 \noften surrounded by  double-membrane tubules (DMTs ), SMVs, and MVBs . Most of 352 \nthe DMVs examined had a closed configuration (i.e. no pores connecting the inside of 353 \nthe DMVs to the cytoplasm were observed; Figure 3B-1), with only one instance (1 out 354 \nof 112 tomograms, consisted of two open DMVs) exhibiting an aperture facing towards 355 \nthe cytosol (Figure 3B-2).  356 \nAs an initial characterisation of the different vesicles, the diameter of all membranous 357 \nstructures that were clearly visible (n = 199) was measured (Figure 3C). The majority 358 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n20 \n \nof SMVs, DMVs and inner vesicles ( InVs, found within DMVs and SMVs)  had a 359 \ndiameter between 50 and 300 nm, whilst LDs displayed a wide r range of diameter , 360 \nranging from 100 to 500 nm. MVBs, the most abundant type of vesicles imaged in the 361 \ndatasets, were much larger and mostly above 400 nm in diameter . However, their 362 \nlarger size rendered them only partially visible within the tomograms, making it 363 \nchallenging to accurately measure their diameter. For this reason, the diameter of only 364 \n20 MVBs was measured. Finally, MMVs were scarce, observed only in 11 tomograms. 365 \nHowever, only 3 could be measured and had a diameter similar to that of MVBs. 366 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n21 \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n22 \n \nFigure 3: HCV membranous structures imaged by cryo-FIB/cryo-ET of Huh7 cells \nharbouring SGR-NS5A-eGFP.  \nA) Representative search map ( magnification 15,000X) of a lamella containing membranous \nstructures around LDs. An area highlighting specific structures is shown in the inset. Scale bars: \nsearch map, 2 µm; inset, 500 nm. B) Representative sections through reconstructed tomograms \nshowing the architecture of HCV MW. Scale bars: 200 nm. The white arrow indicates the opening \nof a DMV towards the cytosol.  C) Graph depicting the diameters of the 199 observed \nmembranous structures . SMV: Single membrane vesicle . DMV: Double membrane vesicle. \nMMV: Multi membrane vesicle. InV: Inner vesicle. LD: Lipid droplets. MVB: Multivesicular bodies. \nMito: Mitochondria. \n \nNext, differences in the general distribution of densities within DMVs and SMVs were 367 \nexamined. Most DMVs (92.8%) either contained faint densities that could be a result 368 \nof the inherent noise within cellular cryo -electron tomograms  or appeared empty  369 \n(Figure 4A-1,2). Only 7.14% DMVs contained patches of densities on the inside of the 370 \nmembrane that could potentially represent an assembly of the replicase component s 371 \n(Figure 4A-3, 4B-1). Finally, 17.8% DMV contained an InV (either a single or double 372 \nmembrane, Figure 4B-1,2,3).  373 \nOn the other hand, most SMVs (51.4%, Figure 4C-1, 4D-1) contained faint densities 374 \nor appeared empty  making it difficult to co nfirm their contents , 28.15% contained 375 \nhomogeneous densities throughout the vesicle (Figure 4 C-2). Only 2.9% SMV 376 \ncontained patches of densities (Figure 4 C-3), which could indicate the presence of 377 \neither cellular or viral proteins . Similar to  DMVs, 19.4% of SMVs contained InVs; 378 \nFigures 4D-1,2,3). Out of 31 InVs measured for diameter (6 inside DMVs and 20 inside 379 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n23 \n \nSMVs), 38% of them contained densities, 32%  of them could not be confirmed but 380 \npossibly have densities, 25% of them appeared empty.  381 \nPrevious reports by i mmunogold labelling suggested that both NS3 and NS5A 382 \nprimarily labelled rER and SMVs with 50-70 nm diameter and to a lesser extent on 383 \nDMVs (Romero-Brey et al., 2012)  and th us at least some of these densities could 384 \npotentially be NS3 and NS5A proteins. Overall, this analysis suggests that DMVs and 385 \nSMVs are similar in terms of content, but a larger percentage of SMVs contained 386 \ninternal densities, which might correspond to viral and/or cellular proteins. Strikingly, 387 \nthe only membranous structure that contained a significant percentage of inner 388 \ndensities were InVs. 389 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n24 \n \n  \nFigure 4: Differences in densities present within DMVs and SMVs . A) Representative \ntomographic slice s showing the differences in the content within DMV s. B) Representative \ntomographic slices showing InVs within DMVs and their contents. C) Representative tomographic \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n25 \n \nslices showing the differences in the content within SMVs. D) Representative tomographic slices \nshowing InVs within the SMV and their content. Scale bars: A1,2; B2,3; C1,2 and D1,2,3: 100 nm, \nA3, B1and C-3: 160 nm. White arrows indicate densities present within the vesicles. SMVs: Single \nmembrane vesicles. DMVs: Double membrane vesicles. InV- Inner vesicle \n 390 \nNS5A-eGFP preferentially locates around MMVs 391 \nSo far, a clear organisation of the replicase machinery within specific structures has 392 \nnot been established using the cryo-FIB milled cryo-ET datasets, due to limitations in 393 \nutilising NS5A -eGFP fluorescence for guided tilt -series collection on the  lamellae. 394 \nTherefore, an alternative workflow  was developed , incorporating CEMOVIS . This 395 \napproach aimed to gather evidence regarding the types of structures close to NS5A, 396 \npotentially housing the replication complex in cells harbouring SGR-NS5A-eGFP 397 \n(Figure 5) . An advantage of this approach is its ability to employ a heterogeneous 398 \npopulation of SGR-NS5A-eGFP cells, thereby eliminating the need for FACS sorting. 399 \nNS5A-eGFP puncta are detectable in cryo-ultrathin sections following cryo-400 \nultramicrotomy and can thus serve as markers for guided tilt-series acquisition. 401 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n26 \n \n \nFigure 5: Schematic sample preparation workflow to generate cryo-ultrathin sections \nby CEMOVIS of Huh7 cells stably harbouring SGR-NS5A-eGFP.  \nA) Cells were initially mixed with a cryoprotectant. B) Subsequently, cells were added to \ngold carriers and high-pressure frozen and imaged by cryo-fluorescence microscopy (C). \nScale bar : 500 µm. Finally, cells were thinned by CEMOVIS (D) to generate ultrathin \nsections (E). Scale bar: 2 µm.  \n 402 \nUsing this approach, ultrathin sections of varying thicknesses (100 nm, 70 nm, and 40 403 \nnm) were evaluated, with the 40 nm sections displaying clear cellular features, 404 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n27 \n \nwhereas the thicker sections exhibited low contrast . Therefore, 12 tilt-series were 405 \ncollected from a single 40 nm section , guided by NS5A-eGFP foci and subsequently 406 \ncorrelated (Figure 6A). 407 \nReconstructed tomograms from 12 NS5A -eGFP tilt-series contained 83.3% MMVs, 408 \n33.3% ER, 25% DMVs, 25% mitochondria, 16.6% LD and 8.3% MVB. 12 tomograms 409 \nprimarily revealed 39 MMVs with notable densities inside (4 of which are illustrated in 410 \nFigure 6B-E) and only 3 DMV identified. Similar densities were observed within and 411 \naround DMVs as noted in the cryo-FIB-milled dataset. Interestingly, the proportion of 412 \nDMVs and MMVs was reversed between tomograms obtained using the CEMOVIS 413 \nand cryo-FIB workflows. In the cryo -FIB datasets, which were not guided by NS5A -414 \neGFP presence, DMVs were more prevalent, comprising 13% of the vesicles (2 7 415 \nDMVs out of 199 structures measured). In contrast, MMVs dominated in the CEMOVIS 416 \ndatasets, comprising 80% of the vesicles  (39 MMVs out of 49 structures identified) , 417 \nwhich were exclusively collected in regions exhibiting NS5A-eGFP signals. Strikingly, 418 \none of the MMVs displayed a heterogeneous arrangement of proteins, potentially 419 \nindicative of a replication complex assembly (Figure 6E). Notably, this represents the 420 \nmost organised assembly of cryo-densities observed across all datasets, underscoring 421 \nthe value of fluorescence-guided tilt-series collection on thin cryo-specimens.  422 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n28 \n \n \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n29 \n \nFigure 6: NS5A-eGFP-guided acquisition of HCV MW observed on Huh7 Cells stably \nexpressing HCV SGR-NS5A-eGFP by CEMOVIS and cryo-ET.  \nA) Left, low magnification search map (125 X) of a 40 nm cryo-ultrathin ribbon with regions \nof NS5A-eGFP signal shown in white boxes. Centre and right, the area of NS5A-eGFP foci \nused for tilt-series collection is shown with green circles and labelled with designated letters \nthat correspond to the search maps and tomograms below. Scale bar: 2 µm. B-E) Left, cryo-\nEM search map (580 X) with highlighted position by a white circle where the tilt series was \nacquired. Centre and right, tomographic sections at low (centre) and high magnification \n(right) of membranous structures observed in the tomograms acquired at the specified \nNS5A-eGFP signals. Scale bars left, 100 nm; centre, 200 nm; right, 100 nm. MMVs: Multi \nmembrane vesicles. DMV: Double membrane vesicle. \n 423 \nDiscussion  424 \nThe use of direct-acting antivirals (DAAs) has dramatically improved the life of HCV-425 \ninfected patients, resulting in a reduction in the global burden of HCV from 170 million 426 \nindividuals 10 years ago to the current estimate of 50 million (WHO, 2024). The targets 427 \nfor DAAs (NS3 protease, NS5A and NS5B RNA -dependent RNA polymerase) are all 428 \ndirectly involved in virus genome replication . It is thus important to understand th e 429 \nmolecular details of this process as this will shed light on the mode of action of DAAs 430 \nand may contribute to an understanding of DAA resistance  which is becoming 431 \nincreasingly common. In this regard one important avenue of research has focused on 432 \nunderstanding how HCV modifies the host membranes to establish its replication 433 \ncomplex. From 2002 to 2019 a number of research groups explored the membranous 434 \nstructures within the MW in relation to HCV infection or the presence of SGR, using a 435 \nmix of light and electron microscopy techniques (Ferraris et al., 2010; Romero-Brey et 436 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n30 \n \nal., 2012; Paul et al., 2014; Berger et al., 2014; Mohl et al., 2016; Pérez -Berná et al., 437 \n2016; Lee et al., 2019). However, these studies relied on chemical fixatives and resin-438 \nembedded sections, limiting direct visualisation of key replication components such as 439 \ndsRNA and protein arrangements within membranous structures. Furthermore, even 440 \nthough DMVs have been identified as the most probable structures to harbour the 441 \nreplication complex (Romero-Brey et al., 2012), the location of the replication complex 442 \nis still under debate  based on direct visualisation of assembly of viral and cellular 443 \nproteins. Here, we aimed to develop a cryo-ET workflow to visualise HCV MW in close-444 \nto-native conditions in the absence of chemical fixation, and to explore the localisation 445 \nof the HCV replication complex within it. A detailed workflow was developed, spanning 446 \nfrom sample preparation to data analysis, incorporating the latest cryo-ET techniques, 447 \nand t wo cryogenic sample preparation techniques were established  for an in-situ 448 \ninvestigation of the membranous structures in cells containing an SGR (HCV genotype 449 \n2a): cryo -FIB milling (Lam and Villa, 2021)  and CEMOVIS (Chlanda and Sachse, 450 \n2014). 451 \nHCV-induced host membrane rearrangements are driven by viral proteins. In SGR 452 \nharbouring cells host membrane modifications similar to those in HCV -infected cells 453 \nwere observed (Romero-Brey et al., 2012). To track viral proteins, we utilised an SGR-454 \nNS5A-eGFP replicon. This construct is the only HCV system with a fluorescently 455 \ntagged non-structural protein. However, one limitation of this approach is that NS5A 456 \nis involved in both replication and assembly (Eyre et al., 2014), suggesting that some 457 \nobserved NS5A-eGFP signals may not be exclusively associated with replication sites. 458 \nDatasets acquired using cryo -FIB milling followed by cryo -ET (Wagner et al., 2020)  459 \nwithout fluorescence guidance enabled the exploration of the MW induced by the 460 \nSGR-NS5A-eGFP in Huh7 cells. Analysis of these datasets revealed numerous DMVs 461 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n31 \n \nand SMVs. While most DMVs appeared empty or contained densities at background 462 \nlevels, SMVs frequently exhibited either patchy densities or were entirely filled. 463 \nAdditionally, both DMVs and SMVs occasionally contained in ner vesicles (InVs) with 464 \ndensities present within . These InVs have been previously described as self -465 \ninvaginations of SMVs (Romero-Brey et al., 2012) or vesicles in cluster (ViCs) (Ferraris 466 \net al., 2013). However, they may also represent exosomes containing dsRNA or non-467 \nstructural proteins that fuse with larger vesicles, potentially serving as replication sites 468 \n(Ramakrishnaiah et al., 2013; Bukong et al., 2014; Yin et al., 2022) . Given that 469 \npositive-sense RNA virus replication sites are expected to contain viral replicase and 470 \nRNA (Wolff et al., 2020) , our findings suggest that SMVs and InVs may serve as 471 \nprimary sites for HCV SGR replication. This is further supported by EM studies of 472 \nchronically infected HCV patient cells, which identified SMV s and not DMV around 473 \nLDs and the ER (Blanchard and Roingeard, 2018). 474 \nIn any case, the m ost frequently observed vesicles in the cryo-FIB/cryo-ET dataset 475 \nwere the MVBs, consistent with EM analysis of replicon cells transfected with NS5A-476 \nmCherry (Grünvogel et al., 2018) , suggesting their involvement in the process of 477 \nreplication. The aggregation of MVBs, found primarily near SMVs, DMVs, and LDs, 478 \nwith diameter sizes ranging from 400–800 nm (though most exceeded 1000 nm) were 479 \npreviously described as single -membrane compartments containing multiple circular 480 \nunits with dense cores (Ferraris et al., 2010). The presence of densities within SMVs 481 \nand DMVs within MVB also aligns with the previous observations of NS5A -mCherry 482 \nlocalised inside MVBs (Grünvogel et al., 2018)  and with the dense lumen within 483 \nconcentric units (Ferraris et al., 2010). The smaller SMVs (30 –150 nm) observed 484 \nwithin MVBs resemble exosomes in size. Previous studies have demonstrated that 485 \nexosomes derived from HCV-infected cells contain viral RNA, proteins, and complete 486 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n32 \n \nvirions (Yin et al., 2022). Notably, these exosomes facilitate the transfer of HCV RNA 487 \nto uninfected cells, even in the presence of neutralising antibodies  (Ramakrishnaiah 488 \net al., 2013). This suggests that MVBs play a role in HCV RNA dissemination. On rare 489 \ninstances, tomograms captured SMVs invaginating into MVBs (not shown), supporting 490 \nthe hypothesis that MVBs may transport vesicles containing replicase machinery or 491 \ndsRNA to neighbouring cells  (Grünvogel et al., 2018) . Perhaps, the accumulation of 492 \nMVBs in SGR harbouring cells is due to the lack of viral assembly in this system, thus 493 \ndepriving nascent genomic RNA of a ‘final destination’? Nonetheless, this observation 494 \nsuggests a potential role for MVBs in HCV replication 495 \nPrevious immunofluorescence studies indicated that HCV replication  vesicle 496 \nmembranes originate from the ER but also colocalise with markers of early and late 497 \nendosomes, coat protein complex (COP) vesicles, mitochondria, and LDs  (Romero-498 \nBrey et al., 2012) , as well as lysosomes (Matsui et al., 2021) . The measured SMV 499 \ndiameter (50–300 nm) aligns with that of endosomes (100 –500 nm) and lysosomes 500 \n(200–300 nm). If these SMVs are derived from early endosomes, their internal 501 \ndensities may represent NS3 and NS5A, while surface -exposed densities may 502 \ncorrespond to GFP-Rab21 (Romero-Brey et al., 2012). Alternatively, if they originate 503 \nfrom lysosomes, internal densities may include NS5A and LAMP -2A (Matsui et al., 504 \n2021). 505 \nNotably, in CEMOVIS datasets guided by NS5A -eGFP fluorescence, M MV 506 \naccumulation was observed. One tomogram revealed densities arranged in a regular 507 \npattern, potentially representing replicase machinery assembly on the inner leaflet of 508 \nan MMV. Overall, this study contributes to the development of cryo in-situ workflows 509 \nfor investigating HCV-induced membranous structures. Our findings show that DMVs 510 \nappear mostly empty, while SMVs and InVs contain significant densities. Additionally, 511 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n33 \n \nMVBs emerge as the most abundant vesicles in MW imaging  at random, reinforcing 512 \ntheir potential role in HCV replication and RNA transfer.  The most abundant vesicles 513 \namong NS5A-eGFP fluorescence-guided cryo-ET were MMVs, suggesting that  are 514 \ninvolved in replication in cells harbouring SGR-NS5A-eGFP. 515 \nIn conclusion, these findings provide new insights into the spatial organi sation of NS 516 \nor cellular proteins within membranous structures in replicon -transfected cells, 517 \nrevealing structural details previously inaccessible through conventional electron 518 \nmicroscopy. As cryo-ET continues to evolve with advancing computational techniques, 519 \nit will further refine our understanding of NS protein interactions with cellular proteins, 520 \nand HCV RNA genome organisation within these membranous structures. 521 \n 522 \nAcknowledgements 523 \nUMS was supported by a Wellcome  PhD studentship (222370/Z/21/Z). HCV studies 524 \nin the MH laboratory w ere supported by a Wellcome Investigator Award 525 \n(096670/Z/11/Z) and an MRC project grant (MR/S001026/1) . JF was supported by 526 \ngrant PID2023-149259NB-I00, funded by MICIU/AEI/10.13039/501100011033 and by 527 \n“ERDF A way of making Europe” . The funders had no role in study design, data 528 \ncollection and analysis, decision to publish, or preparation of the manuscript.  529 \nWe acknowledge the technical contributions of the University of Leeds  Bioimaging 530 \nFacility, especially Dr Ruth Hughes and Dr Sally Boxall, and the Astbury Biostructure 531 \nLaboratory Electron Microscopy  facility, especially Mr Martin Fuller , Dr Rebecca 532 \nThompson; and the Electron BioImaging Centre (eBIC) at Diamond Light Source, 533 \nespecially Dr James Gilchrist. 534 \n 535 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint \n\nHCV replication imaged in close-to-native conditions  \n \n34 \n \nAuthor Contributions 536 \nUMS, JF and MH planned the study. UMS, TJO’S, and YH performed the experiments. 537 \nUMS and JF analysed the data. UMS, JF and MH wrote the main manuscript text. All 538 \nauthors reviewed the manuscript. 539 \nCompeting interests  540 \nThe authors declare no competing interests. 541 \nData availability 542 \nThe datasets generated during and/or analysed during the current study are available 543 \nfrom the corresponding authors on reasonable request. 544 \n  545 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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Nature Methods. 14(4), pp.331–332. 697 \n  698 \n 699 \n.CC-BY 4.0 International licensemade available under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is \nThe copyright holder for this preprintthis version posted April 24, 2025. ; https://doi.org/10.1101/2025.04.24.650446doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}