ER-shaping atlastin proteins act as central hubs to promote flavivirus replication and virion assembly

Abstract

Flaviviruses, including dengue virus and Zika virus, extensively remodel the cellular endomembrane network to generate replication organelles that promote viral genome replication and virus production. However, it remains unclear how these membranes and associated cellular proteins act during the virus cycle. Here, we show that atlastins (ATLs), a subset of ER resident proteins involved in neurodegenerative diseases, have dichotomous effects on flaviviruses-with ATL2 depletion leading to replication organelle defects, and ATL3 depletion to changes in virus production pathways. We characterized non-conserved functional domains in ATL paralogues and show that the ATL interactome is profoundly reprogrammed following dengue virus infection. Screen analysis confirmed non-redundant ATL functions and identified a specific role for ATL3, and its interactor ARF4, in vesicle trafficking and virion maturation. Our data identify ATLs as central hubs targeted by flaviviruses to establish their replication organelle and to achieve efficient virion maturation and secretion.

Extended Data Figure 1. ER protein depletion in virus infection.

a, Phylogenetic analysis of metazoan ATL proteins collected from a subset of publicly available metazoan predicted proteomes. ATL sequences were aligned with MUSCLE and manually trimmed into a 494-site alignment. Phylogenies were reconstructed, and node support values were calculated using MrBayes for posterior probability and RAxML for maximum likelihood and presented as inset (MrBayes/RAxML). The MrBayes tree topology is shown. Scale bar: number of estimated substitutions per site. For genomes used see SI Table 7 b-h, A549 cells were transduced with constructs encoding for shRNAs (b-e, and g-h) or transfected with siRNA (f) targeting indicated mRNA transcripts, or a non-targeting (NT) shRNA or siRNA. b-d, 96 h post transduction, mRNA levels, protein levels and cell viability were evaluated. b, ATLs mRNA transcript levels were evaluated by RT-qPCR and values corrected using HPRT. Graphs show average percent change compared to NT shRNA for 3 independent experiments. c, ATL protein levels evaluated by western blot. Graph shows average protein levels compared to NT shRNA treated cells from 3 experimental replicates. d, Graph shows mean cell viability as percent survival compared to NT shRNA treated cells. e, 48 h after transduction cells were infected with DV (MOI=1) for 48 h. DV titers were determined by PFU assay. Graph show average fold change in PFU/mL (titers). * and ** - p values lower than 0.05 or 0.01, respectively, determined using one-way ANOVA with a Dunnett’s multiple comparison analysis. RTN, reticulon; LNP, lunapark. f, 96 h post transduction, cells were lysed for RNA analysis. Graph shows average percent change vs. NTshRNA for 3 independent experiments g-h, 48 h after transduction cells were infected with DV or RVFV (MOI=1) for 48 h. DV titers were evaluated using a PFU assay and RVFV replication was determined by luciferase assay. Graphs show average fold change in PFU/mL (titers) or average fold change in RLU/mL (replication) relative to NT shRNA expressing cells for three independent experiments. For all graphs, error bars show SEM and N= ≥ 3 biological replicates.

Extended Data Figure 2. Virus protein localization in ATL depleted cells.

48 h after lentiviral transduction of shRNA expression constructs, cells were infected with DV for 48 h. Cells were fixed with paraformaldehyde and viewed by immunofluorescence microscopy using LipidTOX or antibodies directed against capsid, Env, NS4B, NS3, Climp63 or RTN3. Scale bars, 10 μm, inset 5 μm.

Extended Data Figure 3. Effects of ATL depletion on virus-induced membrane alterations.

Cells were transduced with constructs encoding for shRNAs directed against ATL2 or ATL3, or a NT shRNA. a, 48 h after transduction, cells were infected with DV and 48 h later fixed and processed for viewing by TEM. Invaginated vesicles (yellow dots) and viral particles (blue dots) were determined by counting using Fiji software. Inserts display magnified views of the boxed area (scale bars, 2 μm; inset, 200 nm). Note the accumulation of ER tubular networks at the cell periphery in ATL3 depleted cells. b-f, Huh7-Lunet cells stably expressing the bacterial T7 RNA polymerase were transduced for 48 h, followed by transfection with a construct encoding for the viral polyprotein and containing the 3’ untranslated region (UTR) of the DV genome (panel b). c, 24 h post transfection, cells were fixed and stained with antibodies specific to the viral NS3 protein and visualized by confocal light microscopy. Scale bars, 10 μm. d, Graph shows the average relative fluorescence signal of NS3 in shRNA transduced cells. N=7 independent samples. e, Representative EM images of polyprotein expressing cells from a total of 3 independent experiments. Scale bars, 200nm. f, VPs in each cell were counted and mean values are represented in the graph (N=23 cells, error bars represent SEM). For all graphs * and ** represent p values lower than 0.05 or 0.01, respectively as determined by 2-tailed T-test.

Extended Data Figure 4. ATLs associate with distinct DV proteins.

a-c, A549 cells stably expressing HA-ATL2 or HA-ATL3 were infected with DV for 48 h. Cells were then fixed and stained with antibodies directed against NS3 or Env and the HA epitope, respectively. Scale bars, 10 μm. d, Pearson’s colocalization coefficients were calculated for cells from panels a-c. The graph shows the average value from 20 cells for each condition. Error bars, SEM. e, A549 cells stably expressing HA-ATL2, HA-ATL3, HA-CANX, or an empty plasmid were infected with DV for 48 h. Cells were lysed and HA-tagged proteins were immunoprecipitated with anti-HA beads. Inputs and precipitated proteins were analyzed by western blot using NS2B- and NS4B-specific antibodies. Breaks between adjacent blots indicate lanes not relevant to the experiment were removed. f-h, A549 cells stably expressing HA-ATL3 were infected with DV for 48 h. Cells were fixed and stained with antibodies directed against virus proteins or dsRNA (RED) and the HA epitope (green). Pearson’s colocalization coefficients for merge images are given in the top right corners. Images were taken using an Abberior instruments STED microscope. Scale bars, confocal 10 μm, STED 1 μm, inset 100 nm. i, Average Pearson’s colocalization coefficients were calculated for the fluorescent signal corresponding to the HA-tagged ATL3 compared to those from the indicated viral proteins in STED microscopy images. Graph shows the average Pearson’s colocalization coefficients calculated for 10 cells. Error bars, SEM.

Extended Data Figure 5. Production and testing of endogenously tagged ATL3.

a, Schematic representation of the cloning strategy used for endogenous tagging of ATL3. NVD, N-terminal variable domain; 3HB, three-bundle Helix; TM, transmembrane region; CTA, C-terminal amphipathic-helix domain. The tagging cassette includes the 11^th beta-strand of GFP (GFP11) and the FLAG-tag. b, Individual ATL3-ET (Endogenously Tagged) cell clones were lysed and lysates were analysed by western blot using the indicated antibodies. N=2 biological replicates. c, Cells expressing the endogenously tagged ATL3 were fixed and stained for FLAG or the ER marker PDI. Fluorescence signals specific for antibodies or GFP were visualized by confocal microscopy. Scale bar, 10 μm. d-f, A549 cells expressing endogenously-tagged ATL3 were infected with DV for 48 h. d, Cells were fixed and stained with NS3 (red) or FLAG-specific antibodies (green). Scale bars, 10 μm. e, Cells were lysed and FLAG-tagged proteins were precipitated with anti-FLAG magnetic beads. Inputs and captured protein complexes were evaluated by western blot using antibodies of given specificities. Actin served as loading and specificity control. Breaks between adjacent blots indicate lanes not relevant to the experiment were removed. N=2 biological replicates. f, Titers of infectious extracellular DV were determined by PFU assay. Graph shows the average fold change in PFU/mL compared to wild type cells for CRISPR/Cas9 control (Ctrl) or ATL3-ET cells over 3 biological replicates. Error bars, SEM.

Extended Data Figure 6. Production of ATL KO cells and effects of ATL mutations on viral replication and subcellular localization.

a-e, A549 cells were transduced with vectors expressing CRISPR/CAS9 as well as a guide RNA directed towards ATL1, ATL2, ATL3, or a non-target guide RNA (Ctrl). a, Knock out or control cells were lysed and protein levels were determined by western blot using given antibodies. GapDH served as loading control. b, Knock out or control cell pools were infected with ZV or DV for 48 h followed by quantification of extracellular virus titers. Graphs show average fold change in PFU/mL for each cell line compared to control cells over 3 independent experiments. ** and *** represent p values lower than 0.01 or 0.001, respectively as determined by 2-tailed T-test. c-e, Knockout or control cell pools were transduced with lentiviruses encoding for the ATL variant given on the bottom of each. c-d, 72 h after transduction, cell viability was determined using celltiter glow measuring intracellular ATP levels. Graphs show the average fold change in cell viability compared to control A549 cells. Lower dashed line shows the cut off of 80% viability. N=3 biological replicates. e, 24 h after transduction, cells were infected with DV for 48 h followed by evaluation of virus production using PFU assay. Graph shows the average PFU/mL as fold change compared to ctrl cells for 3 independent experiments. Lower dashed line indicates the difference between ATL3 KO cells and ctrl cells, both transduced with an empty plasmid. For all graphs, error bars show SEM. f-g, A549 cells were transduced with constructs encoding for the indicated ATL variants. 72 h after transduction, cells were fixed and stained with antibodies directed against the HA epitope (green) or the ER marker reticulon 3 (RTN3; red). Scale bars, 10 μm.

Extended Data Figure 7. ATL2 and ATL3 overexpression does not alter the cellular proteome.

Proteomic analysis of A549 cells stably expressing HA-ATL2, HA-ATL3, HA-CANX, or an empty plasmid. a, Heat map of log2-transformed LFQ intensities for each individual replicate in rainbow colours (see colour scale). b-c, Volcano plots of the p values vs. the log2 protein abundance differences between HA-tagged ATL2- and ATL3-overexpressing cells compared to HA-Calnexin (CANX) overexpressig cells, with proteins outside the significance lines highlighted (unadjusted two-sided t-test. Permutation based FDR < 0.05, S0 = 1, p<0.05). N=4 independent experiments. For Raw data see Source Data Table 1.

Extended Data Figure 8. ATL interactome and shRNA screen.

a-b, The scatter plot displays ATL2 (a) or ATL3 (b) specific interactors (compared to calnexin (CANX)) in both infected and uninfected cells. Schematics of the variables compared are shown in the bottom right of each scatter plot. Significantly enriched or depleted proteins are shown in red (N = 4 independent experiments. Welch’s T-test unadjusted two-sided P ≤ 0.05; |log₂(fold-change)| ≥ 1). c) Heat map showing imputed log2-transformed iBAQ intensities for each individual replicate in rainbow colors (see color scale). Only the bait proteins and the selected cellular interaction partners used for the RNAi screen are depicted in the plot. d, Knockout or control cells were transduced with lentivirus encoding for shRNAs (3/gene) targeting the genes specified in the left of the panel. 72 h after transduction, cell viability was tested. Graphs show the average change in cell viability, as determined by intracellular ATP quantification, for each treatment compared to control cells that were transduced with the non-target shRNA vectors. N=3 independent experiments. Error bars, SEM e, A549 cells stably expressing HA-ATL2, HA-ATL3, HA-CANX (calnexin) or transduced with the empty vector were infected with DV for 48 h. Cells were lysed and HA-tagged proteins were captured with anti-HA beads. Inputs and precipitated protein were determined by western blot and probing for the indicated proteins. Red numbers below the ARF4 panel indicate efficiency of ARF4 pulldown compared to bait protein over an average of 3 experiments. Breaks between adjacent blots indicate non-relevant lanes were removed.

Extended Data Figure 9. Effects of ATL3 depletion on virus particle maturation and the secretory pathway.

a, Cells were transduced with lentiviruses encoding for ARF4, ARF5 or non-targeting (NT) shRNAs. 72 h later RNA levels were quantified by RT-qPCR. Shown is the average fold change in viral RNA levels, corrected for HRPT. b-c, Cells were transduced with lentiviruses encoding for ATL2, ATL3 or NT shRNAs. b, After 72 h cells were transfected with a construct encoding for Gaussia luciferase and luciferase secretion was measured over 10 h. Graph shows the average levels of secreted luciferase compared to the 0 h time point. c, 72 h post transduction, cells were transfected with VSV-G_ts045_GFP and 8 h later incubated at 40°C. 16 h later temperature was lowered to 32°C and cells were imaged by confocal microscopy. Graph shows the means and SEM of perinuclear fluorescence intensity distribution for each condition. d-h, A549 cells were transduced with constructs of given specificities and cultured for 48 h. d, Cells were infected with ZV, and 48 h later viral RNA levels were determined by RT-qPCR (left panel). Intracellular viral RNA levels were corrected for HRPT. Titers of infectious virus contained in cell lysates and culture supernatants were determined using a PFU assay (right panel). For both panels, average fold changes are shown. e, Levels of prM and NS1 released from DV infected cells were calculated by quantifying western blots using Fiji software. Values were normalized to NT shRNA transduced cells (horizontal line). f, Cells were infected with DV for 48 h. RNA levels were determined by RT-qPCR; graph shows average fold change. g-h, Extracellular proteins were evaluated using western blot. h, Levels of prM released from DV infected cells were calculated using Fiji software. Values are displayed relative to NT shRNA transduced cells. All graphs show means and SEM derived from an average of 3 independent experiments. Significance was determined relative to NT shRNA transduced cells. * or ** represent p values < 0.05 or 0.01, respectively as determined by one-way ANOVA with a Dunnett’s multiple comparison analysis.

Extended Data Figure 10. Effects of ATL3 depletion on specific host protein localization.

A549 cells were transduced with constructs encoding for shRNA directed against the indicated gene, or a NT shRNA. a, 48 h post transduction cells were infected with DV for 48 h. Cells were then fixed and the indicated proteins or structures were visualized by immune staining and confocal microscopy. b-e, 96 h after transduction, cells were fixed and stained with the antibodies of given specificity. After incubation with secondary antibody fluorescence signal was visualized by confocal microscopy. f, shRNA transduced cells expressing the furin reporter protein CD4-Fu were fixed 72 h after transduction. The subcellular localization of the furin reporter was evaluated using immunofluorescence staining and confocal microscopy. Nuclear DNA was stained with DAPI. All scale bars, 10 μm. g, Transduced cells expressing the furin reporter protein were fixed 72 h after transduction and stained with anti-CD4 antibodies. The average total fluorescence levels of CD4-Fu were determined for ≥50 ctrl or ATL3 KD cells. Error bars, SEM.

Figure 1. Impact of atlastin depletion on flavivrus replication.

A549 cells were transduced with the indicated shRNA constructs for 48 h followed by infection with DV (DV-2), ZV (strain H/PF/2013) or WNV (strain NY99) for 48 h. Virus titers (a) and intracellular viral RNA (b) were quantified, protein/dsRNA localization was determined (c-d). a, Graphs show the mean fold change in PFU/mL relative to the non-target (NT) shRNA cells (set to 1), error bars show SEM. N=4 biological replicates. b, Graphs show the mean fold change in viral RNA levels relative to the NT shRNA cells (set to 1), error bars show SEM. N=4 biological replicates. For a and b statistical significance was determined using one-way ANOVA with a Dunnett’s multiple comparison analysis. * , ** , and *** represent p values < 0.05, 0.01 or 0.001, respectively. c-d, Cells were fixed with PFA and viewed by immunofluorescence microscopy using antibodies of given specificities. Images are representative of 3 independent experiments. Scale bars, 10μm. e, Cells were fixed and processed for viewing by TEM. Vesicle packets (VP) and virions (Vi) are highlighted. A VP was defined as array of >2 connected vesicles residing at the same of the ER lumen. Yellow boxes show the region of magnification displayed on the bottom. Images are representative of 3 biological replicates. Scale bars, 200 nm. f, Numbers of virions and vesicle packets were calculated for each condition (N=7 cells/condition). Graphs show the mean number per cell counted, error bars represent SEM. g, Graph shows the mean diameter of vesicles contained in VPs for each treatment. N=200 vesicles for each condition. h, Graph shows the mean distribution of VPs within shRNA transduced cells as measured from a central focus (N=6 cells/treatment). Box plots, box shows 25^th-75^th percentile; whiskers show min to max; line shows the mean value. For f, g, and h, ** and *** represent p values < 0.01 or 0.001, respectively as determined by 2-tailed T-test.

Figure 2. Atlastins associate with DV proteins.

a-b, A549 cells stably expressing HA-ATL2, HA-ATL3, HA-Calnexin (CANX) or an empty plasmid were infected with DV for 48 h. Cells were lysed and HA-tagged proteins were immunoprecipitated with anti-HA beads. Inputs and precipitated proteins were determined by western blot and probing for the indicated proteins. GapDH served as loading and specificity control. Panels are representative of 3 biological replicates. Breaks between adjacent blots indicate lanes not relevant to the experiment were removed. c, A549-derived cells expressing endogenously tagged ATL3 were grown in Mattek gridded dishes and infected with DV. After 48 h, cells were fixed with paraformaldehyde and glutaraldehyde followed by lipidTOX staining to visualize lipid droplets (LD) and imaging by confocal microscopy. Cells were then immediately embedded and processed for EM imaging using grid numbers as a reference. Correlation of light microscopy and EM images was done with Fiji software using lipid droplets as correlation marker. Images are representative of 3 independent samples. Scale bars: panel ii, 1μm; panels 1 - 4, 200 nm. d, A549 cells stably expressing HA-ATL3 were infected with DV for 48 h. Cells were processed using the Tokuyasu protocol and labeled with HA-epitope targeting 10 nm gold beads. For EM images, VP denotes areas containing vesicle packets and Vi denotes regions containing virions. Scale bars, 100 nm. e, Quantification of the number of virions or vesicle packets containing proximal immune-gold staining pertaining to either a control antibody or an HA specific antibody. Graph shows the mean value for 6 cells from 2 biological replicates. For each sample, >100 Vi or VP structures were counted. ***, p values < 0.001 as determined by 2-tailed T-Test.

Figure 3. Mapping of functional domains of ATLs involved in the DV replication cycle.

a, Sequence comparison of human ATL1, ATL2, and ATL3 proteins, showing the amino acid sequence identity of each annotated domain. Sequences were aligned using the NCBI BLAST server. NVD, N-terminal variable domain; 3HB, three-bundle Helix; TM, transmembrane region; CTA, C-terminal amphipathic-helix domain. b, Schematic representation of ATL2 and ATL3 mutant constructs used for rescue experiments. Names of expressed proteins are given on the left. c-f, ATL2 knockout or control cells were transduced with lentivirus encoding for the ATL variant given on the bottom of each panel. 24 h after transduction, cells were infected with DV-R2A (c and e) or synZV-R2A (d and f) for 48 h. Graphs show viral replication expressed as average fold change in RLU/mL relative to control cells transfected with the empty plasmid (Ctrl). Numbers above each bar pair in c and d refers to the number of independent experiments. Lower dashed line indicates the difference between ATL2 KO cells and ctrl cells, both transduced with an empty plasmid. For all graphs, error bars represent SEM; * or ** represent p values < 0.05 or 0.01, respectively calculated by one-way ANOVA with a Dunnett’s multiple comparison analysis.

Figure 4. Comparative ATL interaction networks.

a-b, Network representation of unique and shared ATL2- and ATL3-interacting cellular proteins. AP–LC–MS/MS analysis of HA-tagged ATLs in comparison to Calnexin (CANX). N = 4 independent experiments; significance testing are results from Welch’s T-test. a, Solid lines represent specific interactors identified by AP–MS/MS analysis of naïve A549 cells (log₂(fold change) ≥ 2.5, unadjusted two-sided p ≤ 0.05). b, Solid lines represent specific interactors identified by AP–MS/MS analysis of DV-infected A549 cells (log₂(fold change) ≥ 1), unadjusted two-sided P ≤ 0.05). c, The scatter plot displays ATL2 (x-axis) and ATL3 (y-axis) specific changes in uninfected compared to DV infected cells. Virus proteins (grey), significant enrichment or depletion (red) and specific hits selected for RNAi screen (blue) are shown. Significantly enriched or depleted proteins are shown in red (n = 4 independent experiments; Welch’s T-test unadjusted two-sided p ≤ 0.05; log₂(fold-change) ≥ 1). A schematic of the variables compared is shown on the bottom right.

Figure 5. ATL3 and its interactor ARF4 play important roles in DV maturation.

a, Experimental approach used for the shRNA screen. A549 cells were transduced with shRNA encoding lentivirus (3 different shRNAs/gene). 48 h post transduction, cells were infected with DV-R2A for 48 h followed by quantification of intracellular luciferase levels (replication). Supernatant from infected cells was then used to infect naïve A549 cells for 48 h and luciferase levels were quantified (Assembly/release). b, The heat map shows the mean fold difference between gene specific shRNA treated cells and NT shRNA treated cells. c, Graph shows the mean fold change for genes with the most significant effect on viral replication or assembly. N=6 independent samples; ***, p value < 0.001 calculated by one-way ANOVA with a Dunnett’s multiple comparison analysis. d-h, A549 cells were transduced with constructs encoding for gene-specific or non-targeting (NT) shRNA for 48 h, followed by infection with DV for 48 h. d, Graph shows the mean values for extracellular viral RNA (RT-qPCR) or titers (PFU assay) compared to control cells. e, The left graph shows the average fold change in intracellular or extracellular viral RNA levels over 3 experimental replicates. The right graph shows the average fold change in PFU/mL (PFU assay) for intracellular and extracellular virus over 3 experimental replicas. f-g, Intracellular and extracellular proteins were evaluated using western blot. g, Extracellular protein levels for prM and M were quantified using Fiji software. Graphs show average ratio between M and prM protein for NT shRNA and ATL3 shRNA transduced cells for 3 independent experiments. h, Virus particles in supernatants harvested from infected cells were purified using ultracentrifugation and incubated with purified furin. Graph shows the average fold change in PFU/mL (PFU assay) before and after furin treatment for ATL3 shRNA treated cells compared to NT shRNA treated cells over 3 independent experiments. For all graphs, error bars show SEM; * or **, p values < 0.05 or 0.01, respectively as determined by 2-tailed T-tests.

Figure 6. ATL3 depletion alters retrograde transport.

a, A549 cells were transduced with constructs encoding for shRNA directed against ATL2, ATL3 or a non-targeting (NT) shRNA. 96 h after transduction, cells were serum starved for 30 mins followed by incubation with Alexa fluor 568-conjugated transferrin (Tfn-568) for 40 mins. Cells were then fixed and stained for the trans-Golgi marker TGN46 using specific antibodies. Cells were analyzed by confocal microscopy (upper panel). Scale bars, 10 μm. The relative levels of Tfn-568 signal proximal to the TGN46 signal in control or ATL3 depleted cells is shown in the lower panel. Box, 25^th-75^th percentile; line shows mean value; Whiskers, min to max. N=52 cells were counted per sample over 2 biological replicates. ***, p value < 0.001 based on 2-tailed T-Test,. b-d, A549 cells stably expressing the furin reporter (CD4-Fu) were transduced with constructs encoding for shRNA directed against the indicated gene or a NT shRNA. b, 72 h after transduction, cells were fixed and the subcellular localization of the furin reporter was evaluated using immunofluorescence staining and confocal microscopy. N=3 biological replicates. c, Total cellular fluorescence and Golgi fluorescence were quantified and calculated for ≥20 cells for each condition. The graph shows the mean ratio between Golgi and total cellular fluorescence levels. Error bars show SEM. RTN3, reticulon 3; LNP, lunapark. d, 72 h post transduction, cells were imaged at 1 minute intervals. Following the 2 min time point, anti-CD4 fluor-conjugated antibodies were added and imaging continued for 1 h. Graphs shows mean total cellular fluorescence levels for ≥20 cells at each time point. N=3 biological replicates. e, Model for the function of ATLs in flavivirus infection, highlighting the role of ATL2 in VP formation and ATL3 in virus particle maturation and vesicular transport.