Viral reorganization of the secretory pathway generates distinct organelles for RNA replication

Abstract

Many RNA viruses remodel intracellular membranes to generate specialized sites for RNA replication. How membranes are remodeled and what properties make them conducive for replication are unknown. Here we show how RNA viruses can manipulate multiple components of the cellular secretory pathway to generate organelles specialized for replication that are distinct in protein and lipid composition from the host cell. Specific viral proteins modulate effector recruitment by Arf1 GTPase and its guanine nucleotide exchange factor GBF1, promoting preferential recruitment of phosphatidylinositol-4-kinase IIIbeta (PI4KIIIbeta) to membranes over coat proteins, yielding uncoated phosphatidylinositol-4-phosphate (PI4P) lipid-enriched organelles. The PI4P-rich lipid microenvironment is essential for both enteroviral and flaviviral RNA replication; PI4KIIIbeta inhibition interferes with this process; and enteroviral RNA polymerases specifically bind PI4P. These findings reveal how RNA viruses can selectively exploit specific elements of the host to form specialized organelles where cellular phosphoinositide lipids are key to regulating viral RNA replication.

Graphical abstract

Figure 1

Enteroviral RNA and Replication Machinery Are Localized to the Host Secretory Pathway Organelles (A) Viral RNA and viral replication protein (3A, 3D^pol) subcellular distribution in early stages of CVB3 RNA replication. (B) Viral RNA and viral replication protein (3A, 3D^pol) subcellular distribution at peak stages of CVB3 RNA replication. See also Figure S1. (C) Arf1-RFP and GBF1-YFP dynamics in CVB3-infected HeLa cell. Confocal time-lapse images of single cell are presented. See also Movie S1. (D) GBF1 and viral RNA are colocalized in HeLa cells during CVB3 infection. (E) Replication organelles, at peak replication, form adjacent to ER exit sites. (F) Arf1 and GBF1 facilitate viral RNA replication. CVB3 replicon assays in HeLa cells pretreated with siRNA against Arf1 or GBF1. Bar graph presents maximum replication values for each condition, normalized to control samples transfected with nontargeting siRNA. Error bars are SEM from eight replicates for each condition (^∗∗p < 0.01). See also Figures S2A–S2C and Table S1. (G) Functional ER exit sites facilitate viral RNA replication. PV replicon assays in HeLa cells transiently expressing Sar1[T39N] plasmid. Bar graph presents maximum replication values, normalized to control samples transfected with GFP. Error bars are SEM from eight replicates for each condition (^∗∗p < 0.01). See also Table S1. r.l.u% = relative light unit %. All fluorescence images were confocal images of optical slice thickness ∼1 μm. Scale bar, 10 μm.

Figure S1

Arf1 and Enteroviral Replication Protein Colocalization, Related to Figure 1 (A) Native Arf1 distribution in uninfected and CVB3-infected cells mimics that of Arf1-GFP. (B) Confocal image (optical slice 2 μm) of a single HeLa cell expressing Arf1-CFP immunostained with antibodies to viral 2C protein and CFP, at 4 hr infection with PV.

Figure S2

siRNA-Mediated Reduction of Host Factors and Impact on Enterovirus Replication, Related to Figure 1 and Figure 5 (A) Western blots of HeLa cells treated with nontargeting (lane 1) or Arf1, GBF1, PI4KIIIβ siRNAs (lane 2); equal amounts of protein (confirmed by actin blotting) for nontarget/Arf1, nontarget/GBF1, and nontarget/PI4KIIIβ conditions were loaded on SDS-PAGE gels. Bar graph shows quantification of representative blot, where the extent of siRNA depletion has been normalized to nontargeting siRNA condition. (B and C) CVB3 replicon assays in HeLa cells treated with nontargeting siRNA and Arf1 siRNA (B) or GBF1 siRNA (C). Eight replicate samples were assayed for nontargeting, Arf1, and GBF1 siRNA treatment conditions.

Figure 2

Reorganization of Secretory Pathway Organelles after Enteroviral Infection HeLa cells (A) coexpressing Arf1-RFP/ɛCOP-YFP; expressing Arf1-GFP (B, C, E–I); or coexpressing Arf1-RFP/GalT-YFP (see Movie S2) (D) were infected with CVB3 for 4 hr. Cells in (B), (C), (E)–(I) were fixed and coimmunostained with anti-GFP and (B) anti-clathrin heavy chain; (C) anti-γ-adaptin; (E) anti-GM130; (F) anti-TGN46; (G) anti-GGA1; (H) anti-Rab1b; (I) anti-ERGIC53 antibodies. Arrows in (A), (B), (C), and (D) indicate Arf1-labeled membranes that do not label with ɛCOP-YFP, clathrin, γ-adaptin, and GalT-YFP, respectively. See also Figures S3A–S3H and Figure S4. All fluorescence images were confocal images of optical slice thickness ∼1 μm. Scale bar, 10 μm.

Figure S3

Host Cell Secretory Machinery Distribution and Dynamics in Uninfected and Enterovius-Infected Cells, Related to Figure 2 (A–G) Colocalization in HeLa cells of Arf1-GFP and secretory pathway components in uninfected cells. Antibodies to GFP were utilized to localize Arf1. (H and I) Disruption of secretory trafficking in CVB3 infected cells. HeLa cells expressing ts045VSVG-GFP were infected with CVB3 or mock for 2 hr prior to 32°C switch. Image showing Golgi accumulation of ts045VSVG-GFP 30 min after switch from non-permissive 40°C to permissive 32°C in mock-infected cells (H). Note the lack of ts045VSVG-GFP accumulation at the Golgi after 30 min in CVB3-infected cells (I). (J) Transferrin Receptor is not localized to replication organelles. HeLa cells at 0 hr and 4 hr post CVB3 infection were fixed and coimmunostained with antibodies to native GBF1 to label the replication organelles and Transferrin Receptor. All images unless indicated are confocal sections 1 μm optical slice thickness. Scale bar, 10 μm.

Figure S4

Pearson Correlation Coefficients of Arf1 and Host Secretory Components Pre- and Post-Enterovirus Infection, Related to Figure 2 and Figure 3 Pearson Correlation Coefficients were calculated as a measure of the degree of colocalization between Arf1-labeled membranes and ɛCOP, GalT, GGA, TGN46, GM130, ERGIC53, PI4KIIIβ proteins. The coefficients were calculated at 0 hr post-infection when Arf1 is localized to the Golgi apparatus and at 4 hr post-infection when Arf1 is localized at the replication organelles. In general small Pearson coefficients (<0.5) indicate low or no colocalization.

Figure 3

PI4KIIIβ Is Localized to Enteroviral RNA Replication Organelles (A) PI4KIIIβ and Arf1 are colocalized throughout CVB3 infection. HeLa cells expressing Arf1-GFP infected with CVB3 were immunostained with anti-PI4KIIIβ and anti-GFP antibodies. (B) GBF1/Arf1 inactivation leads to PI4KIIIβ dispersal. HeLa cells were infected with CVB3 for 4 hr and treated with 10 μg/ml of BFA for 30 min. 75% ± 5% (n = 10 cells) of PI4KIIIβ-associated fluorescence was dispersed upon BFA treatment. (C) PI4KIIIβ is in physical complex with viral replication enzymes. HeLa cell lysates from cells infected with CVB3 or mock (−) for 4 hr were immunoprecipitated with anti-PI4KIIIβ antibodies. Input samples verifying the presence of TGN46 in the lysates prior to immunoprecipitation are presented in the bottom panel. All fluorescence images were confocal images of optical slice thickness ∼1 μm. Scale bar, 10 μm.

Figure 4

Enteroviral 3A Proteins Can Promote Selective PI4KIIIβ Recruitment over Coat Proteins (A–F) Impact of 3A-myc ectopic expression on host secretory machinery. HeLa cells were immunostained with antibodies to myc-tag and native: (A) Golgin97, a Golgi resident protein; (B) GBF1; (C) Arf1; (D) βCOP; (E) PI4KIIIβ; (F) PI4KIIIα. Arrows and asterisk in (D) and (E) indicate cells where 3A is either expressed (arrow) or not (^∗). See also Figures S5A–S5D. (G) Quantification of GBF1 (n = 15 cells); Arf1 (n = 9 cells); βCOP (n = 10 cells); and PI4KIIIβ (n = 13 cells) antibody fluorescence associated with 3A-labeled membranes as (%) of their respective values at the Golgi apparatus of cells not expressing 3A. Error bars are SEM (^∗∗p < 0.001). All fluorescence images were confocal images of optical slice thickness ∼1 μm. Scale bar, 10 μm.

Figure S5

High-Level Transient Ectopic Enteroviral 3A Expression, Related to Figure 4 Secretory machinery distribution in cells expressing high levels of 3A-myc. All cells were transfected with 3A-myc and then coimmunostained with antibodies against –myc and (A) βCOP, (B) GalT, (C) sec31, (D) PI4KIIIβ. Scale bar, 5 μm.

Figure 5

PI4KIIIβ Activity Is Required for Enteroviral RNA Replication (A and B) PIK93 block of PI4KIIIβ activity inhibits CVB3 and PV RNA replication. CVB3 and PV replicon assays in cells treated with 500 nM and 1 μM PIK93 are shown. Bar graphs present maximum replication values for CVB3 (A) and PV (B) with PIK93 treatment, normalized to control (DMSO) treatment. See also Figures S6A–S6C. (C) Kinetics of inhibition by PIK93 presented for the CVB3 replicon. (D and E) Reduction of PI4KIIIβ levels with siRNA inhibits CVB3 and PV RNA replication. CVB3 and PV replicon assays in HeLa cells; bar graphs present maximum replication values for each condition, normalized to control (nontargeting) siRNA. See also Figure S2A. (F) Kinetics of inhibition by PI4KIIIβ siRNA presented for PV replicon. (G and H) Expression of ectopic kinase-dead PI4KIIIβ (PI4KIIIβ-KD) inhibits CVB3 and PV replication. CVB3 and PV replicon assays in HeLa cells; bar graphs present maximum replication values for each condition, normalized to control (GFP) plasmid ectopic expression. (I) Kinetics of inhibition by PI4KIIIβ-KD presented for the CVB3 replicon. Error bars in each CVB3 assay are SEM of six CVB3 samples and each PV assay is SEM for eight PV samples. (^∗∗p < 0.01). (J and K) PI4KIIIβ activity regulates viral RNA synthesis. Cell-free PV RNA translation (J) and synthesis (K) assays performed in the presence of PIK93.

Figure S6

PIK93 Impact on Enterovirus RNA Replication and Cell Viability, Related to Figure 5 (A) Impact on PV replication of different concentrations of PIK93 (added at time 0 hr). (B) Impact on replication of PIK93 added at 1 hr and 3 hr post replicon transfection. (C) PIK93 effects on cell viability. Cells were treated with the indicated PIK93 concentrations for 24 hr.

Figure S7

Impact of PI4KIIIα and PI4KIIIβ Reduction on ER Exit Sites in HeLa Cells, Related to Figure 5 HeLa cells were treated with nontargeting, PI4KIIIβ, or PI4KIIIα siRNA's for 72 hr; fixed and immunostained with antibodies to sec31 to label ER exit sites. Note the decrease in the number of ER exit sites in PI4KIIIα siRNA-treated cells whereas it is unaffected in PI4KIIIβ siRNA-treated cells.

Figure 6

PI4P Lipid Microenvironment within Replication Organelles Regulates both Enteroviral and Flaviviral RNA Replication (A) Cellular PI4P lipid levels rise in CVB3 infection. Total cellular PI4P lipids were quantified over time. Error bars are SEM from duplicate samples (^∗∗p < 0.01). (B) Reduction in PI4P lipid levels inhibits enteroviral RNA replication. PV replicon assays in HeLa cells ectopically expressing Sac1 are shown. Inset: western blot showing increase in Sac1 levels after 16 hr of ectopic expression. Bar graph presents maximum replication values, normalized to control (GFP) plasmid ectopic expression. Error bars are SEM from eight replicates cells (^∗∗p < 0.01). (C) PI4P lipids localize to enteroviral replication organelles. Time-lapse confocal images of single HeLa cell infected with CVB3, coexpressing FAPP1PH-GFP/Arf1-RFP (see also Movie S3). (D) PI4KIIIβ activity is responsible for PI4P lipids at enteroviral replication organelles. Time-lapse confocal image of single HeLa cell coexpressing FAPP1-PH-GFP/ARF1-RFP pre- and post-PIK93 treatment at 4 hr post-infection with CVB3. (E) Quantification of FAPP1PH-GFP to Arf1-RFP fluorescence from (D) expressed as a ratio. Error bars are SEM from ten cells for each condition. (F and G) PV RNA polymerase specifically and preferentially binds PI4P lipids. Purified recombinant 3D^pol enzyme was incubated with membrane strips that were previously spotted with different lipids. Antibodies detected 3D^pol binding. Two representative blots are shown. Binding was quantified and plotted in bar graph as a ratio over background non-lipid spotted membrane from three different experiments for each lipid type. (H) Cellular PI4P lipid levels rise in HCV replicating cells. Quantification of PI4P with anti-PI4P primary antibodies of 3-5B(HCV) cells, normalized as % of PI4P lipid quantification within Huh7 cells. Error bars are SEM from ten cells for each cell type. (I) PI4P lipids localize to HCV replication membranes. PI4P lipid and NS5A protein distribution in Huh7 and 3-5B(HCV) cells were determined by immunostaining with anti-PI4P and anti-NS5A antibodies. (J and K) PI4KIIIβ is responsible for a significant fraction of PI4P lipids at HCV replication membranes. 3-5B(HCV) cells were treated with nontargeting, PI4KIIIβ, or PI4KIIIα siRNA. Cells were immunostained and quantified for PI4P lipids. Representative images of groups of siRNA-treated cells are shown (J). Quantification was done on 20 cells for each siRNA treatment condition. (L) Reduction of PI4P lipids inhibits HCV replication. HCV replicon assays conducted with J6/JFH (p7-Rluc2A) replicons in Huh7 cells ectopically expressing Sac1, PI4KIIIβ-KD, or both plasmids are shown. Bar graph presents maximum replication value normalized to control (GFP) plasmid ectopic expression. Error bars are SEM from eight replicates of cells for each treatment condition (^∗∗p < 0.01). All fluorescence images were confocal images of optical slice thickness ∼1 μm. Scale bar, 10 μm.

Figure 7

Model for Secretory Pathway Reorganization in Enteroviral Infections (A) Tail-anchored membrane protein 3A by binding and modulating GBF1/Arf1 promotes PI4KIIIβ recruitment to the membrane bilayer at the expense of coat protein COPI. Recruited PI4KIIIβ will catalyze the production of a PI4P lipid microenvironment (red lipids) that will in turn facilitate the recruitment of 3D^pol from the cytosolic pool to the membrane and promote the synthesis of viral RNA. (B) Uninfected (0 hr): Steady-state exchange of membranes, Golgi enzymes, and cargo through bidirectional trafficking between the ERGIC and Golgi/TGN compartments. Golgi enzymes and cargo are sorted out of Sar1/COPII-labeled ER exit sites into ERGIC compartments whereupon GBF1/Arf1 and COPI coats mediate the trafficking to and from the Golgi/TGN compartments. PI4KIIIβ enzymes are recruited to Golgi/TGN membranes by Arf1 and catalyze the production of PI4P lipids at these membranes. 2 hr: Upon infection, newly synthesized viral replication enzymes such as the membrane-bound 3A target to and assemble on secretory organelle membranes but concentrate and initiate viral RNA synthesis on the Golgi/TGN membranes, where the pre-existing (i.e., prior to infection) steady-state pool of PI4P lipids facilitates viral replication protein assembly and RNA replication. Rising levels of 3A combined with its modulation of effector recruitment by GBF1/Arf1 will enhance the recruitment of PI4KIIIβ over COPI, leading to a decreased rate of anterograde transport out of the ERGIC and subsequent disassembly of the Golgi/TGN organelles. 4 hr: Enhanced recruitment of PI4KIIIβ over COPI results in the formation of uncoated PI4P lipid-enriched organelles adjacent to ER exit sites. The PI4P lipid-enriched microenvironment of these organelles facilitates the ongoing assembly of newly synthesized viral replication proteins such as RdRp 3D^pol and viral RNA replication.