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. 2018 Aug 27;14(8):e1007280.
doi: 10.1371/journal.ppat.1007280. eCollection 2018 Aug.

Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles

Ekaterina G Viktorova  1 , Jules A Nchoutmboube  1 , Lauren A Ford-Siltz  1 , Ethan Iverson  2 , George A Belov  1
Affiliations

Phospholipid synthesis fueled by lipid droplets drives the structural development of poliovirus replication organelles

Ekaterina G Viktorova et al. PLoS Pathog. .

Abstract

Rapid development of complex membranous replication structures is a hallmark of picornavirus infections. However, neither the mechanisms underlying such dramatic reorganization of the cellular membrane architecture, nor the specific role of these membranes in the viral life cycle are sufficiently understood. Here we demonstrate that the cellular enzyme CCTα, responsible for the rate-limiting step in phosphatidylcholine synthesis, translocates from the nuclei to the cytoplasm upon infection and associates with the replication membranes, resulting in the rerouting of lipid synthesis from predominantly neutral lipids to phospholipids. The bulk supply of long chain fatty acids necessary to support the activated phospholipid synthesis in infected cells is provided by the hydrolysis of neutral lipids stored in lipid droplets. Such activation of phospholipid synthesis drives the massive membrane remodeling in infected cells. We also show that complex membranous scaffold of replication organelles is not essential for viral RNA replication but is required for protection of virus propagation from the cellular anti-viral response, especially during multi-cycle replication conditions. Inhibition of infection-specific phospholipid synthesis provides a new paradigm for controlling infection not by suppressing viral replication but by making it more visible to the immune system.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Overexpression of CCTα phenotypically recapitulates retargeting of long chain FAs from neutral to membrane lipid synthesis observed in poliovirus-infected cells.
A. HeLa cells were transfected with a CCTα-RFP expressing plasmid, and the next day they were incubated for 1 h with Bodipy C4/C9 in the medium. Yellow arrows indicate cells expressing the fusion protein, blue arrows show non-transfected cells. B. HeLa cells were infected (mock-infected) with poliovirus at an MOI of 10 PFU/cell, and at 4 h p.i. they were incubated with Bodipy C4/C9 in the medium for 1 h.
Fig 2
Fig 2. CCTα translocates from the nuclei to the cytoplasm in infected cells.
A. Translocation of CCTα into the cytoplasm upon infection. Confocal images of HeLa cells infected with poliovirus at an MOI of 10 PFU/cell, fixed, and processed for imaging of endogenous CCTα at 4 hp.i. Replication complexes are visualized by staining for a viral antigen 3A. B. Translocation of CCTα upon replication-independent expression of the fragments of poliovirus polyprotein. HeLa cells were transfected with plasmids coding for the indicated polyprotein fragments under control of T7 promotor and infected with a vaccinia virus expressing T7 RNA polymerase. Control cells were transfected with an empty vector. The next day, the cells were fixed and processed for imaging of endogenous CCTα and viral antigen 2B. C. Expression of the whole viral polyprotein fragments in the samples produced as in (B) is confirmed by western blot with antibodies against the viral protein 3D encoded in the very 3’-terminal part of the genome. The lack of 2A proteolytic activity upon expression of the fragment with mutant 2A*-3D and 2B-3D is confirmed by the lack of processing of eIF4G (arrow). Actin is shown as a loading control.
Fig 3
Fig 3. CCTα associates with the replication organelles and is important for controlling the activation of phospholipid synthesis upon infection.
A. Western blot showing changes in phosphorylation status and localization of CCTα upon infection. HeLa cells infected with 10 PFU/cell of poliovirus were treated with digitonin and collected for Western blot at the indicated times p.i. (lanes 6–8); control cells (lanes 2–4) underwent the same treatment but without the detergent. Arrow indicates dephosphorylated activated form of CCTα. Mock-infected cells (lanes 1 and 5) were collected at 6 h. Proteins were detected on the same membrane after stripping previous antibodies. B. Scheme of poliovirus genome with the sites of HA antigen insertions in the 2A or 3A sequence indicated. C. Top panel: CCTα signal from co-IP with anti-HA antibodies from lysates of HeLa cells infected at an MOI of 10 PFU/cell with either wt poliovirus (lanes 1 and 5), 2A-HA (lanes 2 and 6), 3A-HA (lane 3 and 7), or cells transfected with a plasmid expressing ACSL3-HA (lanes 4 and 8). Infected cells were processed at 6 h p.i. Bottom panel shows western blot of the same membrane probed with anti-HA antibodies. Multiple products of the viral polyprotein processing containing HA antigens (lanes 2, 3, and 6, 7) as well as a single band of ACSL3-HA (lanes 4 and 8) are detected. Positions of heavy and light antibody chains in co-IP samples are indicated. Input material on the western constitutes 5% of the lysate taken for co-IP. D. Cells with siRNA-knockdown expression of CCTα were infected with poliovirus at 10 PFU/cell and at 4 h post-infection, they were provided with a fluorescent long chain FA analog. The accumulation of fluorescence reflects the activation of lipid synthesis. The data are normalized to incorporation of the fluorescent long chain FA in corresponding mock-infected controls.
Fig 4
Fig 4. FAs from lipid droplets sustain activated phospholipid synthesis in infected cells.
A. Incorporation of a choline analog propargylcholine into the membranes of infected cells. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell, and were incubated with the indicated inhibitors after infection in balanced Earle solution. Orlistat is an inhibitor of de novo FA synthesis by FASN, DEUP is an inhibitor of mobilization of neutral lipids stored in lipid droplets. At 5 h p.i., the incubation medium was replaced with fresh pre-warmed balanced Earle solution containing propargylcholine. The cells were fixed at 6 h p.i. and processed for click-chemistry-based detection of incorporated propargylcholine. B. Quantitation of propargylcholine incorporation by HeLa cells infected and processed as in A.
Fig 5
Fig 5. Poliovirus infection stimulates hydrolysis of neutral lipids in lipid droplets.
A. Scheme of the experiments shown on panels B and C. HeLa cells were pre-incubated with a fluorescent FA analog before infection so that the molecule is incorporated into the neutral lipids stored in lipid droplets. B. Incorporation of a fluorescent long chain FA into lipid droplets of non-infected cells upon 1 h of incubation. C. HeLa cells pre-treated with Bodipy 500/510 C4/C9 for 1h were infected (mock-infected) with poliovirus at an MOI of 10 PFU/cell and incubated without the fluorescent FA for 6 h p.i. Red arrows indicate cells with redistribution of the fluorescent FA into the membranes of replication organelles, blue arrows indicate cells with residual fluorescence in lipid droplets. D. Disappearance of lipid droplets by the end of the poliovirus replication cycle. HeLa cells were infected (mock-infected) with poliovirus at an MOI of 10 PFU/cell and at 6 h p.i. they were fixed and processed for immunofluorescent analysis of a viral antigen 2B and stained with a lipid droplets-specific dye Bodipy 493/503. Arrows indicate infected cells where lipid droplets are no longer detectable. E. Recruitment of HSL to lipid droplets early during the poliovirus replication cycle. HeLa cells were infected (mock-infected) with poliovirus at an MOI of 10 PFU/cell and at 3 h p.i. they were fixed and processed for immunofluorescent analysis of a viral antigen 2B and HSL. Arrows indicate recruitment of HSL to lipid droplets. Inset shows a high magnification confocal image of HSL recruitment to lipid droplets in poliovirus-infected cells.
Fig 6
Fig 6. Inhibition of PC synthesis restores neutral lipid synthesis in infected cells but does not significantly affect the first cycle of poliovirus replication.
A. Scheme of the experiments shown on panels B-D. HeLa cells pre-incubated in choline-free medium for ~72h were infected with poliovirus and were incubated after infection either in choline-free or choline-supplemented medium. B. Incorporation of the fluorescent long chain FA analog added to choline-deprived HeLa cells infected with an MOI of 10 PFU/ml of poliovirus at 4 h p.i. for 1 h. In cells incubated in choline-free medium, it redistributes to neutral lipids in lipid droplets (left panel), while in cells incubated with choline-supplemented medium it is used for membrane synthesis (right panel). C. Accumulation of the viral proteins 2C and 2BC (left panel) at 4 h p.i. and total virus yield (right panel) at 6 h p.i. did not depend on whether previously choline-deprived cells were incubated in choline-free or choline-supplemented medium after infection. Actin shown as a loading control. D. Total virus yield at 6 h p.i. upon infection of choline-deprived cells at low MOIs of 0.05 and 0.5 does not depend on whether the cells were incubated in choline-free or choline-supplemented medium after infection. E. Presence of choline in the incubation medium also did not affect the dynamics of accumulation of extracellular (extra) and intracellular (intra) progeny virus upon infection of choline-deprived HeLa cells. Virus yield at the indicated time points was determined after infection of choline-deprived HeLa cells with an MOI of 10 and incubation after infection in choline-free or choline-supplemented media.
Fig 7
Fig 7. Inhibition of phospholipid synthesis impedes structural development of replication organelles.
HeLa cells were infected with 10 PFU/cell of poliovirus and processed for transmission EM imaging at 4 h p.i. The cells were incubated in either normal serum-supplemented medium before and after infection (control), or were pre-incubated for ~72h in choline-free medium, and after infection were incubated in choline-free (choline-), or choline-supplemented (choline +) medium. Areas outlined on low magnification images on the left are shown at high magnification on the right. Arrows on high magnification choline- panel indicate enlarged ER-like structures.
Fig 8
Fig 8. Inhibition of phospholipid synthesis affects the accessibility of the replication complexes.
HeLa cells after choline deprivation were infected with poliovirus at an MOI of 10 PFU/cell and incubated in a choline-free or a choline-supplemented medium after infection. A. Confocal images of the cells permeabilized with either 0.2% Triton X100 (strong permeabilization) or 0.02% saponin (mild permeabilization) and stained for a viral antigen 2B (red). Nuclear DNA is stained with Hoechst 33342 (blue). Arrows indicate areas protected upon mild permeabilization in cells incubated with choline-supplemented medium. B. Visualization of dsRNA in HeLa cells processed and infected as in A after Triton X100 permeabilization. Red arrows indicate dsRNA association with the nuclear envelope (PN) in choline-deprived cells; white arrows show big perinuclear blobs (CYT) of dsRNA signal reflecting normal development of replication membranes in choline-supplemented cells. C. HeLa cells were infected with poliovirus at an MOI of 10 PFU/cell after choline deprivation and incubated in a choline-free or a choline-supplemented medium for 4 h p.i. The cells were permeabilized with digitonin and treated (lines 3 and 4) or non-treated (lines 1 and 2) with proteinase K. * indicates protein fragments generated upon proteinase K treatment. Actin is shown as a loading control.
Fig 9
Fig 9. Inhibition of membrane synthesis promotes cellular anti-viral signaling and impedes poliovirus propagation in multiple cycles of infection especially in conditions of pre-activated anti-viral response.
A and B. HeLa cells were pre-incubated in choline-free medium for ~72h, they were infected with poliovirus at an MOI of 10 PFU/cell and incubated in either a choline-free- or a choline-supplemented medium for the indicated time p.i. Phosphorylated IRF3 is indicated by the arrow. Actin is shown as a loading control. C. HeLa cells were pre-incubated in choline-free medium for ~72 h, infected with poliovirus at an MOI of 0.1 or 0.01 PFU/cell, and were incubated in either a choline-free or a choline-supplemented medium for another 24 h. Total virus yield is shown. D. HeLa cells were pre-incubated in choline-free medium for ~60h followed by ~12 h of incubation with 20u of universal type I interferon in choline-free medium. After that, the cells were infected with poliovirus at an MOI of 0.1 or 0.01 PFU/cell and were incubated in either choline-free or choline-supplemented medium for another 24 h. Total virus yield is shown.
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References

    1. Romero-Brey I, Bartenschlager R. Membranous replication factories induced by plus-strand RNA viruses. Viruses. 2014;6(7):2826–57. 10.3390/v6072826 ; PubMed Central PMCID: PMC4113795. - DOI - PMC - PubMed
    1. Belov GA, van Kuppeveld FJ. (+)RNA viruses rewire cellular pathways to build replication organelles. Current opinion in virology. 2012;2(6):740–7. 10.1016/j.coviro.2012.09.006 . - DOI - PMC - PubMed
    1. Nagy PD, Strating JRPM, van Kuppeveld FJM. Building Viral Replication Organelles: Close Encounters of the Membrane Types. PLoS pathogens. 2016;12(10). doi: ARTN e1005912 10.1371/journal.ppat.1005912 WOS:000387666900023. - DOI - PMC - PubMed
    1. Miller S, Krijnse-Locker J. Modification of intracellular membrane structures for virus replication. Nat Rev Microbiol. 2008;6(5):363–74. Epub 2008/04/17. 10.1038/nrmicro1890 nrmicro1890 [pii]. . - DOI - PMC - PubMed
    1. Knowlton KU. CVB infection and mechanisms of viral cardiomyopathy. Curr Top Microbiol Immunol. 2008;323:315–35. Epub 2008/03/25. . - PubMed

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