Hijacking of multiple phospholipid biosynthetic pathways and induction of membrane biogenesis by a picornaviral 3CD protein

Figures

• 

  Fig 1. Ectopic expression of poliovirus 3CD causes induction and redistribution of PI4P. (A) Schematic representation of the major pathway for processing of the P3 polyprotein. 3C-encoded protease activity cleaves at the Gln-Gly junction between the 3B and 3C proteins to produce 3AB and 3CD proteins. (B) Effect of 3AB, 3CD, 3CDpro+, and 3A expression on PI4P levels and localization in HeLa cells. Cells were transfected with in-vitro synthesized mRNA for EGFP, 3AB, 3CD, 3CDpro+, or 3A. Four hours post-transfection, cells were immunostained for the presence of PI4P (red) and the expressed protein (green). The nucleus was stained with DAPI (blue). Mock represents cells that were taken through the transfection protocol in the absence of mRNA. (C) Quantification of PI4P intensity. The PI4P fluorescence intensity was determined using 11 z-stacks per transfected cell (n = 30). The average PI4P intensity of mock-transfected cells was used to normalize the value measured in each cell, the results of which are plotted in the graph. The averages of the normalized values were: 1.00 ± 0.03 (SEM) in mock-transfected cells, 0.81 ± 0.04 (SEM) in 3AB transfected cells, 4.98 ± 0.43 (SEM) in…

• 

  Fig 2. 3CD-mediated induction of PI4P does not require host transcription but uses GBF1 and a type III PI4 kinase(s). (A) Effect of actinomycin D (AMD) on nucleolin localization. HeLa cells were treated with AMD (5 μg/mL) for 4 h then immunostained for nucleolin (red) [37]. (B) Effect of actinomycin D (AMD) on 3CD-mediated induction of PI4P. HeLa cells were maintained in the absence or presence of AMD (5 μg/mL) prior to transfection with 3CD mRNA. Cells were immunostained for PI4P (red) or 3CD (green); the nucleus was stained with DAPI (blue). Subsequent experiments were performed as described for AMD but used the following inhibitors: (C) brefeldin A (BFA, 2 μg/mL), (D) golgicide A (GCA, 10 μM), and (E) PIK93 (15 μM). All of these treatments interfered with induction of PI4P. PIK93 was the least effective. (F) Quantification of the PI4P levels per cell in the absence (1.8 ± 0.1 x 108) and presence (7.1 ± 0.5 x 107) of PIK93. Error bars represent SEM; Student’s t-test gave a P value of <0.0001.

• 

  Fig 3. Ectopic expression of 3CD causes activation of Arf1. (A) Schematic of assay used to detect GTP-bound (activated) Arf1. HeLa cells were transfected with or without 3CD mRNA. A lysate was prepared using transfected cells and combined with magnetic beads to which the GGA3 adaptor protein was immobilized. Arf1-GTP is uniquely bound by the GGA3 adaptor protein [41]. Beads were isolated, washed, and boiled in SDS-PAGE sample buffer to elute proteins. Eluted proteins were resolved by SDS-PAGE and detected by Western blotting using an anti-Arf1 antibody. (B) Detection of Arf1-GTP (bead-eluted material), total Arf1 (from unfractionated lysate), and 3CD (from unfractionated lysate) by Western blot using the indicated antibodies and enhanced chemifluorescence. (C) The magnitude of Arf1 activation was determined by quantifying the fluorescence intensity of Arf1-GTP and total Arf1 and reporting the quotient thereof. The averages of the values for the quotients (n = 3) were: 0.43 ± 0.03 (SEM) in EGFP-transfected cells and 1.77 ± 0.03 (SEM) in 3CD-transfected cells. The difference was significant based on a Student’s t-test, which gave a P value of <0.0001.

• 

  Fig 4. 3CD expression causes redistribution of Arf1, GBF1, and PI4KB to sites of PI4P synthesis without affecting Golgi or ER integrity. (A) HeLa cells expressing 3CD were immunostained for the presence of the following: (i) Arf1, (ii) GBF1, or (iii) PI4KB (green). 3CD expression was monitored (red); the nucleus is stained witth DAPI (blue). (B) The same experiment as performed in panel A, with immunostaining for PI4P (red) instead of 3CD. (C) HeLa cells expressing 3CD were immunostained for the presence of the following: (i) Giantin or (ii) Calnexin (green). 3CD expression was monitored (red); the nucleus is stained witth DAPI (blue).

• 

  Fig 5. Changes to Arf1, GBF1, and PI4KB caused by 3CD are also observed during PV infection in the absence of replication. (A) HeLa cells were infected with PV at an MOI of 10 in the presence of 3 mM GuHCl, a replication inhibitor. Four hours post-infection, cells were immunostained for the following: (i) PI4P (red) (ii) Arf1 (green), (iii) GBF1 (green), or (iv) PI4KB (green). Under these conditions, PV proteins are expressed at a level below our limit of detection. Infected cells can be identified by changes to pattern of PI4P expression (red). The nucleus was stained with DAPI (blue). (B) HeLa cells were infected with PV at an MOI of 10 in the presence of 3 mM GuHCl. Four hours post-infection, cells were immunostained for the following: (i) Giantin or (ii) Calnexin (green). PI4P is labeled red; DAPI is shown in blue.

• 

  Fig 6. Identification of 3CD variants with substitutions in 3C or 3D domain that impair induction of PI4P biosynthesis at distinct steps. (A) 3CD derivatives with amino acid substitutions in 3C (3CmD) or 3D (3CDm) were expressed individually or together in HeLa cells and immunostained for PI4P (red) and 3CD (green). The nucleus was stained with DAPI (blue). (B) Quantification of PI4P intensity per cell was performed as described in the legend to Fig 1C. The averages of the normalized values were: 1.00 ± 0.04 (SEM) in mock-transfected cells; 1.06 ± 0.04 (SEM) in 3CDm-transfected cells; 1.10 ± 0.04 (SEM) in 3CmD-transfected cells; and 3.08 ± 0.13 (SEM) in 3CDm and 3CmD co-transfected cells. The level of PI4P induction observed in co-transfected cells was significant when compared to any other experiment based on a Student’s t-test, which gave a P value of <0.0001. (C) Activation of Arf1 by the 3CD derivatives was determined as described in the legend to Fig 3A. (D) The magnitude of Arf1 activation was determined as described in the legend to Fig 3B. The averages of the values for the quotients (n = 3) were: 0.43 ± 0.02 (SEM) in mock-transfected cells; 0.37 ± 0.07 (SEM) in 3CDm-transfected…

• 

  Fig 7. Inhibitory activity of 3AB on PI4P biosynthesis can be overcome by 3CD but that of 3A cannot. (A) HeLa cells expressing 3CD in the absence or presence of 3AB or 3A were immunostained for the presence of PI4P (red), 3CD (green), and 3A(B) (magenta). The nucleus is stained with DAPI (blue). (B) Quantification of PI4P intensity per cell was performed as described in the legend to Fig 1C. The averages of the normalized values were: 1.00 ± 0.03, n = 40 (SEM) in mock cells; 2.61 ± 0.13, n = 30 (SEM) in 3CD-transfected cells; 2.37 ± 0.10, n = 30 (SEM) in 3CD and 3AB co-transfected cells; and 0.83 ± 0.03, n = 35 (SEM) in 3CD and 3A co-transfected cells. The level of PI4P induction observed in 3CD and 3AB co-transfected cells was significant when compared to mock or 3CD and 3A co-transfected cells based on a Student’s t-test, which gave a P value of <0.0001 in both instances. There was no significant difference between 3CD-transfected cells and 3AB and 3CD co-transfected cells based on a Student’s t-test, which gave a P value of 0.1490. The diminished level of PI4P observed in 3CD and 3A co-transfected cells was significant when compared to mock based on a Student’s t-test, which gave a P…

• 

  Fig 8. 3CD derivatives exhibit modulated binding to PI4P-containing membranes. A microfluidics-based, membrane-binding assay was used to measure membrane binding by 3CD and derivatives thereof [47]. A bilayer is formed in the channels of the microfluidics device. The bilayer contains a phosphatidylcholine (POPC) and a fluorescent derivative of phosphatidylethanolamine (0.5 mol%) in the presence or absence of PI4P (7.5 mol%). Binding of the protein to the membrane quenches fluorescence, which enables measurement of the dissociation constant, Kd,app. (A) Image of the fluorescence observed in the channels of the device as a function of 3CD concentration using a 10X objective. (B) The change in fluorescence was converted to percent of bilayer saturated (see Materials and Methods) and plotted as a function of protein concentration. Data were fit to a Langmuir binding isotherm with an n = 1 to obtain values for Kd,app. Three independent experiments were performed. Error bars represent standard error of the mean. (C) Values for Kd,app measured for 3CD and its derivatives on the indicated bilayer. When using a bilayer consisting of POPC and the fluorescence probe, no change in fluorescence was…

• 

  Fig 9. 3CD uses distinct mechanisms to induce different lipids. (A) PV infection induces PIP2. HeLa cells were infected with WT PV expressing EGFP at an MOI of 10 and immunostained for PIP2 (red). EGFP (green) indicates infected cells, and DAPI (blue) marks the nuclei. PIP2 was quantified as described for PI4P in the legend to Fig 1C. The averages of the normalized values were: 1.00 ± 0.05 (SEM) in mock-infected cells and 6.62 ± 0.37 (SEM) in PV-infected cells. The difference was significant based on a Student’s t-test, which gave a P value of <0.0001. (B) PV infection induces PC. Experiment was performed as described in panel A. Cells were immunostained for PC (red). PC was quantified as described for PI4P in the legend to Fig 1C. The averages of the normalized values were: 1.00 ± 0.04 (SEM) in mock-infected cells and 3.03 ± 0.15 (SEM) in PV-infected cells. The difference was significant based on a Student’s t-test, which gave a P value of <0.0001. (C) Induction of PIP2 by 3CD, 3CDm and 3CmD. HeLa cells were transfected with various 3CD mRNAs and fixed 4 h post-transfection. Cells were immunostained for PIP2 (red) and 3CD (green). Nucleus was stained with DAPI (blue). Quantification of…

• 

  Fig 10. 3CD induces membrane biogenesis in the perinuclear region of the cell in the vicinity of the Golgi. HeLa cells were transfected with EGFP or the indicated 3CD mRNA and then processed for transmission microscopy (TEM) 4 h post-transfection. Electron micrograph of a EGFP-transfected cell (left panel) shows the normal ultrastructure of the cell. The nucleus is indicated (N). The Golgi apparatus is located in the perinuclear region of the cell (black box). The comparable view of 3CD-transfectd cell shows proliferation of membranes in the area normally occupied by the Golgi apparatus (red, blue, and green boxes). The second row of images shows magnifications of the areas highlighted above by the colored boxes. Scale bar indicates 1 μM.

• 

  Fig 11. Roles of poliovirus P3-encoded proteins in biogenesis and phospholipid composition of the replication organelle. (A) The P3 precursor polyprotein is cleaved by the 3C-encoded protease activity by two distinct pathways: major and minor. Therefore, proteins 3AB and 3CD are present at substantially higher levels in an infected cell than products of the minor pathway. (B) 3CD hijacks the GBF1-Arf1-PI4KB pathway to induce PI4P biosynthesis. How 3CD recruits GBF1 to the membrane remains unclear. This recruitment event leads to Arf1 activation and is sensitive to golgicide A (GCA). Alteration of the back of the thumb of the 3D domain of 3CD (3CDm) interferes with Arf1 activation. Post-Arf1 activation, PI4KB is recruited to membranes and is itself activated for synthesis of PI4P. PI4P biosynthesis is inhibited by the PI4KB inhibitor, PIK93. Steps leading to recruitment and/or activation of PI4KB require the 3C domain of 3CD and are defective in 3CmD. The ability of 3CDm and 3CmD to complement the other (Fig 6) suggests that 3CD functions minimally at two discrete steps (black boxes). Importantly, these black boxes represent the least understood aspects of the normal pathway of PI4P…