Multiple poliovirus-induced organelles suggested by comparison of spatiotemporal dynamics of membranous structures and phosphoinositides

<div><p>At the culmination of poliovirus (PV) multiplication, membranes are observed that contain phosphatidylinositol-4-phosphate (PI4P) and appear as vesicular clusters in cross section. Induction and remodeling of PI4P and membranes prior to or concurrent with genome replication has not been well studied. Here, we exploit two PV mutants, termed EG and GG, which exhibit aberrant proteolytic processing of the P3 precursor that substantially delays the onset of genome replication and/or impairs virus assembly, to illuminate the pathway of formation of PV-induced membranous structures. For WT PV, changes to the PI4P pool were observed as early as 30 min post-infection. PI4P remodeling occurred even in the presence of guanidine hydrochloride, a replication inhibitor, and was accompanied by formation of membrane tubules throughout the cytoplasm. Vesicular clusters appeared in the perinuclear region of the cell at 3 h post-infection, a time too slow for these structures to be responsible for genome replication. Delays in the onset of genome replication observed for EG and GG PVs were similar to the delays in virus-induced remodeling of PI4P pools, consistent with PI4P serving as a marker of the genome-replication organelle. GG PV was unable to convert virus-induced tubules into vesicular clusters, perhaps explaining the nearly 5-log reduction in infectious virus produced by this mutant. Our results are consistent with PV inducing temporally distinct membranous structures (organelles) for genome replication (tubules) and virus assembly (vesicular clusters). We suggest that the pace of formation, spatiotemporal dynamics, and the efficiency of the replication-to-assembly-organelle conversion may be set by both the rate of P3 polyprotein processing and the capacity for P3 processing to yield 3AB and/or 3CD proteins.</p></div

WT PV induces tubules in the presence of a replication inhibitor.

<p>(<b>A</b>) GuHCl has no impact on cell ultrastructure. HeLa cells were grown for 10 h at 37 ^oC in the presence of 3 mM GuHCl, and the cell ultrastructure was visualized by TEM. Bar = 1 μm. N denotes nucleus. (<b>B</b>) Ultrastructural changes are observed in the absence of replication. HeLa cells were infected with WT PV at an MOI of 10 in the presence of 3 mM GuHCl at 37 ^oC. Ten hours post-infection, cell ultrastructure was visualized by TEM. Bar = 1 μm. Representative images are shown in panels i, ii, and iii; the lower panels are enlargements of the boxed fields in the panels above. Some of the tubular-reticular structures are marked by the dotted line and/or arrowheads in the various panels to highlight the structures to which we refer but not to be exhaustive in our labeling. N denotes nucleus.</p

Poliovirus genome organization and P3-polyprotein processing.

<p>(<b>A</b>) Schematic of the poliovirus genome. The 7.5 kb long genome consists of a 5’-nontranslated region (NTR), an open reading frame, a 3’-NTR and a poly(rA) tail. The 5’-end of the genome is covalently linked to a peptide (VPg) encoded by the 3B gene. The 5’-NTR contains a cis-acting replication element (CRE) termed oriL or cloverleaf followed by a type II internal ribosome entry site (IRES). Two additional CREs exist: oriI and oriR, located within the 2C gene and 3’-NTR, respectively. IRES mediated translation yields a single polyprotein comprised of three functional domains of structural (P1) and the non-structural (P2 and P3) proteins. (<b>B</b>) Processing of the P3 region by WT and mutant PVs. Two pathways, major and minor, exist for P3 processing. Major pathway used for WT PV is shown and produces only 3AB and 3CD because of cleavage at Gln-Gly junction between 3B and 3C. EG PV changes the 3B-3C junction to Glu-Gly, producing the normal products at a reduced rate. GG PV changes the 3B-3C junction to Gly-Gly, which is uncleavable, inducing aberrant processing and producing 3ABC and 3D instead 3AB and 3CD.</p

Differences in the ultrastructural changes caused by GG PV when compared to WT PV are caused by differences in processing of the P3 polyprotein but not the levels of proteins produced.

<p>(<b>A</b>) 5-nitrocytidine (5-NC) inhibits the viral RdRp and reduces the rate and yield of viral protein and RNA. HeLa cells were transfected with a firefly luciferase-expressing subgenomic replicon in the absence (- 5-NC) or presence (+ 5-NC) of 2 mM 5-NC. Replication was monitored as a function of time post-transfection by monitoring luciferase activity (relative light units per microgram, RLU/μg). (<b>B</b>) Ultrastructural analysis was performed of cells in the absence or presence of 5-NC at 7 h post-infection. Bar = 1 μm. N denotes nucleus. (<b>C</b>) mRNA coding for WT or GG versions of the 2BCP3 polyprotein in frame with a GFP protein that could be released from the polyprotein by 3C protease activity was transfected into HeLa cells and processed 2C protein was detected by Western blotting. (<b>D</b>) Ultrastructural analysis was performed on cells prepared as described in panel C. The WT polyprotein produces vesicular-tubular structures (highlighted by the blue and red boxes). The GG polyprotein produces mostly tubular structures (some of which are marked by an arrowhead). Bar = 1 μm. N denotes nucleus.</p

EG PV exhibits a delay in the onset of RNA synthesis.

<p>(<b>A</b>) Kinetics of replication of subgenomic replicon by WT (■) and EG (□) monitored indirectly by luciferase activity. HeLa cells were transfected with <i>in vitro</i> transcribed replicon RNA, placed at 37°C and luciferase activity (RLU/μg) measured at the indicated times post-transfection. A control for translation of transfected replicon RNA was performed in the presence of 3 mM GuHCl (●). (<b>B</b>) Kinetics of replication of subgenomic replicon by WT (●) and EG (○) monitored by Northern blotting. HeLa cells were transfected with <i>in vitro</i> transcribed replicon RNA, placed at 37°C, and at the indicated times post-transfection, cells were harvested for total RNA isolation. Total RNA was separated on a 0.6% agarose gel containing 0.8 M formaldehyde, transferred to nylon membrane and hybridized with a ³²P-labeled DNA probe. (<b>C</b>) Image of a representative blot visualized by phosphorimaging. <i>In vitro</i> transcribed RNA (<b>*</b>) is shown as reference.</p

Kinetics of genome replication precedes the kinetics of vesicular cluster formation for EG PV.

<p>(<b>A</b>) Kinetics of RNA synthesis (○) and virus production (●) by EG PV. HeLa cells were infected with EG PV at an MOI of 10, placed at 37°C, and at the indicated times post-infection, total RNA was isolated and subjected to either Northern blotting or assayed for virus production by standard plaque assay. (<b>B</b>) Image of a representative blot visualized by phosphorimaging. <b>(C)</b> Kinetics of formation of virus-induced vesicular clusters by transmission electron microscope (TEM). Vesicular clusters begin to form at 4 h post-infection and continue throughout the time course are indicated by white dotted circles. HeLa cells were infected with EG PV at MOI of 10, placed at 37°C, and at the indicated times post-infection, infected cells were fixed and visualized by TEM, bar = 1 μm. UN denotes uninfected control; N denotes nucleus.</p

EG PV exhibits delayed induction and redistribution of PI4P.

<p><b>(A)</b> Immunostaining of PI4P in mock-infected HeLa cells. <b>(B)</b> Time-course of PI4P-staining in HeLa cells infected with WT or EG PV. HeLa cells were infected with WT or EG virus at an MOI of 10, fixed at indicated times post-infection, and immunostained for PI4P. <b>(C)</b> Impact of GuHCl on PI4P induction by WT and EG PVs. HeLa cells were either incubated with PBS or infected with WT or EG virus (MOI 10) in presence of 3 mM GuHCl and immunostained. In all cases, PI4P was stained using anti-PI4P antibody (red) and nuclei were stained with DAPI (blue).</p

GG PV induces and redistributes PI4P in spite of impaired formation of vesicular clusters.

<p>(<b>A</b>) Kinetics of RNA synthesis by WT (●) and GG (Δ) subgenomic replicon RNA. HeLa cells were co-transfected with two different replicon RNAs, luciferase replicon (2 μg) and EGFP replicon (4 μg), placed at 34°C and at the indicated times post-transfection, luciferase activity was measured. (<b>B</b>) Cell sorting to isolate PV replicon-positive cells. WT replicon RNA-transfected cells were 61% positive in pre-sort cells (top-left) and 98% positive in post-sort cells (bottom-left). GG replicon RNA transfected cells were 18% positive in pre-sort cells (top-right) and 94% positive in post-sort cells (bottom-right). (<b>C</b>) WT and GG PV-induced membranes visualized by TEM. Vesicular clusters that form with the WT replicon are indicated by a white dotted circle. Vesicular clusters are not observed with the GG replicon. The right most panel is an enlargement of the area indicated by the black box in the middle panel for the GG replicon. The tubular/reticular network that forms with the GG replicon is indicated by white arrows. HeLa cells were transfected with either WT or GG replicon RNA, placed at 34°C for 5 h or 14 h, respectively, at which time cells were fixed and visualized by TEM. Bar = 1 μm. N denotes nucleus. <b>(D)</b> Kinetics of PI4P induction and redistribution by the GG PV subgenomic replicon. HeLa cells were transfected with replicon RNA expressing EGFP and samples were fixed at the indicated time post-transfection and subjected to IFM using anti-PI4P antibody (red) and nuclei were stained with DAPI (blue).</p

Induction and redistribution of PI4P are not sufficient to disrupt the Golgi.

<p><b>(A)</b> Immunostaining of PI4P and Giantin in mock-infected HeLa cells. <b>(B,C)</b> PI4P and Giantin-staining in HeLa cells infected with WT or EG PV at the indicated times post-infection. (<b>D</b>) Immunostaining of PI4P and Giantin in mock-infected HeLa in the presence of 3 mM GuHCl. <b>(E,F)</b> PI4P and Giantin-staining in HeLa cells infected with WT or EG PV in the presence of 3 mM GuHCl. In all cases, PI4P was stained using anti-PI4P antibody (red), Giantin (green) and nuclei were stained with DAPI (blue).</p

Kinetics of genome replication precedes the kinetics of vesicular cluster formation for WT PV.

<p>(<b>A</b>) Kinetics of RNA synthesis (○) and virus production (●) by WT PV. HeLa cells were infected with WT PV at an MOI of 10, placed at 37°C, and at the indicated times post-infection, total RNA was isolated and subjected to either Northern blotting or assayed for virus production by standard plaque assay. (<b>B</b>) Image of a representative blot visualized by phosphorimaging. (<b>C</b>) Kinetics of formation of WT PV-induced vesicular cluster formation was visualized by TEM. Vesicular clusters begin to form at 3 h post-infection and continue throughout the time course are indicated by white dotted circles. HeLa cells were infected with WT PV at an MOI of 10, placed at 37°C, and at the indicated times post-infection, the infected cells were fixed and visualized by TEM, bar = 1 μm. UN denotes uninfected control; N denotes nucleus.</p