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

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

Charged Residues in Hepatitis C Virus NS4B Are Critical for Multiple NS4B Functions in RNA Replication ▿

The nonstructural 4B (NS4B) protein of hepatitis C virus (HCV) plays a central role in the formation of the HCV replication complex. To gain insight into the role of charged residues for NS4B function in HCV RNA replication, alanine substitutions were engineered in place of 28 charged residues residing in the N- and C-terminal cytoplasmic domains of the NS4B protein of the HCV genotype 1b strain Con1. Eleven single charged-to-alanine mutants were not viable, while the remaining mutants were replication competent, albeit to differing degrees. By selecting revertants, second-site mutations were identified for one of the lethal NS4B mutations. Second-site mutations mapped to NS4B and partially suppressed the lethal replication phenotype. Further analyses showed that three NS4B mutations disrupted the formation of putative replication complexes, one mutation altered the stability of the NS4B protein, and cleavage at the NS4B/5A junction was significantly delayed by another mutation. Individual charged-to-alanine mutations did not affect interactions between the NS4B and NS3-4A proteins. A triple charged-to-alanine mutation produced a temperature-sensitive replication phenotype with no detectable RNA replication at 39°C, demonstrating that conditional mutations can be obtained by altering the charge characteristics of NS4B. Finally, NS4B mutations dispensable for efficient Con1 RNA replication were tested in the context of the chimeric genotype 2a virus, but significant defects in infectious-virus production were not detected. Taken together, these findings highlight the importance of charged residues for multiple NS4B functions in HCV RNA replication, including the formation of a functional replication complex

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

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

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

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

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