Activation of PKR by tRNAs from various sources.

<p>(A) Activation assays for total tRNAs isolated from <i>E. coli</i>, bovine liver, yeast, wheat, and mitochondrial bovine liver. RNA concentrations are 0.63, 1.25, 2.5, and 5 µM. (B) PKR activation for T7 transcript and natively isolated mitochondrial tRNAs specific for methionine. RNA concentrations are 0.31, 0.63, 1.25, 2.5, and 5 µM. For each panel, a control of no RNA and a positive control of 0.1 µM dsRNA-79 are included. Phosphorylation activities are provided below each gel lane. Phosphorylation activities were normalized to dsRNA-79 and rounded to the nearest integer.</p

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

PKR is activated by and binds tightly to A14G mt-tRNA^Leu dimers.

<p>(A) Activation assays for native gel purified WT monomer and A14G dimer of mt-tRNA^Leu. RNA concentrations are 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, and 5 µM. A no RNA control and a positive control of 0.1 µM dsRNA-79 are included. Phosphorylation activities are provided below each gel lane. Phosphorylation activities were normalized to dsRNA-79 and rounded to the nearest integer. (B, C) Binding assays for dsRBD of PKR (P20) and WT and A14G mt-tRNA^Leu. Trace amount of 5′-³²P-labeled tRNA was used in the binding experiments. Protein concentrations are 0, 0.04, 0.08, 0.16, 0.32, 0.63, 1.25, 2.5, 5, 10 and 20 µM and are present in Lanes 1 to 11, respectively. For one set of binding experiments (B) herring sperm DNA competitor was used (0.1 mg/mL) and for the other (C) tRNA competitor was used (0.1 mg/mL). Binding constants for A14G (○) and WT (•) in DNA competitor were 1.3 and 5.1 µM with Hill coefficients of 3.7 and 2.9 respectively, and in the presence of tRNA competitor they were 3.1 and >20 µM with Hill coefficients of 3.1 and undetermined, respectively. Higher Hill coefficients correlate with multiple bands of lower mobility on native gels, especially in Panel C (14G); this may relate to the dimer having enough binding registers to accommodate multiple copies of P20 at one time.</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

Effect of tRNA modifications on PKR activation.

<p>(A) Secondary structure of tRNA^Phe in the cloverleaf representation. Positions of modifications are depicted in bold font, where m2G = <i>N</i>²-methylguanosine, D = dihydrouridine, m22G = <i>N</i>²,<i>N</i>²-dimethylguanosine, Cm = 2′-<i>O</i>-methylcytidine, Gm = 2′-<i>O</i>-methylguanosine, ψ = pseudouridine, m7G = 7-methylguanosine, m5C = 5-methylcytidine, and m1A = 1-methyladenosine, Y = wybutosine. (B) Activation assays for modified tRNAs (10% SDS-PAGE). RNA concentrations are 0.31, 0.63, 1.25, 2.5, 5, and 10 µM. A no RNA control and a positive control of 0.1 µM dsRNA-79 are included. Positions of modifications are depicted, where 5′-ppp = triphosphate at 5′-terminus (present on all transcripts, unless otherwise noted), 5′-OH = hydroxyl at 5′-terminus, s2U = 2-thiouridine, m5C = 5-methylcytidine, 2′-FC = 2′-fluorocytidine, and, m1A, and Cm are as defined in panel A. Phosphorylation activities are provided below each gel lane. Phosphorylation activities were normalized to dsRNA-79 and rounded to the nearest integer.</p

Activation of PKR by yeast tRNA <i>in vivo</i>.

<p>Cells were plated 8 h before transfection. Cells were transfected for 16 h with 2 µg of either 79 bp dsRNA (lane 2), native tRNA^Phe (Sigma) (lane 3), or unmodified yeast tRNA^Phe (T7) (lane 4), except in the ‘mock’ lane (lane 1) which had no RNA. The tRNA and dsRNA-79 were prepared as per <i>in vitro</i> activation experiments. The ‘mock’ and ‘dsRNA-79′ lanes serve as negative and positive controls, respectively, for PKR phosphorylation. Proteins were denatured in SDS buffer and resolved in 10% SDS-PAGE. Phosphorylated PKR (PKR-p), phosphorylated eIF2α (eIF2α-p), and GAPDH (loading control) were identified by Western blotting.</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

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

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