Distribution of the decay time in (A) PACAP and (B) L-DOPA treated PC12 cells.

<p>Histograms indicate the existence of two populations of spikes that can be well fitted with two Gaussian functions. PACAP reduces the proportion of rapid spikes of the decay time. Moreover, PACAP shortens the decay time of both fast and slow spikes. In contrast, distributions of decay time of both fast and slow spikes are shifted to the right by the treatment of L-DOPA.</p

Representative amperometric foot current transients (A) and summary of foot duration, foot quantal size and mean catecholamine flux (B).

<p>Flux was computed as foot area divided by duration. Error bars represent mean ± SEM (control, 93 events; PACAP, 146 events and L-DOPA, 52 events). *** p<0.001 and ** p<0.01 vs. control, respectively (ANOVA test).</p

Representative TEM images of (A) control and (B) PACAP-treated cells.

<p>Large dense core vesicles are distributed near and far from the plasma membranes. A portion of the nucleus can be seen in the cells. Scale bars = 200 nm. (C) Mean vesicle sizes of control and PACAP-treated cells (n = 17 cells from control group, n = 24 cells for PACAP-treated group; **<i>p</i><0.01 vs. control cells, <i>t-</i>test).</p

Summary of rise time and decay time in PACAP and L-DOPA treated cells.

<p>Error bars represent mean ± SEM (control, 1767 events; PACAP, 1436 events and L-DOPA, 2246 events). Significance: *** p<0.001 vs. control, respectively (ANOVA test).</p

Histograms of the cube root of quantal size, amplitude and half-width for control and PACAP-treated PC12 cells (n = 482 events for control cells, n = 1436 events for PACAP-treated cells).

<p>Distribution of cube root transforms of the quantal sizes can be fitted with one Gaussian function in both control and PACAP-treated cells.</p

Summary of the shape characteristics of individual release events from control, PACAP-treated, and L-DOPA-treated cells.

<p>*<i>p</i><0.05 vs. control,</p><p>**<i>p</i><0.01 vs. control.</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

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