Genome-wide diversity and selective pressure in the human rhinovirus

BACKGROUND: The human rhinoviruses (HRV) are one of the most common and diverse respiratory pathogens of humans. Over 100 distinct HRV serotypes are known, yet only 6 genomes are available. Due to the paucity of HRV genome sequence, little is known about the genetic diversity within HRV or the forces driving this diversity. Previous comparative genome sequence analyses indicate that recombination drives diversification in multiple genera of the picornavirus family, yet it remains unclear if this holds for HRV. RESULTS: To resolve this and gain insight into the forces driving diversification in HRV, we generated a representative set of 34 fully sequenced HRVs. Analysis of these genomes shows consistent phylogenies across the genome, conserved non-coding elements, and only limited recombination. However, spikes of genetic diversity at both the nucleotide and amino acid level are detectable within every locus of the genome. Despite this, the HRV genome as a whole is under purifying selective pressure, with islands of diversifying pressure in the VP1, VP2, and VP3 structural genes and two non-structural genes, the 3C protease and 3D polymerase. Mapping diversifying residues in these factors onto available 3-dimensional structures revealed the diversifying capsid residues partition to the external surface of the viral particle in statistically significant proximity to antigenic sites. Diversifying pressure in the pleconaril binding site is confined to a single residue known to confer drug resistance (VP1 191). In contrast, diversifying pressure in the non-structural genes is less clear, mapping both nearby and beyond characterized functional domains of these factors. CONCLUSION: This work provides a foundation for understanding HRV genetic diversity and insight into the underlying biology driving evolution in HRV. It expands our knowledge of the genome sequence space that HRV reference serotypes occupy and how the pattern of genetic diversity across HRV genomes differs from other picornaviruses. It also reveals evidence of diversifying selective pressure in both structural genes known to interact with the host immune system and in domains of unassigned function in the non-structural 3C and 3D genes, raising the possibility that diversification of undiscovered functions in these essential factors may influence HRV fitness and evolution

Tauopathic Changes in the Striatum of A53T α-Synuclein Mutant Mouse Model of Parkinson's Disease

Tauopathic pathways lead to degenerative changes in Alzheimer's disease and there is evidence that they are also involved in the neurodegenerative pathology of Parkinson's disease [PD]. We have examined tauopathic changes in striatum of the α-synuclein (α-Syn) A53T mutant mouse. Elevated levels of α-Syn were observed in striatum of the adult A53T α-Syn mice. This was accompanied by increases in hyperphosphorylated Tau [p-Tau], phosphorylated at Ser202, Ser262 and Ser396/404, which are the same toxic sites also seen in Alzheimer's disease. There was an increase in active p-GSK-3β, hyperphosphorylated at Tyr216, a major and primary kinase known to phosphorylate Tau at multiple sites. The sites of hyperphosphorylation of Tau in the A53T mutant mice were similar to those seen in post-mortem striata from PD patients, attesting to their pathophysiological relevance. Increases in p-Tau were not due to alterations on protein phosphatases in either A53T mice or in human PD, suggesting lack of involvement of these proteins in tauopathy. Extraction of striata with Triton X-100 showed large increases in oligomeric forms of α-Syn suggesting that α-Syn had formed aggregates the mutant mice. In addition, increased levels of p-GSK-3β and pSer396/404 were also found associated with aggregated α-Syn. Differential solubilization to measure protein binding to cytoskeletal proteins demonstrated that p-Tau in the A53T mutant mouse were unbound to cytoskeletal proteins, consistent with dissociation of p-Tau from the microtubules upon hyperphosphorylation. Interestingly, α-Syn remained tightly bound to the cytoskeleton, while p-GSK-3β was seen in the cytoskeleton-free fractions. Immunohistochemical studies showed that α-Syn, pSer396/404 Tau and p-GSK-3β co-localized with one another and was aggregated and accumulated into large inclusion bodies, leading to cell death of Substantia nigral neurons. Together, these data demonstrate an elevated state of tauopathy in striata of the A53T α-Syn mutant mice, suggesting that tauopathy is a common feature of synucleinopathies

Synucleins Antagonize Endoplasmic Reticulum Function to Modulate Dopamine Transporter Trafficking

<div><p>Synaptic re-uptake of dopamine is dependent on the dopamine transporter (DAT), which is regulated by its distribution to the cell surface. DAT trafficking is modulated by the Parkinson's disease-linked protein alpha-synuclein, but the contribution of synuclein family members beta-synuclein and gamma-synuclein to DAT trafficking is not known. Here we use SH-SY5Y cells as a model of DAT trafficking to demonstrate that all three synucleins negatively regulate cell surface distribution of DAT. Under these conditions the synucleins limit export of DAT from the endoplasmic reticulum (ER) by impairment of the ER-Golgi transition, leading to accumulation of DAT in this compartment. This mechanism for regulating DAT export indirectly through effects on ER and Golgi function represents a previously unappreciated role for the extended synuclein family that is likely applicable to trafficking of the many proteins that rely on the secretory pathway.</p></div

Synucleins limit the immobile fraction of DAT.

<p>SH-SY5Y cells were transfected with DAT-mCherry and α-Syn-GFP, β-Syn-GFP, or γ-Syn-GFP and DAT-mCherry mobility was analyzed by FRAP in live Syn-positive and Syn-negative (DAT alone) cells. (A) Guide images were captured (GFP and mCherry columns) and a region of interest (120×80 pixels) was defined (white rectangles) and three pre-bleach images (I_PRE) were collected followed by bleaching a 30×15 pixel area (red rectangles) so that between 20% and 40% of DAT signal was retained (I_POST). Representative confocal images of photo-bleached cells (I_POST) are shown with bleached areas and corresponding reference areas indicated (yellow rectangles), with recovery shown at 27 s post-bleach (I∞). (B) Representative recovery curve fits of FRAP data from DAT-alone cells or DAT-mCherry co-transfected with α-Syn-GFP, β-Syn-GFP, or γ-Syn-GFP. Pre-bleach (I_PRE) and post-bleach (I_POST) intensities are indicated with dashed lines. Recovery curves normalized to I_PRE are fit to background- and bleach-corrected DAT-mCherry intensity at each time point, with plateau indicated at I∞. The immobile fraction of DAT (FI; I_PRE - I∞) in representative curves is indicated by vertical red lines at t = 27 s, and was calculated from a single exponential curve fit: I(t) = I_POST–((I∞–I_POST)*exp^(−t*K)). (C) FI data were compiled from three experiments conducted independently on five cells per condition (n = 15; mean ± SEM; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070872#pone-0070872-t001" target="_blank">Table 1</a>). FI data were analyzed by one-way ANOVA followed by Dunnet's post-hoc analysis for comparison to DAT-alone cells (***p<0.001).</p

Attenuation of the ER-Golgi transition by α-Syn, β-Syn, and γ-Syn.

<p>The ER-Golgi transition was analyzed by the tsVSVG-GFP transport assay (see Materials and methods). (A) Representative tsVSVG-GFP images from 0, 6, and 12 min after temperature shift are shown for each condition. 12 min images are paired with Gpp130 images used to define Golgi area, and images of the respective stains for α-Syn, β-Syn, or γ-Syn. (B) Transport index was calculated from 5–10 cells per condition from three independent experiments (15–30 total cells per condition). Values are presented as mean ± SEM and are analyzed by one-way ANOVA with Dunnett's post-hoc analysis at each time point for comparison to DAT alone control (*p<0.05, **p<0.01, ***p<0.001). (C) ER microsomes (ER/M) were prepared from SH-SY5Y cells transfected with DAT (100 ng/cm²) and (400 ng/cm²) of α-Syn, β-Syn, or γ-Syn and analyzed by immunoblot for expression of DAT relative to Calnexin (loading control). (D) Data are presented as percent of DAT alone control (n = 3–4; mean ± SEM). Means were analyzed by one sample t-tests for difference from a theoretical mean of 100 (* = p<0.05, ** = p<0.01).</p

Kinetic analysis of DAT-mCherry FRAP^a.

a<p>Performed on transfected SH-SY5Y cells as described (Materials and methods; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070872#pone-0070872-g002" target="_blank">Fig. 2</a>) with Kinetic Analysis module of Zeiss AIM software. ^bK of fluorescence recovery (presented as mean ± SEM) calculated for a single exponential from background corrected fluorescence intensity at each time (I(t)) according to: [I(t) = I_POST–((I∞–I_POST)*exp^(−t*K))] where I_POST  =  fluorescence intensity immediately following bleaching period and I∞  =  plateau of fitted recovery curve (I∞ normalized to 1 for calculations). ^cMobile fraction of DAT-mCherry (FM) determined according to FM  =  I∞ – I_POST. ^dImmobile fraction of DAT-mCherry (FI) determined according to FI  =  I_PRE – I∞, where I_PRE  =  fluorescence intensity immediately prior to initiation of bleaching. Data are compiled from three experiments conducted independently on five cells per condition (n = 15) and are presented as mean ± SEM and analyzed by one-way ANOVA followed by Dunnet's post-hoc analysis for difference from DAT alone control (*** = p<0.001).</p

Contribution of synuclein-dependent attenuation of ER export to synuclein modulation of DAT.

<p>Under normal conditions (A) where the Syns are absent or at low levels, DAT (red ovals) export is maintained at normal levels, with a relatively increased amount of plasma membrane (PM) distribution. When Syn expression is elevated (B) ER export and the ER-Golgi transition are attenuated, leading to retention of DAT and other secreted proteins in the ER. Thus, although overall expression of the transporter remains unchanged, DAT distribution to the PM is decreased.</p

Function and cell-surface expression of DAT is reduced by synucleins.

<p>(A) Uptake of [³H]-DA into SH-SY5Y cells co-transfected with DAT (100 ng/cm²) and increasing amounts (100–400 ng/cm²) of β-Syn (orange open circles) or γ-Syn (red open squares) was measured over 10 min. Values (n = 6; mean ± SEM) are presented as percent of DAT alone (line at 100%). Non-specific uptake in the presence of 10 μM indatraline was subtracted. As a positive control for Syn modulation of DAT, uptake was also measured in cells co-transfected with DAT and 400 ng/cm² of α-Syn (yellow triangle). Total amount of transfected DNA was kept constant at 500 ng/cm² with the addition of empty pcDNA3.1 vector. (B) Expression levels of α-Syn, β-Syn, γ-Syn, and DAT in cells used for uptake assays were confirmed by immunoblot (IB). Representative blot images are shown and quantified relative to actin in the adjacent graphs (n = 3; mean ± SEM). (C) Viability was assessed by MTT assay in SH-SY5Y cells transfected under conditions identical to the uptake experiment. Results from three independent experiments assayed in quadruplicate are expressed as absorbance (OD) at 570 nm ± SEM. (D) Cell surface protein was biotinylated in SH-SY5Y cells co-transfected at the 4∶1 ratio as above. Biotinylated DAT (IB DAT Biotin) captured with streptavidin beads and total DAT (IB DAT Total) were measured by immunoblot. DAT biotinylation was quantified as optical density (OD) of DAT Biotin divided by OD of DAT Total relative to actin (n = 4; mean ± SEM). Molecular mass (M_r) of nearest protein ladder bands is indicated. Data in were analyzed by t-test for difference from a theoretical mean of 100 (*p<0.05, **p<0.01, ***p<0.001) and corrected for multiple t-tests <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070872#pone.0070872-Bland1" target="_blank">[44]</a>.</p