Mouse Hepatitis Coronavirus A59 Nucleocapsid Protein Is a Type I Interferon Antagonist

The recent emergence of several new coronaviruses, including the etiological cause of severe acute respiratory syndrome, has significantly increased the importance of understanding virus-host cell interactions of this virus family. We used mouse hepatitis virus (MHV) A59 as a model to gain insight into how coronaviruses affect the type I alpha/beta interferon (IFN) system. We demonstrate that MHV is resistant to type I IFN. Protein kinase R (PKR) and the alpha subunit of eukaryotic translation initiation factor are not phosphorylated in infected cells. The RNase L activity associated with 2′,5′-oligoadenylate synthetase is not activated or is blocked, since cellular RNA is not degraded. These results are consistent with lack of protein translation shutoff early following infection. We used a well-established recombinant vaccinia virus (VV)-based expression system that lacks the viral IFN antagonist E3L to screen viral genes for their ability to rescue the IFN sensitivity of the mutant. The nucleocapsid (N) gene rescued VVΔE3L from IFN sensitivity. N gene expression prevents cellular RNA degradation and partially rescues the dramatic translation shutoff characteristic of the VVΔE3L virus. However, it does not prevent PKR phosphorylation. The results indicate that the MHV N protein is a type I IFN antagonist that likely plays a role in circumventing the innate immune response

Cellular localization of epigenetic and other transcription-related molecules in CA1 of the hippocampus from AD and ND cases.

<p>All tissue samples were counterstained with neutral red for cell layer and cell landmark verification. (a1) high power micrograph of CA1 labeled with an antibody to DNMT1 in a typical ND case, showing appropriate cellular distribution. Comparable field in a typical AD case shows cytoplasmic accumulation and nuclear loss of DNMT1 immunoreactivity (a2). Figures (c1) and (c2) show normal nuclear immunoreactivity for both ND and AD respectively in the pathologically spared cerebellum. Similar patterns of immunoreactivity were observed for RNA pol2 in ND (b1) and AD (b2) in CA1 of hippocampus and ND (d1) and AD (d2) in cerebellum. (Scale bars = 15 µM). (e) Quantification of the cellular distribution of immunoreactivity in CA1 of the hippocampus in 5 AD and 5 ND samples. 100 neurons from CA1 of AD and ND cases were quantified and the mean signal/group/area (i.e. signal intensity/AD or ND/nuclear, cytoplasmic or both) was analyzed. Neurons were evaluated by delineating the nucleus (dashed circle) from cytoplasm (solid black line). (f) DNMT1 and RNA pol II affymetrix array data from AD (n = 10) and ND (n = 10) hippocampal CA1 neurons (500 neurons/case). * Indicates significant difference compared to control, 2-tailed t-test p = <.05.</p

Reduced RAN Expression and Disrupted Transport between Cytoplasm and Nucleus; A Key Event in Alzheimer’s Disease Pathophysiology

<div><p>Transcription of DNA is essential for cell maintenance and survival; inappropriate localization of proteins that are involved in transcription would be catastrophic. In Alzheimer’s disease brains, and <em>in vitro</em> studies, we have found qualitative and quantitative deficits in transport into the nucleus of DNA methyltransferase 1 (DNMT1) and RNA polymerase II (RNA pol II), accompanied by their abnormal sequestration in the cytoplasm. RAN (<em>RA</em>s-related <em>N</em>uclear protein) knockdown, by siRNA and oligomeric Aβ42 treatment in neurons, replicate human data which indicate that transport disruption in AD may be mechanistically linked to reduced expression of RAN, a pivotal molecule in nucleocytoplasmic transport. <em>In vitro</em> studies also indicate a significant role for oligomeric Aβ42 in the observed phenomena. We propose a model in which reduced transcription regulators in the nucleus and their increased presence in the cytoplasm may lead to many of the cellular manifestations of Alzheimer’s disease.</p> </div

mRNA expression from laser captured neurons in the CA1 of the hippocampus, superior frontal gyrus, and visual cortex using Affymetrix Human Genome U133 plus 2.0 microarrays.

<p>mRNA data from all three brain regions show a significant decreases in RAN and RAN binding proteins, with lesser amounts in the visual cortex, an area with only modest AD pathology <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053349#pone.0053349-Braak1" target="_blank">[16]</a>. With the exception of RANBP6 in the hippocampus, all other RAN binding proteins were significantly down in AD compared to controls.</p

The antibodies used, sources, dilutions and recognition motif.

<p>The antibodies used, sources, dilutions and recognition motif.</p

RAN knock down <i>in vitro</i> replicates neuronal distribution of nuclear proteins.

<p>Neuroblastoma cells were treated with 100 nM RAN SNArpol siRNA, a concentration that reduced RAN levels by 71% (a), but did not materially affect cell viability (b). Untreated, tranfection reagent only (c1, d1) and treated, RAN siRNA (c2–d2) cells were immunoreacted for RNA polII (c1, c2), and DNMT1 (d1, d2). After 48 hours, RAN knockdown induced cytoplasmic accumulation and nuclear losses in both target proteins, recapitulating <i>in vivo</i> observations in AD brain. DNMT1 IR after siRNA treatment was largely localized in axons (d2, arrows), RNA pol2 immunoreactivity was chiefly located in the cytosol (c1, arrows).</p

Decreased Ran protein and message in CA1 of AD hippocampus.

<p>Confocal micrographs (488 nm bandwidth, green fluorphore) reveals robust nuclear and cytoplasmic RAN immunoreactivity in ND hippocampal CA1 neurons (a), compared to AD (b). Pathologically spared cerebellum show similar patterns of immunoreactivity in both ND (c) and AD (d) samples in all but the larger Purkinje neurons (asterisks). (e) Significant AD decrements in RAN protein were confirmed in Western blot analyses (p<0.0001). (f) 40X micrographs show normal cytoplasmic and nuclear distributions of RAN message by fluorescence <i>in situ</i> hybridization in ND. By contrast, there was a significant decrease (p = 0.00001) in overall signal in comparable fields from AD, with limited reactivity in the cytoplasm and slightly greater reactivity in the nucleus (g). (h) Mean RAN expression, and fold change (i) from Affymetrix array data looking at AD (n = 10) and ND (n = 10) hippocampal CA1 neurons (500neurons/case). *Indicates significant difference between AD and control at p<0.05. Scale bars = 15 µm.</p

<i>In vitro</i> Aβ42 treatment replicates human neuronal distributions of nuclear proteins.

<p>Micrographs of DNMT1 and RNA pol II in neurons (SK-N-Be(2)) treated with higher molecular weight (MW) oligomers of Aβ42 (1 uM) or lower MW oligomers of Aβ42 (1 uM) for 36 hours. High power micrograph (40X) of cultures labeled with an antibody to RAN before (a1) and after treatment with higher MW oligomeric Aβ42 (a2) or lower MW oligomeric Aβ42 (a3); shows nuclear and cytoplasmic loss with nuclear envelope accumulation (arrows), similar to that seen <i>in vitro</i>. Western blot analysis confirms these data of an overall reduction in basal RAN protein levels when treated with oligomeric abeta (d). Normal distributions for nuclear molecules DNMT1 (b1), and RNA pol II (c1) was readily apparent in the nucleus of untreated neurons, but translocation to the cytoplasm is seen in both molecules when treated with high or lower MW oligomeric Aβ42; Dnmt1 (b2, b3), and RNA pol II (c2, c3). e) Mean nuclear fluorescence intensity and mean cytoplasmic fluorescence intensity (f) of nerve cells treated with either low MW Aβ42 oligomers, or high MW Aβ42 oligomers. Asterisk (*) signifies a significant difference compared to control samples (p<0.05). Data are presented as mean +/− S.E.M. (g) Western blot analysis using oligomeric antibody A11, revealed the presence of oligomers in both preparations, with higher MW oligomers in lane 1 (96 hour aggregation), compared to lane 2 (immediately frozen). (Scale bars = 15 um).</p