Inhibition of Borna disease virus replication by an endogenous bornavirus-like element in the ground squirrel genome.

Animal genomes contain endogenous viral sequences, such as endogenous retroviruses and retrotransposons. Recently, we and others discovered that nonretroviral viruses also have been endogenized in many vertebrate genomes. Bornaviruses belong to the Mononegavirales and have left endogenous fragments, called "endogenous bornavirus-like elements" (EBLs), in the genomes of many mammals. The striking features of EBLs are that they contain relatively long ORFs which have high sequence homology to the extant bornavirus proteins. Furthermore, some EBLs derived from bornavirus nucleoprotein (EBLNs) have been shown to be transcribed as mRNA and probably are translated into proteins. These features lead us to speculate that EBLs may function as cellular coopted genes. An EBLN element in the genome of the thirteen-lined ground squirrel (Ictidomys tridecemlineatus), itEBLN, encodes an ORF with 77% amino acid sequence identity to the current bornavirus nucleoprotein. In this study, we cloned itEBLN from the ground squirrel genome and investigated its involvement in Borna disease virus (BDV) replication. Interestingly, itEBLN, but not a human EBLN, colocalized with the viral factory in the nucleus and appeared to affect BDV polymerase activity by being incorporated into the viral ribonucleoprotein. Our data show that, as do certain endogenous retroviruses, itEBLN potentially may inhibit infection by related exogenous viruses in vivo

Signaling Dependent and Independent Mechanisms in Pemphigus Vulgaris Blister Formation

<div><p>Pemphigus vulgaris (PV) is an autoimmune epidermal blistering disease caused by autoantibodies directed against the desmosomal cadherin desmoglein-3 (Dsg3). Significant advances in our understanding of pemphigus pathomechanisms have been derived from the generation of pathogenic monoclonal Dsg3 antibodies. However, conflicting models for pemphigus pathogenicity have arisen from studies using either polyclonal PV patient IgG or monoclonal Dsg3 antibodies. In the present study, the pathogenic mechanisms of polyclonal PV IgG and monoclonal Dsg3 antibodies were directly compared. Polyclonal PV IgG cause extensive clustering and endocytosis of keratinocyte cell surface Dsg3, whereas pathogenic mouse monoclonal antibodies compromise cell-cell adhesion strength without causing these alterations in Dsg3 trafficking. Furthermore, tyrosine kinase or p38 MAPK inhibition prevents loss of keratinocyte adhesion in response to polyclonal PV IgG. In contrast, disruption of adhesion by pathogenic monoclonal antibodies is not prevented by these inhibitors either in vitro or in human skin explants. Our results reveal that the pathogenic activity of polyclonal PV IgG can be attributed to p38 MAPK-dependent clustering and endocytosis of Dsg3, whereas pathogenic monoclonal Dsg3 antibodies can function independently of this pathway. These findings have important implications for understanding pemphigus pathophysiology, and for the design of pemphigus model systems and therapeutic interventions.</p> </div

Model for PV pathogenicity.

<p>Monoclonal pathogenic antibodies, such as AK23 and monovalent PV mAbs (scFv) cloned from patients, cause loss of adhesion primarily through steric hindrance, which does not require signaling through p38 MAPK. In contrast, PV IgG induce two separate pathogenic events. In addition to the steric hindrance caused by a portion of antibodies contained in PV IgG, the polyclonal nature of PV IgG causes clustering and endocytosis of Dsg3 in a p38 MAPK dependent manner.</p

Genistein and SB202190 prevent adhesion loss in skin treated with PV IgG but not AK23.

<p>(<b>A–B</b>) Human skin explant injected with PBS following a pre-treatment with DMSO. Epidermal morphology is normal (A) with no detectable bound IgG in the epidermis (B). (<b>C</b>–<b>H</b>) Human skin explants injected with PV IgG following a pre-treatment with DMSO (C,F), genistein (D,G) or SB202190 (E,H). PV IgG caused epidermal acantholysis (loss of cell-cell adhesion) typical of that observed in PV patients (C). Genistein and the p38MAPK inhibitor (SB202190) both prevented acantholysis in skin sections injected with PV patient IgG (D and E respectively). (<b>I–N</b>) Human skin explants injected with AK23 following a pre-treatment with DMSO (I,L), genistein (J,M) or SB202190 (K,N). AK23 also caused epidermal acantholysis typical of PV (I). Neither genistein nor the p38MAPK inhibitor (SB202190) prevented acantholysis in skin sections injected with AK23 (J and K respectively). PV IgG (F,G,H) and AK23 (L,M,N) deposition in the basal and immediate suprabasal layers of the epidermis was verified by direct immunofluorescence using goat anti-human and goat anti-mouse antibodies respectively. D = dermis. Scale bar, 20 µm.</p

Loss of adhesion does not cause Dsg3 endocytosis.

<p>(<b>A</b>) The Dsg3 cytoplasmic domain was fused to the non-adhesive extracellular domain of the IL-2 receptor (IL2R) alpha chain. Carboxyl terminal deletion mutants were also generated to remove various Dsg3 cytoplasmic domains as illustrated. Immunoprecipitation and western blot analysis verified expression of each construct and demonstrated that deletion of the ICS domain abolished plakoglobin binding. (<b>B–I</b>) Antibody labeling of cells incubated at 4°C demonstrated that each of the chimeric IL2R polypeptides were delivered to the cell surface (B,D,F,H). After 6 h at 37°C, cell surface levels of IL-2R were significantly diminished (C). In contrast, the non-adhesive IL-2R-Dsg3_cyto was stable at the cell surface over the 6 hr time course (E). Deletion of the DTD and RUD domains resulted in more rapid loss of surface pools of the Dsg3 chimera (F–G, J), and further removal of the IPL and ICS domains enhanced this effect (H–I, J). (<b>J</b>) Quantitative analysis of cell surface levels remaining at 6 hr normalized to chimera levels at 0 h. IA, intracellular anchor; ICS, intracellular cadherin-like sequence; IPL, intracellular proline-rich linker; RUD, repeat unit domain; DTD, desmoglein terminal domain.</p

Genistein prevents PV IgG-induced clustering and loss of adhesion.

<p><b>A–F</b>) Cell surface Dsg3 was labeled with biotin-conjugated AK23 monoclonal antibody followed by addition of NH IgG (A,B) PV IgG (C,D) or goat anti-mouse IgG (E,F) in the absence (A,C,E) or presence (B,D,F) of the pan-tyrosine kinase inhibitor genistein. Treatment of cells with genistein prevented PV IgG- and goat anti-mouse IgG induced clustering of the AK23-Dsg3 complex (D,F). (<b>G</b>) Quantitative assessment of clustering. (<b>H</b>) In the dispase-based cell dissociation assay, genistein potently prevented loss of adhesion (monolayer fragmentation) in keratinocytes treated with PV IgG. In contrast, genistein treatment was unable to prevent loss of adhesion induced by AK23. *Indicates statistically significant differences with genistein treatment (P<0.05). Scale bar, 10 µm.</p

Polyclonal and monoclonal pathogenic Dsg3 antibodies cause distinct morphological changes in desmosomes.

<p>(<b>A–C</b>) Desmoplakin (DP) localization is unchanged upon addition of NH IgG. (<b>D–I</b>) PV IgG bound to cell-cell borders in a linear and punctate pattern in cells incubated at 4°C (time 0). After 6 h at 37°C, PV IgG staining at cell borders became discontinuous (E), and was markedly disrupted by 24 h (F). DP staining also became disorganized by 6 h (H) and deteriorated further by 24 h (I). (<b>J–O</b>) In contrast, when cells were treated with AK23, the linear border staining pattern remained largely unchanged even after 24 h (J–L). Similarly, DP staining showed little disruption, although small gaps were occasionally noted between cells after 24 h (M–O). (<b>P–U</b>) Desmosomes in cells treated with NH IgG exhibit normal desmosomal morphology (P,Q). Desmosomes in PV IgG-treated cells (R, S) were smaller in size, disorganized and exhibited reduced keratin association. Desmosomes of AK23-treated cells appeared morphologically indistinguishable from control cells (T, U). (<b>V</b>) In the dispase-based dissociation assay, keratinocyte sheets treated with NH IgG remained nearly intact, whereas cells treated with either PV IgG or AK23 IgG exhibited extensive fragmentation. (<b>W–X</b>) Human skin explants injected with PV IgG (W) or AK23 (X) were analyzed by structured-illumination microscopy (SIM) to further examine PV IgG and AK23 distribution. Images are oriented with dermis down and the basal layer horizontal across the bottom of each panel. Loss of adhesion (acantholysis) is evident by the empty space above the basal layer (a portion of the suprabasal layer is still visible in panel X). See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050696#pone-0050696-g006" target="_blank">Figure 6</a> for additional histological analysis of these samples. As observed by SIM, Dsg3 clustering is apparent in skin explants in response to PV IgG (W) but not AK23. *Indicates statistical significance compared to NH IgG (P<0.05). Scale bar for A-O, 10 µm. Scale bar for P-U, 0.5 µm. Scale bar for W-X, 5 µm.</p