Two Types of Functionally Distinct Fiber Containing Structural Protein Complexes Are Produced during Infection of Adenovirus Serotype 5

<div><p>Adenoviruses are common pathogens. The localization of their receptors coxsackievirus and adenovirus receptor, and desmoglein-2 in cell-cell junction complexes between polarized epithelial cells represents a major challenge for adenovirus infection from the apical surface. Structural proteins including hexon, penton base and fiber are excessively produced in serotype 5 adenovirus (Ad5)-infected cells. We have characterized the composition of structural protein complexes released from Ad5 infected cells and their capacity in remodeling cell-cell junction complexes. Using T84 cells as a model for polarized epithelium, we have studied the effect of Ad5 structural protein complexes in remodeling cell-cell junctions in polarized epithelium. The initial Ad5 infection in T84 cell culture was inefficient. However, progressive distortion of cell-cell junction in association with fiber release was evident during progression of Ad5 infection. Incubation of T84 cell cultures with virion-free supernatant from Ad5 infected culture resulted in distortion of cell-cell junctions and decreased infectivity of Ad5-GFP vector. We used gel filtration chromatography to fractionate fiber containing virion–free supernatant from Ad5 infected culture supernatant. Fiber containing fractions were further characterized for their capacity to inhibit the infection of Ad5-GFP vector, their composition in adenovirus structural proteins using western blot and LC-MS/MS and their capacity in remolding cell-cell junctions. Fiber molecules in complexes containing penton base and hexon, or mainly hexon were identified. Only the fiber complexes with relatively high content of penton base, but not the fiber-hexon complexes with low penton base, were able to penetrate into T84 cells and cause distortion of cell-cell junctions. Our findings suggest that these two types of fiber complexes may play different roles in adenoviral infection.</p></div

Remodeling of cell-cell junction between T84 epithelial cells by virion-free supernatant from Ad5 infected culture.

<p>Virion-free supernatant from Ad5-infected A549 culture at day 3 post infection with Ad5 at a MOI of 1 were prepared as conditioned media (CM) by ultra-centrifugation at 108 000 g. (A) T84 cells cultured on the insert of transwell until the development of TJ were apically treated with 100 μl Ad5 CM (corresponding to 4 functional units of fiber molecules) for 6 hr. CAR and DSG-2 were immuno-labeled with rabbit anti-CAR antibody and mouse anti-DSG-2 antibody followed by Alexa Flour 488 conjugated donkey anti-rabbit IgG and Alexa Flour 568 conjugated donkey-anti mouse IgG. The localization of CAR (green) and DSG-2 (red) in the control (ctrl, treated with supernatant from non-infected A549 cultures prepared in parallel to the CM from Ad5 infected A549 cultures) and Ad5 CM-treated T84 cultures are documented. Data in the XZ planes show the rearrangement of CAR and DSG2 localization. (B) T84 cells cultured as in (A) were basolaterally treated with Ad5 CM for 6 hr. Data on XY plane show diffuse CAR staining at cell-cell junction and cytoplasmic staining of CAR, as well as fiber staining following supernantant treatment; data on XZ plane show the apex-localization of CAR. Internalization of CAR and fiber molecules were found following either apical or basolateral application of Ad5 CM. Cells were visualized using Zeiss confocal microscopy LSM510. Bar: 10 μm.</p

Identification and characterization of structure protein complexes in the Ad5 CM.

<p>(A) Elution profile of gel filtration chromatography. One ml of concentrated Ad5 CM was separated by Superdex 200 column at a flow rate of 0.5 ml/min. Protein concentration of each fraction was monitored with absorbance at 280 nm (Y axis). The indicated fractions (arrow), named according to their positions on the collection rack, were selected for the analyses performed in panels B to E. (B) Upper panel: western-blot analysis for the detection of fiber (F) and penton base (PB) in the fractions from (A); lower panel: SimplyBlue staining in the corresponding SDS-PAGE gel. Fiber molecules were abundant in B12 and C5 fractions, penton base mainly in B12 fraction. (C) Identification of Ad structure proteins in fiber-containing fractions by cell surface binding assay. A549 cells were incubated with 200 μl of the indicated fractions for 15 min on ice. Cells were then washed and incubated with the 4D2 anti-fiber mAb in combination with rabbit anti-penton base antibody, followed by staining with Alexa Flour 647 conjugated goat anti-mouse IgG and Alexa Flour 488 conjugated goat anti-rabbit antibody. Parallel samples stained with 4D2 mAb in combination with FITC-conjugated goat anti-hexon antibody were then stained with Alexa Flour 647 conjugated goat anti-mouse IgG. The cells were analyzed in FACS Calibur for the double stained cells. The dot-plots shown are representative of three independent experiments. Fiber, penton base and hexon were detected in B12 fraction, whereas only hexon and fiber were detected in C5 fraction. (D) Characterization of structural protein complexes by native PAGE and western-blot analysis. Proteins in different fractions were separated by pre-casted 4–20% native PAGE gel and blotted onto PVDF membrane. The membrane was probed with anti-fiber, anti-penton base and anti-hexon antibodies simultaneously and then further probed with the secondary antibodies used in (C). The membrane was scanned by a PharosFX Plus system (Bio-Rad) using 3-laser channels for the visualization of specific binding bands for different antigens. SimplyBlue protein staining of the native PAGE gel (left panel) depicts that B12 and C5 fraction contain protein complexes of about 720 kDa and 440 kDa, respectively. The area on the gel corresponding to the western-blot data shown in the right panel is indicated by the rectangle. Fiber, hexon and penton base were detected in the B12 fraction, whereas fiber and hexon were detected in the C5 fraction. (E) Capacity of fiber containing fractions in inhibition of Ad5-GFP infection in A549 cells. The fractions containing fiber molecule (B10, B12, C5 and D7) were first concentrated by a factor of 5 and subsequently diluted as indicated with fresh cell culture media, 200 μl of each dilution were applied to each well of A549 cells cultured in 24-well plates for 30 min at 37°C. Following two washes with fresh media, cells were infected with Ad5-GFP virus at a MOI of 100 for 1 hr at 37°C. The percentage of infected cells was analyzed with FACS assay as GFP positive cells at 24 hpi. NC: negative control samples (mock infection); PC: positive control samples (infection without pre-treatment with Ad5 CM).</p

Consequences of cell-cell junction remodeling on Ad infection.

<p>(A) T84 cultures with well-developed cell-cell junctions were pre-treated with Ad5 CM (corresponding to 4 functional units of fiber molecules) or with supernatant from mock infected culture from the apical surface for 6 hr, and subsequently infected with Ad5-GFP or Ad5F35-GFP at a MOI of 200 and 100, respectively. The infected cells were visualized by GFP at 48 hpi. Pre-treatment with Ad5 CM inhibited the infection of Ad5-GFP but promoted the infection of Ad5F35-GFP. Images were taken using an Olympus IX70 microscopy with a 20x objective. (B) Average fluorescent focus forming units (FFU) per slide of 6 individual photos as shown in (A). Each treatment was preformed in triplicate and 2 photos were taken from each trial. The FFU were analyzed and calculated by ImageJ program (n = 6, *: P < 0.05, ***: P < 0.001, <i>t</i>-test). The data shown are representative of three experiments performed.</p

No detectable reduction in the expression of CAR and DSG-2 following treatment with B12 fraction.

<p>Images of merged optical sections of the CAR and DSG-2 stainings shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0117976#pone.0117976.g007" target="_blank">Fig. 7</a> are shown. No obvious reduction in the staining of CAR and DSG-2 following treatment with B12 fraction was detected.</p

Fiber is released and TJs exhibit progressive distortion over time after Ad5 infection of polarized T84 cultures from the apical surface.

<p>(A) Fiber molecules were already released prior to cell lysis from infected cells. T84 cells cultured on transwell inserts with well-developed TJs were infected with Ad5 at a MOI of 20 pfu/cell from the apical surface. Cultures were washed twice with the DMEM-F12 medium at 4 hrs post infection. Merged and separated images of hexon (green) and fiber (red) staining show that at 30 hpi, fiber molecules were released from infected cells positively stained with hexon and bound to non-infected bystander cells. Bar: 20 μm. (B) Progressive distortion of TJs. At 30 hpi, CAR staining (green) was predominantly localized within distinct TJs at the apex of T84 cell layer. At 60 hpi, distorted staining pattern of CAR was detected at the apex, basolateral membrane and in cytoplasma of T84 cells. Internalized fiber molecules (red dots, arrow heads) and co-localization of internalized fiber and CAR (yellow dots, arrows) in non-infected cells are indicated. Bar: 10 μm. Cells were visualized using Zeiss confocal microscopy LSM510. Images are representative of multiple experiments performed in this project. The crosshairs in the plots indicate the positions for the XZ plane.</p

Only penton base-containing fiber complexes can penetrate into host cells.

<p>T84 cells cultured on 8-well chamber slides were incubated with protein fractions B12, C5 and D7 at 5x dilutions in fresh media for 1h at 37°C. Cells were then fixed, permeabilized and double labeled with anti-fiber and anti-penton base antibodies or with anti-fiber and anti-hexon antibodies. (A) Data from co-staining of penton base and fiber. (B) Data from co-staining of fiber and hexon. Fiber, penton base and hexon were co-localized intracellularly in cells incubated with fraction B12. Fiber molecule clearly labeled the cell-cell borders after incubation with fraction C5. The nuclei were stained with DAPI using Vectachield mounting medium. Cells were visualized using Zeiss confocal microscopy LSM700. Data shown are representative of the results with the fractions from three independent gel filtration chromatography experiments.</p

Status of glucose intolerance in female normal population and female adult breast cancer patients at initial diagnosis and during chemotherapy.

<p>Abbreviation: NP- Normal Population <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0093630#pone.0093630-Yang1" target="_blank">[14]</a>, BCID- breast cancer patients at initial diagnosis, BCDC- breast cancer patients during chemotherapy, NGT: Normal glucose tolerance, IIFG: Isolated impaired fasting glucose, IIGT: Isolated impaired glucose tolerance, CIFGIGT: combined impaired fasting glucose and impaired glucose tolerance, PUD: Previously undiagnosed diabetes, PDD: Previously diagnosed diabetes.</p

Component of diabetes and prediabetes among female breast cancer patents after initial diagnosis & during chemotherapy.

<p>Abbreviation: IIFG: Isolated impaired fasting glucose, IIGT: Isolated impaired glucose tolerance, CIFGIGT: Combined impaired fasting glucose and impaired glucose tolerance, PUD: Previously undiagnosed diabetes, PDD: Previously diagnosed diabetes.</p

U0126 treatment reduces the dwelling of PDGFRA in recycling system.

<p>A. Representative confocal images of PDGFRA and Rab11 staining in glioma tissues of sample #2. B. The same condition as in A, but EEA-1 was detected. C. Immunostaining of PDGFRA and Rab11 with or without U0126 treatment in cell line #2. Arrows indicate the colocalized clusters. D. Average of PDGFRA co-localization to Rab11 detected under conditions as in C (18 cells in each condition, ***p<0.001, <i>t</i>-test). E. Immunostaining of PDGFRA and EEA-1 before and after U0126 treatment in cell line #2. Arrows indicate the colocalized clusters. F. Average of PDGFRA co-localization to EEA-1 under the same conditions as in E (28 cells in each condition, **p<0.01, <i>t</i>-test). G. Same as in E but Giantin was detected. The three separated images (right), the top, middle and bottom indicating Giantin, PDGFRA and respectively, are the zoom-in from box in left picture. H. Average of PDGFRA co-localization to Giantin under the same conditions as in G (42 cells in each condition. **p<0.01, <i>t</i>-test).</p