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. 2015 Feb 27;10(2):e0117976.
doi: 10.1371/journal.pone.0117976. eCollection 2015.

Two types of functionally distinct fiber containing structural protein complexes are produced during infection of adenovirus serotype 5

Bo Zhang  1 , Yuhua Yan  1 , Jie Jin  1 , Hongyu Lin  1 , Zongyi Li  2 , Xiaoyan Zhang  1 , Jin Liu  1 , Chao Xi  1 , Andre Lieber  2 , Xiaolong Fan  1 , Liang Ran  1
Affiliations

Two types of functionally distinct fiber containing structural protein complexes are produced during infection of adenovirus serotype 5

Bo Zhang et al. PLoS One. .

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Fiber is released and TJs exhibit progressive distortion over time after Ad5 infection of polarized T84 cultures from the apical surface.
(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.
Fig 2
Fig 2. Remodeling of cell-cell junction between T84 epithelial cells by virion-free supernatant from Ad5 infected culture.
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.
Fig 3
Fig 3. Consequences of cell-cell junction remodeling on Ad infection.
(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, t-test). The data shown are representative of three experiments performed.
Fig 4
Fig 4. Identification and characterization of structure protein complexes in the Ad5 CM.
(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).
Fig 5
Fig 5. Only penton base-containing fiber complexes can penetrate into host cells.
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.
Fig 6
Fig 6. Only penton base-containing fiber complexes can penetrate into host cells.
T84 cells cultured and treated as in Fig. 5 were co-stained with fiber and sodium potassium ATPase. The staining results were visualized using the same conditions as in Fig. 5. Fiber molecules were found to be predominantly intracellularly following B12 treatment.
Fig 7
Fig 7. Remodeling of cell-cell junction between T84 cells by penton base-containing fiber complexes.
T84 cells cultured on the insert of transwell with well developed TJs were basolateraly treated with media (Ctrl), Ad5 fiber knob (Ad5K, 100 ng/μl in fresh media), B12 or C5 fractions (5x diluted in fresh media) for 6 hr. Cells were then fixed, permeabilized and double stained for CAR and DSG-2. The localization of CAR (green) and DGS-2 (red) staining in consecutive optical sections taken from top to a depth of 5 μm below the apical surface is depicted. Individual optical sections show weak staining for both CAR and DSG-2 following incubation with B12 fraction. Cells were visualized using Zeiss confocal microscopy LSM510. The nuclei were stained with DAPI using Vectachield mounting medium. Data shown are representative of the results with the fractions from three independent gel filtration chromatography experiments.
Fig 8
Fig 8. No detectable reduction in the expression of CAR and DSG-2 following treatment with B12 fraction.
Images of merged optical sections of the CAR and DSG-2 stainings shown in Fig. 7 are shown. No obvious reduction in the staining of CAR and DSG-2 following treatment with B12 fraction was detected.
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