Protein crystals in adenovirus type 5-infected cells: requirements for intranuclear crystallogenesis, structural and functional analysis

Intranuclear crystalline inclusions have been observed in the nucleus of epithelial cells infected with Adenovirus serotype 5 (Ad5) at late steps of the virus life cycle. Using immuno-electron microscopy and confocal microscopy of cells infected with various Ad5 recombinants modified in their penton base or fiber domains, we found that these inclusions represented crystals of penton capsomers, the heteromeric capsid protein formed of penton base and fiber subunits. The occurrence of protein crystals within the nucleus of infected cells required the integrity of the fiber knob and part of the shaft domain. In the knob domain, the region overlapping residues 489–492 in the FG loop was found to be essential for crystal formation. In the shaft, a large deletion of repeats 4 to 16 had no detrimental effect on crystal inclusions, whereas deletion of repeats 8 to 21 abolished crystal formation without altering the level of fiber protein expression. This suggested a crucial role of the five penultimate repeats in the crystallisation process. Chimeric pentons made of Ad5 penton base and fiber domains from different serotypes were analyzed with respect to crystal formation. No crystal was found when fiber consisted of shaft (S) from Ad5 and knob (K) from Ad3 (heterotypic S5-K3 fiber), but occurred with homotypic S3K3 fiber. However, less regular crystals were observed with homotypic S35-K35 fiber. TB5, a monoclonal antibody directed against the Ad5 fiber knob was found by immunofluorescence microscopy to react with high efficiency with the intranuclear protein crystals in situ. Data obtained with Ad fiber mutants indicated that the absence of crystalline inclusions correlated with a lower infectivity and/or lower yields of virus progeny, suggesting that the protein crystals might be involved in virion assembly. Thus, we propose that TB5 staining of Ad-infected 293 cells can be used as a prognostic assay for the viability and productivity of fiber-modified Ad5 vectors

Engineered expression of the Coxsackie B and adenovirus receptor (CAR) in human dendritic cells enhances recombinant adenovirus-mediated gene transfer

Dendritic cells (DCs) are key antigen-presenting cells (APCs) that act as central modulators of cellular immune responses. Genetic modification of DCs has considerable therapeutic potential in the treatment of a wide spectrum of diseases, including cancer and persistent viral infection. In this report, we show that pre-treatment of DCs with a recombinant adenovirus encoding the major adenovirus receptor, Coxsackie B and adenovirus receptor (CAR), significantly increased the uptake of recombinant adenoviruses (Ads) by primary immature monocyte-derived DCs. This could be correlated with CAR mRNA and surface protein expression. Transduction of DCs by recombinant adenoviruses did not significantly alter cellular viability. Therefore, we propose that pre-treatment of DCs with Ad5-CAR is one strategy to increase the susceptibility of DCs to transduction by recombinant Ads

EM analysis of 293 cells infected with dual fiber-expressing recombinant Ad5/R7-ZZ_wt/E1:WTFib.

<p>(A), Portion of nucleoplasm showing adenovirions clustering around a protein crystal (Cr) viewed in cross-section. (B), Another area of nucleoplasm showing Ad particles packed into clusters. Note that several particles have an electron-luscent centre (arrows).</p

(A), Schematic representation of penton base and fiber mutant constructs in Ad5 vectors.

<p>The acronyms of the recombinants are indicated on the left side of the figure. The penton base and its RGD loops is represented by a five pointed star. The different structural domains and ligands of the fibers are shown by various symbols, as indicated in the figure. In Ad5ΔR8-21 and Ad5R7ΔKnob fibers, the extrinsic trimerization motif is represented by a stippled bar. On the right side of the figure, ‘Crystals’ indicates the occurrence of nuclear protein crystals : P, positive for crystals; N, negative; A, altered in crystal lattice arrangement. The shaded areas represent the regions of the fiber knob and shaft domains which are crucial for crystal formation. (B), Schematic representation of the Ad5 fiber knob domain and mutation positions. The end of the shaft domain is represented by a dotted line on the left side, and the knob domain by a solid line, with the amino acid numbering starting at residue 400. The β-strands regions are represented as open boxes, the flexible loops as hatched boxes, and the regions involved in CAR receptor binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894#pone.0002894-Law1" target="_blank">[31]</a> as shaded boxes. The positions of mutations are indicated by solid bars for substitutions, by open triangles for deletions, and by a solid triangle for the RGD4C insertion.</p

Model of the crystal lattice of Ad penton intranuclear inclusions.

<p>(A), 2D averaging of the Ad5/F3 crystal cross-section was generated from the crystal cross-section shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894#pone-0002894-g008" target="_blank">Fig. 8 d</a>. A total of 40 overlapping fields were cut out using the X3d program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894#pone.0002894-Conway1" target="_blank">[72]</a>, and averaged after cross-correlation. (B), 2D projection of an Ad3 penton dodecamer (dodecahedron) with its 12 fiber projections, shown at the same magnification as in (a). The 3D map of a dodecahedron with 12 fibers was filtered to 45 Å and reprojected along its 3-fold axis using SPIDER <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894#pone.0002894-Frank1" target="_blank">[73]</a>. (C), Schematic 3D representation of a portion of penton crystal, showing its three axes (arrows). (D), Dodecahedron array, presented along the XY axes. (E), Arrangement of the dodecahedron units along the YZ axes. The 3D isosurface representation shown in (D) and (E) was visualized using WEB <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894#pone.0002894-Frank1" target="_blank">[73]</a>.</p

EM (A), and immuno-EM (B) analyses of Ad5R7Δknob-infected 293 cells at 48 h pi.

<p>In panel (A), an area of nucleoplasm shows small and twinned protein crystals seen in longitudinal, oblique and cross-sections. Virions are seen dispersed in the neighbourhood. Epon-embedded specimens. In panel (B), is shown a model of the crystal lattice derived from data obtained from as in (A). In panel (C), a section of metacrylate-embedded specimen was reacted with rabbit anti-penton base antibody followed by 10-nm colloidal gold-labeled anti-rabbit IgG goat antibody. Note the accumulation of gold-labeling in the protein crystalline inclusions. Some gold grains are also seen associated with virion clusters.</p

IF microscopy of Ad-infected 293 cells at 48 h pi.

<p>(A–D) and (H–J). Conventional IF microscopy using anti-fiber monoclonal antibody TB5 and secondary Alexa Fluor®633-labeled anti-mouse IgG. (A), Ad5WTFib; (B), Ad5PbEGD; (C), Ad5H508A; (D), Ad5(HI)RGD4C (with DAPI counterstaining). (E–G), Confocal analysis of Ad5ßGalWTFib-infected 293 cells with TB5. (E), Sample reacted with TB5 Alexa Fluor®633-labeled anti-mouse IgG; (F), sample reacted with anti-penton base antibody and secondary FITC-labeled anti-rabbit IgG; (G), image overlay with DAPI nuclear staining. (H–J), Kinetics of appearance of intranuclear crystals in Ad-infected cells. 293 cells infected with Ad5WTFib were harvested at different times pi and reacted with TB5 and Alexa Fluor®633-labeled anti-mouse IgG, with DAPI counterstaining. (H), 12 h pi; (I), 16 h pi; (J), 20 h pi. Note that IF pictures are presented at low magnification to show large field of view including several crystal-containing cells (A, B ; H–J), or at higher magnification to show the mass and number of crystals within a single nucleus (C–G).</p

EM analysis of Ad5WTFib-infected 293 cells at 48 h pi.

<p>(A–C). Portions of cell nucleoplasm showing crystalline inclusions of viral proteins (Cr) seen in longitudinal sections. Note the presence of virions in close contact with crystal elements. Arrows on top of panel (A) indicate virions which seem to emerge from the edge of the crystal. Arrows on panel (B) and (C) point to filaments connecting virions to the crystal. (D), Portion of cell nucleoplasm showing a viral protein crystalline inclusion seen in cross section. (E), Model of the crystal lattice viewed in cross section, with its main parameters, diameter of the tubular unit (TD = 21.8) and inter-tubular distance (IT = 27.7), indicated in nm. In (F), a longitudinally sectioned crystal showed adenovirions included within the crystal lattice. Epon-embedded specimens.</p