Abstract
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.
Highlights
In order to redirect adenovirus (Ad) virions to desired cell targets and transform them into cell-specific vectors suitable for biotherapy, diverse modifications of the adenoviral capsid have been designed and tested experimentally in various laboratories
Since the nucleoplasm of Ad-infected cells is the compartment of virion assembly [1], we investigated the Adenovirus serotype 5 (Ad5) assembly process in situ, using electron microscopy (EM), immuno-EM and immunofluorescence (IF) microscopy of cells infected with Ad5WT and a panel of Ad vectors with genetic modifications in the fiber or penton base genes
In longitudinal and cross-sections observed under different angles (Fig. 1 A–D), they appeared to be formed by the regular arrangement of a pair of concentric tubules of two different shades, a dark inner tubule surrounded by a lighter shaded one (Fig. 1 E ; see [25])
Summary
In order to redirect adenovirus (Ad) virions to desired cell targets and transform them into cell-specific vectors suitable for biotherapy, diverse modifications of the adenoviral capsid have been designed and tested experimentally in various laboratories. These modifications have mainly concerned the projecting capsomer referred to as the fiber, and more its distal globular domain (called the ‘knob’) involved in cell receptor recognition and attachment (reviewed in [1]). At the late phase of the virus life cycle, the fiber knob-CAR interaction is considered as being responsible for the disruption of the tight junctions between epithelial cells [11]. In the absence of available or accessible CAR molecules, Ad5 fiber shaft has been considered as the capsid component involved in cellular attachment through the interaction of the KKTK motif in its third repeat with cellular heparan sulfate proteoglycans [14]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
