Fusion of Large Polypeptides to Human Adenovirus Type 5 Capsid Protein IX Can Compromise Virion Stability and DNA Packaging Capacity.

The human adenovirus (HAdV) protein IX (pIX) is a minor component of the capsid that acts in part to stabilize the hexon-hexon interactions within the mature capsid. Virions lacking pIX have a reduced DNA packaging capacity and exhibit thermal instability. More recently, pIX has been developed as a platform for presentation of large polypeptides, such as fluorescent proteins or large targeting ligands, on the viral capsid. It is not known whether such modifications affect the natural ability of pIX to stabilize the HAdV virion. In this study, we show that addition of large polypeptides to pIX does not alter the natural stability of virions containing sub-wild-type-sized genomes. However, similar virions containing wild-type-sized genomes tend to genetically rearrange, likely due to selective pressure caused by virion instability as a result of compromised pIX function.IMPORTANCE Human adenovirus capsid protein IX (pIX) is involved in stabilizing the virion but has also been developed as a platform for presentation of various polypeptides on the surface of the virion. Whether such modifications affect the ability of pIX to stabilize the virion is unknown. We show that addition of large polypeptides to pIX can reduce both the DNA packaging capacity and the heat stability of the virion, which provides important guidance for the design of pIX-modified vectors.

Characterization of an antigenic chitosanase from the cellulolytic fungus Chaetomium globosum.

We are interested in identifying human fungal allergens and antigens from species common on water-damaged or damp building materials for use as marker proteins and diagnostic tests. The cellulolytic fungus Chaetomium globosum is common on damp materials in the building environment worldwide. ELISA and immunoblotting tests identified two related proteins of molecular weights 45 and 47 kDa which were identified as fungal antigens found on spore surfaces and in culture filtrate. The sequences were determined by liquid chromatography tandem mass spectrometry (LC-MS/MS), which indicated that the two proteins were chitosanases, confirmed by enzyme assay. The 47 kDa protein was not glycosylated and had an acidic pI of 4.5. These proteins have not been reported from other fungi and similar antigens were not seen in other fungi common in buildings. The production of polyclonal antibodies in rabbits showed the antigenicity of the target proteins and confirmed they were not artifacts of the isolation process. The proteins isolated are useful biomarkers for the detection of C. globosum in the building environment.

Quantification of C. globosum spores in house dust samples.

Chaetomium globosum is one of the most common fungi that grows in damp buildings and occurs in agricultural and forestry workplaces. Using sera from atopic patients, we characterized and purified an extracellular chitosanase (Chg47) from C. globosum that is antigenic to humans. The study reports the production of monoclonal antibodies to the protein. Three capture ELISAs were developed for Chg47 for detection of spores and spore and mycelial fragments in dust samples using different mono- and polyclonal antibody combinations. One method is based on an enhanced biotinylated polyclonal antibody as the secondary antibody and coating anti-IgM to capture one of two clones of IgM monoclonal antibodies as the capture antibody. The other method makes use of an enhanced rabbit polyclonal antibody as both the primary and capture antibody. The detection limit of the double PAb method for the Chg47 antigen was 7.6 pg/ml. When the anti-IgM+10B3 clone was used, the detection limit was 61 pg/ml and for anti-IgM+5F12, 122 pg/ml. The detection limit of double PAb method is comparable to methods for the allergen and spores of Aspergillus versicolor in house dust and is more sensitive than other immunoassays for allergens in house including for Stachybotrys chartarum, Aspergillus fumigatus and Alternaria alternata. All three methods had limited cross-reactivity to fungi common in house dust representing a diverse array of taxa.