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

Corrective GUSB transfer to the canine mucopolysaccharidosis VII brain

Severe deficiency in lysosomal ?-glucuronidase (?-glu) enzymatic activity results in mucopolysaccharidosis (MPS) VII, an orphan disease with symptoms often appearing in early childhood. Symptoms are variable, but many patients have multiple organ disorders including neurological defects. At the cellular level, deficiency in ?-glu activity leads to abnormal accumulation of glycosaminoglycans (GAGs), and secondary accumulation of GM2 and GM3 gangliosides, which have been linked to neuroinflammation. There have been encouraging gene transfer studies in the MPS VII mouse brain, but this is the first study attempting the correction of the \textgreater200-fold larger and challenging canine MPS VII brain. Here, the efficacy of a helper-dependent (HD) canine adenovirus (CAV-2) vector harboring a human GUSB expression cassette (HD-RIGIE) in the MPS VII dog brain was tested. Vector genomes, ?-glu activity, GAG content, lysosome morphology and neuropathology were analyzed and quantified. Our data demonstrated that CAV-2 vectors preferentially transduced neurons and axonal retrograde transport from the injection site to efferent regions was efficient. HD-RIGIE injections, associated with mild and transient immunosuppression, corrected neuropathology in injected and noninjected structures throughout the cerebrum. These data support the clinical evaluation of HD CAV-2 vectors to treat the neurological defects associated with MPS VII and possibly other neuropathic lysosomal storage diseases.Molecular Therapy (2013); doi:10.1038/mt.2013.283

Membrane Dynamics and Signaling of the Coxsackievirus and Adenovirus Receptor

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Lysosomal and network alterations in human mucopolysaccharidosis type VII iPSC-derived neurons

Mucopolysaccharidosis type VII (MPS VII) is a lysosomal storage disease caused by deficient β-glucuronidase (β-gluc) activity. Significantly reduced β-gluc activity leads to accumulation of glycosaminoglycans (GAGs) in many tissues, including the brain. Numerous combinations of mutations in GUSB (the gene that codes for β-gluc) cause a range of neurological features that make disease prognosis and treatment challenging. Currently, there is little understanding of the molecular basis for MPS VII brain anomalies. To identify a neuronal phenotype that could be used to complement genetic analyses, we generated two iPSC clones derived from skin fibroblasts of an MPS VII patient. We found that MPS VII neurons exhibited reduced β-gluc activity and showed previously established disease-associated phenotypes, including GAGs accumulation, expanded endocytic compartments, accumulation of lipofuscin granules, more autophagosomes, and altered lysosome function. Addition of recombinant β-gluc to MPS VII neurons, which mimics enzyme replacement therapy, restored disease-associated phenotypes to levels similar to the healthy control. MPS VII neural cells cultured as 3D neurospheroids showed upregulated GFAP gene expression, which was associated with astrocyte reactivity, and downregulation of GABAergic neuron markers. Spontaneous calcium imaging analysis of MPS VII neurospheroids showed reduced neuronal activity and altered network connectivity in patient-derived neurospheroids compared to a healthy control. These results demonstrate the interplay between reduced β-gluc activity, GAG accumulation and alterations in neuronal activity, and provide a human experimental model for elucidating the bases of MPS VII-associated cognitive defects

The influence of blood on in vivo adenovirus bio-distribution and transduction

Intravascular delivery of adenovirus (Ad) vectors is being developed for liver-directed gene therapy for targeting disseminated disease in cancer therapeutics and for targeting non-hepatic tissues and organs through vector engineering strategies. The utility of Ad vectors is not limited to serotype 5 (Ad5), and many alternate human serotypes and non-human serotypes of Ad are currently being investigated. Critical to intravascular delivery of Ad is the interaction of the virus with host blood cells and plasma proteins, because immediate contact is observed following injection. Although incompletely understood, recent studies suggest that these interactions are critical in dictating the particle bio-distribution and resulting transduction properties of Ad in vivo. For example, plasma proteins—in particular, vitamin K–dependent coagulation zymogens—are able to directly bind to Ad, and "bridge" the virus to receptors in the liver. Unraveling and characterizing these mechanisms will be of fundamental importance both for understanding basic Ad biology in vivo and for refinement and optimization of Ad vectors for human gene therapy