Engineering polypeptide coatings to augment gene transduction and in vivo stability of adenoviruses.

We sought to modify adenoviral (Ad) particles by incorporating the advantageous characteristics of non-viral gene delivery vehicles to complement the viral vectors. α-Amino acid-N-carboxyanhydride chemistry was used to synthesize homopolypeptides and diblock copolypeptides that possess well-defined secondary structures. Using cryo-electron and fluorescence microscopy, we showed that these polypeptides can coat the surfaces of Ad particles in a non-covalent manner to modify their transduction properties. The coated Ad particles were found to bind to and be internalized by cells. In contrast to reports using covalently PEGylated Ad particles, we found that our physically coated Ad hybrid complexes facilitate gene transfer both in vitro and in vivo. We showed that our polypeptide coating was able to shield the Ad particles from the neutralizing effect of antibodies and mitigate the binding of blood coagulation factor (Factor X) in vitro. The coating also reduced the antigenicity of Ad in immunocompetent mice. The biodistribution of the systemically administered hybrid complexes mirrored the behavior of both viral and non-viral vectors, exhibiting liver tropism as well as enhanced lung transduction. These data demonstrated that our non-covalent modification was able to alter Ad's interactions with cells and organs with retention of transduction efficiency. Advantages such as facile coating of the Ad vector, design flexibility and ease of attaching ligands to the polypeptides make this system potentially useful as a platform for adding functionalities to Ad to target cancer metastasis.

AlphaB-crystallin is found in detergent-resistant membrane microdomains and is secreted via exosomes from human retinal pigment epithelial cells.

αB-crystallin (αB) is known as an intracellular Golgi membrane-associated small heat shock protein. Elevated levels of this protein have been linked with a myriad of neurodegenerative pathologies including Alzheimer disease, multiple sclerosis, and age-related macular degeneration. The membrane association of αB has been known for more than 3 decades, yet its physiological import has remained unexplained. In this investigation we show that αB is secreted from human adult retinal pigment epithelial cells via microvesicles (exosomes), independent of the endoplasmic reticulum-Golgi protein export pathway. The presence of αB in these lipoprotein structures was confirmed by its susceptibility to digestion by proteinase K only when exosomes were exposed to Triton X-100. Transmission electron microscopy was used to localize αB in immunogold-labeled intact and permeabilized microvesicles. The saucer-shaped exosomes, with a median diameter of 100-200 nm, were characterized by the presence of flotillin-1, α-enolase, and Hsp70, the same proteins that associate with detergent-resistant membrane microdomains (DRMs), which are known to be involved in their biogenesis. Notably, using polarized adult retinal pigment epithelial cells, we show that the secretion of αB is predominantly apical. Using OptiPrep gradients we demonstrate that αB resides in the DRM fraction. The secretion of αB is inhibited by the cholesterol-depleting drug, methyl ß-cyclodextrin, suggesting that the physiological function of this protein and the regulation of its export through exosomes may reside in its association with DRMs/lipid rafts.

Subnanometer-resolution structures of the grass carp reovirus core and virion.

Grass carp reovirus (GCRV) is a member of the Aquareovirus genus of the family Reoviridae, a large family of double-stranded RNA (dsRNA) viruses infecting plants, insects, fishes and mammals. We report the first subnanometer-resolution three-dimensional structures of both GCRV core and virion by cryoelectron microscopy. These structures have allowed the delineation of interactions among the over 1000 molecules in this enormous macromolecular machine and a detailed comparison with other dsRNA viruses at the secondary-structure level. The GCRV core structure shows that the inner proteins have strong structural similarities with those of orthoreoviruses even at the level of secondary-structure elements, indicating that the structures involved in viral dsRNA interaction and transcription are highly conserved. In contrast, the level of similarity in structures decreases in the proteins situated in the outer layers of the virion. The proteins involved in host recognition and attachment exhibit the least similarities to other members of Reoviridae. Furthermore, in GCRV, the RNA-translocating turrets are in an open state and lack a counterpart for the sigma1 protein situated on top of the close turrets observed in mammalian orthoreovirus. Interestingly, the distribution and the organization of GCRV core proteins resemble those of the cytoplasmic polyhedrosis virus, a cypovirus and the structurally simplest member of the Reoviridae family. Our results suggest that GCRV occupies a unique structure niche between the simpler cypoviruses and the considerably more complex mammalian orthoreovirus, thus providing an important model for understanding the structural and functional conservation and diversity of this enormous family of dsRNA viruses.