PubMed · 8924990
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F A Sagliocco, P A Moore, A J Brown. 1996. Polysome analysis.. https://doi.org/10.1385/0-89603-319-8%3A297
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Understanding the primary steps of viral entry can have important implications for strategies to prevent infection of known viral pathogens as well as determining parameters for efficient gene delivery using viral vectors. Recently, a two-step process for viral infection involving attachment of virus to a primary receptor (coxsackievirus adenovirus receptor and heparan sulfate proteoglycan) and subsequent mediation of virus entry by a co-receptor (alphaV integrins and HVEM) has been determined for both adenovirus and HSV, respectively. Heparan sulfate proteoglycan serves as a primary attachment receptor for adeno-associated virus type 2 (AAV-2)(ref. 5). Here we determined that alphaVbeta5 integrin plays a part in efficient AAV infection. Experiments using the chelating agent EDTA to disrupt integrin function resulted in a corresponding decrease in AAV infection, consistent with the possibility that integrin mediates infection. Viral overlay experiments on purified plasma membrane proteins as well as immunoprecipitated integrin beta5 subunit demonstrated that AAV directly associates with the beta5 subunit of alphaVbeta5 integrin. Genetically defined cells expressing alphaVbeta5 integrin showed increased susceptibility to AAV infection, demonstrating a biological role of this integrin in AAV infection. Finally, viral binding and internalization studies indicate that alphaVbeta5 integrin is not a primary attachment receptor for AAV-2, but is instead involved in facilitating virus internalization. This study supports the idea that alphaVbeta5 integrin serves as a co-receptor for AAV-2 virions, and should have a substantial effect on the use of AAV vectors in human gene therapy.
A method for the purification of lysosomes from fibroblasts has been developed which uses endocytosis of superparamagnetic colloidal iron dextran particles followed by separation of the iron-containing lysosomes in a magnetic field. This permitted isolation of lysosomes from fibroblasts from patients with infantile sialic acid storage disorder and other lysosomal storage diseases in which a shift in lysosomal density induced by the storage material prevents purification by centrifugation in a Percoll gradient. The magnetic lysosomes isolated from these cells are very similar to those from normal cells as judged by lysosomal marker enzyme activity and 2D-PAGE analysis of the enriched proteins.
We have developed a protocol for the sequential release of the intermembrane space (IMS) content of Saccharomyces cerevisiae mitochondria. Two distinct fractions were obtained: a soluble IMS with cytochrome b2 as key marker and a salt-extractable IMS with cytochrome c as key marker. The identity of several proteins was determined by amino-terminal amino acid sequencing. The IMS fractions were devoid of contaminations from cytosol and mitochondrial outer and inner membranes. By subtraction analysis, the protein profiles of soluble and salt-extractable IMS fractions were depleted of contaminating bands derived from matrix proteins. The fractionation method will provide the basis for the further analysis of IMS proteins and characterization of their functions in bioenergetics, mitochondrial biogenesis, and regulatory processes.