RESUMO
Glycosylphosphatidylinositol anchoring is a type of post-translational modification that allows proteins to be presented on the exterior side of the cell membrane. Purified glycosylphosphatidylinositol-anchored protein can spontaneously re-insert into lipid bilayer membranes in a process termed Molecular Painting. Here, we demonstrate the possibility of inserting purified, recombinant CD59 into virus particles produced from a murine retroviral producer cell line. CD59 is a regulator of the complement system that helps protect healthy cells from the lytic activity of the complement cascade. In this study, we could show that Molecular Painting confers protection from complement activity upon murine retroviral vector particles. Indeed, increased infectivity of CD59-modified virus particles was observed upon challenge with human serum, indicating that Molecular Painting is suitable for modulating the immune system in gene therapy or vaccination applications.
Assuntos
Antígenos CD59/metabolismo , Glicosilfosfatidilinositóis/química , Bicamadas Lipídicas/metabolismo , Vírion/imunologia , Animais , Antígenos CD59/química , Antígenos CD59/genética , Linhagem Celular , Ativação do Complemento , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Retroviridae/genéticaRESUMO
In this study, we describe a versatile, flexible, and quick method to label different families of enveloped viruses with glycosylphosphatidylinositol-modified green fluorescent protein, termed fluorescence molecular painting (FMP). As an example for a potential application, we investigated virus attachment by means of flow cytometry to determine if viral binding behavior may be analyzed after FMP of enveloped viruses. Virus attachment was inhibited by using either dextran sulfate or by blocking attachment sites with virus pre-treatment. Results from the FMP-flow cytometry approach were verified by immunoblotting and enzyme-linked immunosorbent assay. Since the modification strategy is applicable to a broad range of proteins and viruses, variations of this method may be useful in a range of research and applied applications from bio-distribution studies to vaccine development and targeted infection for gene delivery.
Assuntos
Citometria de Fluxo/métodos , Fluorescência , Ligação Viral , Animais , Sulfato de Dextrana/metabolismo , Cães , Ensaio de Imunoadsorção Enzimática , Proteínas Ligadas por GPI/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Herpesviridae/crescimento & desenvolvimento , Herpesviridae/isolamento & purificação , Immunoblotting , Vírus da Influenza A/crescimento & desenvolvimento , Vírus da Influenza A/isolamento & purificação , Lentivirus/crescimento & desenvolvimento , Lentivirus/isolamento & purificação , Células Madin Darby de Rim Canino , Filogenia , Vírion/metabolismoRESUMO
Gene delivery vectors based on retroviral or lentiviral particles are considered powerful tools for biomedicine and biotechnology applications. Such vectors require modification at the genomic level in the form of rearrangements to allow introduction of desired genes and regulatory elements (genotypic modification) as well as engineering of the physical virus particle (phenotypic modification) in order to mediate efficient and safe delivery of the genetic information to the target cell nucleus. Phenotypic modifications are typically introduced at the genomic level through genetic manipulation of the virus producing cells. However, this paper focuses on methods which allow modification of viral particle surfaces after they have exited the cell, that is, directly on the viral particles in suspension. These methods fall into three categories: (i) direct covalent chemical modification, (ii) membrane-topic reagents, and (iii) adaptor systems. Current applications of such techniques will be introduced and their advantages and disadvantages will be discussed.