title: Chemical Modulation of Endocytic Sorting Augments Adeno-associated Viral Transduction.
abstract: Intracellular trafficking of viruses can be influenced by a variety of inter-connected cellular sorting and degradation pathways involving endo-lysosomal vesicles, the ubiquitin-proteasome system, and autophagy-based or endoplasmic reticulum-associated machinery. In the case of recombinant adeno-associated viruses (AAV), proteasome inhibitors are known to prevent degradation of ubiquitinated AAV capsids, thereby leading to increased nuclear accumulation and transduction. However, the impact of other cellular degradation pathways on AAV trafficking is not well understood. In the current study, we screened a panel of small molecules focused on modulating different cellular degradation pathways and identified eeyarestatin I (EerI) as a novel reagent that enhances AAV transduction. EerI improved AAV transduction by an order of magnitude regardless of vector dose, genome architecture, cell type, or serotype. This effect was preceded by sequestration of AAV within enlarged vesicles that were dispersed throughout the cytoplasm. Specifically, EerI treatment redirected AAV particles toward large vesicles positive for late endosomal (Rab7) and lysosomal (LAMP1) markers. Notably, MG132 and EerI (proteasomal and endoplasmic reticulum-associated degradation inhibitors, respectively) appear to enhance AAV transduction by increasing the intracellular accumulation of viral particles in a mutually exclusive fashion. Taken together, our results expand on potential strategies to redirect recombinant AAV vectors toward more productive trafficking pathways by deregulating cellular degradation mechanisms.
authors.affiliation: From the Gene Therapy Center, Department of Genetics, Curriculum in Genetics and Molecular Biology, and.
authors.forename: Garrett E
authors.initials: GE
authors.lastname: Berry
authors.affiliation: From the Gene Therapy Center, Department of Genetics, Department of Biochemistry and Biophysics, The University of North Carolina, Chapel Hill, North Carolina 27599-7352 aravind@med.unc.edu.
authors.forename: Aravind
authors.initials: A
authors.lastname: Asokan
mesh_terms: D000818:Animals; D002213:Capsid / Q000378:metabolism; D045744:Cell Line, Tumor; D002645:Chickens; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D011992:Endosomes / Q000187:drug effects / Q000378:metabolism; D016679:Genome, Viral; D057809:HEK293 Cells; D006801:Humans; D006835:Hydrazones / Q000494:pharmacology; D006918:Hydroxyurea / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000378:metabolism; D008954:Models, Biological; D012703:Serotyping; D014161:Transduction, Genetic; D020691:rab GTP-Binding Proteins / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins; D052118:Lysosomal-Associated Membrane Protein 1
chemical_list: D006835:Hydrazones; D006918:Hydroxyurea; D007976:Leupeptins; D051907:Lysosomal Membrane Proteins; D020691:rab GTP-Binding Proteins; D000090802:rab7 GTP-Binding Proteins; C492847:1-(4-chlorophenyl)-3-(3-(4-chlorophenyl)-5,5-dimethyl-1-(3-(5-nitrofuran-2-yl)allyldienehydrazinocarbonylmethyl)-2-oxoimidazolidin-4-yl)-1-hydroxyurea; C489116:LAMP1 protein, human; D052118:Lysosomal-Associated Membrane Protein 1; C000716463:rab7 GTP-binding proteins, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde
keywords: adeno-associated virus; confocal microscopy; gene therapy; intracellular trafficking; proteasome; transduction; vesicle; virus