title: Visualization of DC-SIGN-mediated entry pathway of engineered lentiviral vectors in target cells.
abstract: Dendritic cells (DCs) are potent antigen-presenting cells and therefore have enormous potential as vaccine targets. We have previously developed an engineered lentiviral vector (LV) that is pseudotyped with a mutated Sindbis virus glycoprotein (SVGmu), which is capable of targeting DCs through Dendritic Cell-specific ICAM3-grabbing Nonintegrin (DC-SIGN), a receptor that is predominantly expressed by DCs. In this study, we aimed to elucidate the internalization and trafficking mechanisms of this viral vector system through direct visualization of GFP-Vpr-tagged viral particles in target DCs, which was further corroborated by drug inhibition and dominant-negative mutants of cellular proteins that regulate the endocytic traffic. We demonstrated that our engineered LVs enter the cell via receptor-mediated clathrin- and dynamin-dependent endocytosis. Microtubule networks were also involved in a productive infection. Viral vector fusion was low-pH-dependent and occurred in the early endosomal stage of the intracellular transport. Autophagy was also examined for its effect on transduction efficiency, and we observed that enhanced autophage activity reduced vector infectivity, while suppressed autophagy boosted transduction efficiency. This study shed some light on the internalization and trafficking mechanisms of DC-directed LVs and offers some strategies to further improve the efficiency of LV-mediated gene therapy.
authors.affiliation: Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, California, United States of America.
authors.forename: Yarong
authors.initials: Y
authors.lastname: Liu
authors.forename: April
authors.initials: A
authors.lastname: Tai
authors.forename: Kye-Il
authors.initials: KI
authors.lastname: Joo
authors.forename: Pin
authors.initials: P
authors.lastname: Wang
mesh_terms: D001343:Autophagy; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism; D003713:Dendritic Cells / Q000378:metabolism* / Q000821:virology; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005818:Genetic Engineering; D005822:Genetic Vectors; D057809:HEK293 Cells; D054884:Host-Pathogen Interactions; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037181:Lectins, C-Type / Q000378:metabolism*; D016086:Lentivirus / Q000235:genetics* / Q000502:physiology; D008247:Lysosomes / Q000502:physiology; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D059010:Single-Cell Analysis; D014161:Transduction, Genetic; D053586:Virus Internalization; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin
chemical_list: D015815:Cell Adhesion Molecules; D002966:Clathrin; D037181:Lectins, C-Type; D011956:Receptors, Cell Surface; D000099215:DC-Specific ICAM-3 Grabbing Nonintegrin