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.
Highlights
Vaccines are incredibly potent tools in immunology, capable of greatly reducing, and even eradicating as in the case of smallpox, vaccine-preventable infections
Dendritic cells (DCs)-SIGN-mediated and Clathrin-dependent Virus Entry We sought out imaging methods to elucidate intracellular dynamics of interactions between engineered lentiviral vector (LV) and target cells and to interrogate the molecular mechanism of viral transduction
To visualize the intracellular trafficking of individual LV-SVGmu particles within target cells, LVs were labeled with green fluorescent protein (GFP) fused to the N-terminus of the human immunodeficiency virus (HIV) accessory protein viral protein R, designated as GFP-Vpr, which was described previously [19]
Summary
Vaccines are incredibly potent tools in immunology, capable of greatly reducing, and even eradicating as in the case of smallpox, vaccine-preventable infections. Dendritic cells (DCs) are excellent targets for vaccine therapy because they are considered to be the most potent antigen-presenting cells of the immune system [1,2,3,4]. Lentiviral vectors (LVs) have been considered as an attractive vehicle for gene therapy because they are able to transduce nondividing cells and permanently integrate into the target cell genome [9,10,11]. These vectors can be efficiently pseudotyped with other viral glycoproteins to alter their tropism and allow them to target specific cell types [12,13]
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