TY - JOUR
T1 - Visualization of DC-SIGN-Mediated Entry Pathway of Engineered Lentiviral Vectors in Target Cells
AU - Liu, Yarong
AU - Tai, April
AU - Joo, Kye Il
AU - Wang, Pin
N1 - Funding Information:
This work was supported by grants from the National Institutes of Health (R01AI68978 and P01CA132681) and a translational acceleration grant from the Joint Center for Translational Medicine.
PY - 2013/6/28
Y1 - 2013/6/28
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84879538190&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0067400
DO - 10.1371/journal.pone.0067400
M3 - Article
C2 - 23840690
AN - SCOPUS:84879538190
VL - 8
JO - PLoS ONE
JF - PLoS ONE
SN - 1932-6203
IS - 6
M1 - e67400
ER -