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.

Rabies Virus Envelope Glycoprotein Targets Lentiviral Vectors to the Axonal Retrograde Pathway in Motor Neurons

“…A recent study indicated that RV-G bound to an antibody transfected in a neuroblastoma cell line is targeted via early and late endosomes to lysosomes (58). In addition, lentiviral vector transduction of dendritic cells involved endosomal trafficking, and its efficiency was enhanced by suppressing autophagy (59). Indeed, we have recently reported an increase in retrograde transduction in the brain of lentiviral vectors pseudotyped with a chimeric RV-G bearing a portion of gp41 for its cytoplasmic tail (60).…”

Visualization of intracellular pathways of engineered baculovirus in mammalian cells

“…It fuses with lysosomes and subsequently matures into autolysosomes, where viruses are degraded (Shoji-Kawata and Levine, 2009). Although it has been shown that autophagy aids in the degradation of other viruses in infected cells, such as vesicular stomatitis virus (VSV) (Shelly et al, 2009) and lentivector (Liu et al, 2013), no evidence has shown that autophagy is involved in the degradation process of baculoviruses in mammalian cells. Through a colocalization study using autophagosomal and lysosomal markers and a viral transduction study using the autophagy inhibitor (3-MA), we demonstrated that the autophagosome-lysosome network is, indeed, involved in the degradation process of baculoviruses during transduction.…”

Breast cancer vaccines delivered by dendritic cell-targeted lentivectors induce potent antitumor immune responses and protect mice from mammary tumor growth

“…LV were produced as previously described [25]. Briefly, 293T cells were transiently transfected with a standard calcium phosphate precipitation method.…”