erization and microtubule polymerization, respectively, before transduction with LVSVGmu encoding a reporter GFP gene. FACS analysis of the GFP+ cells 3 days post-transduction revealed that the actin blocking by Cyto-D had no effect on viral infectivity, while microtubule blocking by nocodazole reduced infection levels to about 40% of the control, suggesting the involvement of microtubules in intracellular trafficking of LV-SVGmu particles. To further elucidate the functional involvement of microtubules in the viral transport within target cells, we conducted a colocalization experiment using a-tubulin-specific antibodies in 293T/hDC-SIGN cells. Visualization of the stained cells revealed that 53% of viral particles were detected on the microtubule structures, confirming the results of the drug treatment. Taken together, these results suggest that a successful viral infection is dependent on the microtubule, 23696131 but not actin, networks in the cell. Visualization of Viral 
Membrane Fusion Engineered Lentivector Trafficking in Target Cells LV-SVGmu particles and fusion signals . The images obtained after 30 min of incubation showed significantly lower colocalization of GFP-Vpr with DiD signals, suggesting that certain viral particles were presumably released into cytosol after the endosomal fusion. This quantitative colocalization analysis indicated that virus-endosome fusion of LV-SVGmu peaked at 20 min of incubation in DCSIGN-expressing cells. It is also reported that viral fusion proteins 22619121 undergo conformational change, which is induced by receptor-binding and/or pH change. To investigate the pH-dependency of viral membrane fusion of LV-SVGmu, drug treatment with bafilomycin A1, which specifically inhibits vacuolar proton ATPases, was utilized to block low pH-associated endosomal processes in DC-SIGNexpressing cells. The viral transduction efficiency was then measured in cells treated with different concentrations of the drug. The FACS analysis of GFP+ cells showed that the transduction of LV-SVGmu was remarkably inhibited by bafilomycin A1 treatment in a dose-dependent manner. Similarly, the transduction of LV-SVGmu was also significantly decreased in cells treated with NH4Cl that can neutralize acidic endosomal compartments. To reaffirm the low-pH dependency of the viral fusion, viral membrane fusion events were visualized in the presence of NH4Cl 4 Engineered Lentivector Trafficking in Target Cells visualized 293T/hDC-SIGN cells incubated with GFP-Vprlabeled LV-SVGmu virions at different time points, which were then antibody-stained for early endosome antigen 1 and cation-independent mannose-6-phosphate receptor for the early and late endosomal markers, respectively. Images were taken at 0, 10, 20, and 30 min, correlating directly with the time points taken for the fusion study, and quantified. At 0 min, none of the viral particles was colocalized with the endosomal markers, and most were only bound to the cell surfaces. At 10 min, more viruses had moved into the endosomes, with 25% of the viruses colocalized with the early endosomal marker, whereas no significant colocalization of viruses with the late endosomes was buy Oleandrin observed. At 20 min of incubation, which was also the time when most of the fusion events occurred,.60% of viruses were colocalized with the early endosomes, with about 27.4% in late endosomes. Correlation of this data with that of the fusion study leads us to believe that fusion occurs mostly at 20 min during the early endos