Lentiviral vectors hold great promise for the development of gene therapy due to their ability to produce long-term transgene expression and transduce nondividing cells. Although many attempts have been made to develop more efficient gene delivery systems by using lentivral vectors, the underlying mechanisms of the viral entry still remain elusive. Intracellular trafficking of the viral vectors is essential to understand a variety of virus- host cell interactions. Previously, we developed the targeting lentiviral vector by incorporation of antibody and fusogenic protein as two different molecules on the viral surface1. To elucidate the endocytic pathway of the engineered lentivirus, we have tracked the individual lentiviruses in the targeted cells by labeling the virus with the GFP-Vpr fusion protein. By combining the GFP-Vpr tagging with other labeling techniques, we visualized the surface-displayed proteins on a single virus and the antibody induced targeting to a desired cell type. We also revealed the dynamics of the virus fusion with an endosome, and endosome-mediated transport of the viruses in the infected cells. Our results suggest that the fusion between the engineered lentivirus and endosomes takes place in the early endosomal compartment, and the completion of the virus-endosome fusion to release the viral core into the cytosol is correlated with the endosome-mediated maturing processes. These visualization approaches can be extended to study the virus entry process of other engineered lentiviral vectors in living cells. The current method with other virus-labeling techniques can also be used for exploring the infection pathway of HIV.

1. Yang, L., Bailey, L., Baltimore, D., and Wang, P. Targeting lentiviral vectors to specific cell types in vivo. Proc. Natl. Acad. Sci. USA 103, 11479-11484. (2006)