Dissecting the entry mechanism of targeting lentiviral vectors in living cells and developing quantum dot labeling of viruses for single virus tracking

A strategy to target lentiviral vectors to specific cell types holds great promise for future clinical applications of gene therapy. We have previously developed an efficient method to target lentivirus-mediated gene transduction by introducing a targeting antibody and pH-dependent fusogenic protein as two distinct molecules on the lentiviral surface. However, the molecular mechanism that controls the targeted infection needs to be defined. To elucidate the endocytic pathway of the engineered lentivirus, we have monitored intracellular trafficking of the individual lentiviruses in the targeted cells by various direct visualization approaches. This study proposed that the fusogen-mediated membrane fusion could be a rate-limiting step of targeting lentivirus transduction. However, the specific features of the fusogen-associated membrane fusion that control the targeted infection still remain largely unknown. Therefore, we further demonstrated the intracellular behaviors of two engineered lentiviruses displaying a class I fusogen derived from Sindbis virus glycoprotein or a class II fusogen derived from influenza virus hemagglutinin by tracking the individual viral particles in target cells. Our results suggest that both engineered lentiviruses enter target cells through clathrin-dependent endocytosis. However, the different kinetics of virus-endosome fusion as well as the distinct requirement of endosomal traffic for viral fusion of two engineered lentiviruses was suggested by imaging multiple sequential steps of fusion event in target cells. These imaging studies shed some light on the infection mechanism of the engineered lentivirus and is beneficial to the design of more efficient gene delivery vectors.;Good photostability of the labeling fluorophores is always desirable for the continuous tracking of individual viruses or any other biomolecules, and also, with the development of new imaging techniques requiring rapid and continuous excitation of fluorophores for z-stack image acquisition (3D reconstruction) and time-lapse imaging, greater photostability is necessary for detailed trafficking studies. Traditionally, organic fluorophores are used to detect and track biomolecules, such as antibodies, peptides, and viruses. However, problems with metabolic degradation, or photobleaching, have limited their applicability for long-term imaging of biological processes. The use of quantum dots can potentially mitigate these concerns, as well as allow for the development of novel detection techniques. Quantum dots are resistant to metabolic degradation and posses a wide absorption spectrum and a narrow emission spectrum. Here, we report general strategy to label enveloped and nonenveloped viruses with quantum dots for single-virus tracking. The results indicated that quantum dot labeling holds several advantages over conventional organic dyes, such as greatly improved photostability against photobleaching and detection sensitivity, which leads to the enhanced and detailed monitoring of viral behaviors in cells. We believe that these labeling methods can take advantage of the excellent fluorescence property of QDs and may represent an attractive tool for elucidating the molecular details of entry and intracellular transport of many kinds of enveloped and non-enveloped viruses.