Visualization of intracellular pathways of engineered baculovirus in mammalian cells.
Baculoviruses are a promising gene delivery vector. They have the ability to express large transgenes in mammalian cells without displaying pathogenicity in humans; however, little is known about their transduction mechanisms in target cells. In this study, we use colocalization and live-cell imaging studies to elucidate the internalization and intracellular trafficking pathways of baculoviruses through direct visualization of VP39-GFP-labeled viral particles and various endocytic structures within target cells. Drug inhibition and confocal microscopy results suggested that baculoviruses enter the cells via clathrin-mediated endocytosis in a dynamin-dependent manner. Viral particles were shown to traffic through early endosomes, triggering a low-pH-dependent endosomal fusion process of viruses. Suppressed autophagy activity enhanced viral transduction and overexpression of autophagosomes reduced viral transduction, suggesting that autophagy is involved in degradation process of viral particles. Actin filaments were involved in the viral transduction, while microtubules negatively regulated viral transduction by facilitating the fusion of autophagosomes with lysosomes to form autolysosomes, where degradation of viral particles occurs. These results shed some light on the essential cellular factors limiting viral transduction, which can be used to improve the use of baculoviral vectors in cell and gene therapy.
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA.
Yarong
Y
Liu
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA.
Kye-Il
KI
Joo
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA.
Yuning
Y
Lei
Mork Family Department of Chemical Engineering and Materials Science, University of Southern California, Los Angeles, CA 90089, USA; Department of Biomedical Engineering, University of Southern California, Los Angeles, CA 90089, USA; Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, CA 90089, USA. Electronic address: pinwang@usc.edu.
Pin
P
Wang
D000199:Actins / Q000378:metabolism; D000818:Animals; D001343:Autophagy; D016367:Baculoviridae / Q000502:physiology*; D002460:Cell Line; D002966:Clathrin / Q000378:metabolism; D011992:Endosomes / Q000821:virology; D006801:Humans; D042541:Intracellular Space / Q000821:virology*; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism; D014161:Transduction, Genetic; D053586:Virus Internalization
D000199:Actins; D002966:Clathrin
Autophagosome; Baculovirus; Endosomal fusion; Intracellular trafficking; Microtubule