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Visualization of intracellular pathways of engineered baculovirus in mammalian cells.

ABSTRACT: 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.

SUBMITTER: Liu Y

PROVIDER: S-EPMC4439417 | biostudies-literature | 2014 Mar

REPOSITORIES: biostudies-literature

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Visualization of intracellular pathways of engineered baculovirus in mammalian cells.

Liu Yarong Y Joo Kye-Il KI Lei Yuning Y Wang Pin P

Virus research 20140120

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 wit ...[more]

PMID: 24457070

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Project description:Baculoviruses, although they infect insects in nature, can transduce a wide variety of mammalian cells and are therefore promising gene therapy vectors. However, baculovirus transduction into many mammalian cells is very inefficient, and the limiting stages and factors remain unknown. An important finding is that a short-duration trigger with low pH can significantly enhance virus transduction efficiency, but the mechanism is poorly understood. Herein, we performed a detailed comparative study on entry mechanisms of the prototypical baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) into insect and mammalian cells. The results showed that AcMNPV could be internalized into mammalian cells efficiently, but fusion in early endosomes (EEs) appeared to be the major obstacle. Measurement of endosomal pH suggested that virus fusion might be restricted under relatively high-pH conditions in mammalian cells. Interestingly, mutations of the major viral fusion protein GP64 that conferred decreased fusogenicity did not affect virus infection of insect cells, whereas virus transduction into mammalian cells was severely impaired, suggesting a more stringent dependence on GP64 fusogenicity for AcMNPV entry into mammalian cells than into insect cells. An increase in the fusogenicity of GP64 mutants resulting from low pH triggered the rescue of fusion-deficient recombinant virus transduction efficiency. Based on the above-described findings, the pH of EEs was specifically reduced with a Na+/K+-ATPase inhibitor, and the AcMNPV transduction of many mammalian cells indeed became highly efficient. This study not only revealed the roadblocks to mammalian cell entry of baculovirus but also provides a new strategy for improving baculovirus-based gene delivery and therapy.IMPORTANCE Baculoviruses can transduce a wide variety of mammalian cells but do so with low efficiency, which greatly limits their practical application as potential gene delivery vectors. So far, the understanding of baculovirus entry into mammalian cells is obscure, and the limiting stages and factors are unclear. In this study, by comparatively analyzing the mechanisms of baculovirus entry into mammalian and insect cells, virus fusion during the early stage of endocytosis was revealed as the major obstacle for efficient baculovirus transduction into mammalian cells. A higher fusogenicity of the major viral fusion protein GP64 was found to be required for virus entry into mammalian cells than for entry into insect cells. Interestingly, by decreasing the pH of early endosomes with a specific agent, virus transduction of a wide range of mammalian cells was greatly enhanced. This study uncovers the roadblocks to mammalian cell entry of baculoviruses and presents mechanisms to overcome the roadblocks.

Dataset Information

Visualization of intracellular pathways of engineered baculovirus in mammalian cells.

Publications

Visualization of intracellular pathways of engineered baculovirus in mammalian cells.

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