Visualization of intracellular pathways of engineered baculovirus in mammalian cells

Liu, Yarong; Joo, Kye-Il; Lei, Yuning; Wang, Pin

doi:10.1016/j.virusres.2014.01.006

Cited by 15 publications

(8 citation statements)

References 54 publications

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“…2). This is consistent with a previous report indicating that perturbation of the function of the EE by a dominant-negative Rab5 mutant, but not of the LE by that of Rab7, significantly reduced AcMNPV transduction in HeLa cells (30).…”

Section: Discussionsupporting

confidence: 93%

The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

Ning

et al. 2019

J Virol

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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 fusiondeficient recombinant virus transduction efficiency. Based on the above-described findings, the pH of EEs was specifically reduced with a Na ϩ /K ϩ -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. RESULTSAcMNPV enters mammalian cells efficiently, but further nucleocapsid transport into the nucleus is blocked. AcMNPV can enter many mammalian cells, but only with low efficiency. As expected, the transduction rates of Ac-BPegfp (a recombinant v...

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Section: Discussionsupporting

confidence: 93%

The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

Ning

et al. 2019

J Virol

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“…Hence, the assumption that the pH of early endosomes is 6.0 to 6.5 is probably not applicable for endosomes in insect host Sf9 cells. Our study is further confirmed by the findings that AcMNPV nucleocapsid release in mammalian cells occurs also primarily in early endosomes but not late endosomes (31,75,76).…”

Section: Discussionsupporting

confidence: 87%

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis

Qin

et al. 2019

J Virol

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The budded virus of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) infects insect cells through mainly clathrin-mediated endocytosis. However, the cell entry pathway of AcMNPV remains unclear. In this study, by using population-based analysis of single-virus tracking and electron microscopy, we investigated the internalization, fusion behavior, and endocytic trafficking of AcMNPV. AcMNPV internalization into host insect cells was facilitated by actin polymerization and dynamin. After incorporation into early endosomes, the AcMNPV envelope fused with the membranes of early endosome, allowing for nucleocapsid release into the cytoplasm. Microtubules were implicated in the bidirectional and long-range transport of virus-containing endosomes. In addition, microtubule depolymerization reduced the motility of virus-bearing early endosomes, impairing the progression of infection beyond enlarged early endosomes. These findings demonstrated that AcMNPV internalization was facilitated by actin polymerization in a dynamin-dependent manner, and nucleocapsid release occurred in early endosomes in a microtubule-dependent manner. This study provides mechanistic and kinetic insights into AcMNPV infection and enhance our understanding of the infection pathway of baculoviruses. IMPORTANCE Baculoviruses are used widely as environmentally benign pesticides, protein expression systems, and potential mammalian gene delivery vectors. Despite the significant application value, little is known about the cell entry and endocytic trafficking pathways of baculoviruses. In this study, we demonstrated that the alphabaculovirus AcMNPV exhibited actin-and microtubule-dependent transport for nucleocapsid release predominantly from within early endosomes. In contrast to AcMNPV transduction in mammalian cells, its infection in host insect cells is facilitated by actin polymerization for internalization and microtubules for endocytic trafficking within early endosomes, implying that AcMNPV exhibits cell type specificity in the requirement of the cytoskeleton network. In addition, experimental depolymerization of microtubules impaired the progression of infection beyond enlarged early endosomes. This is the first study that dissects the cell entry pathway of baculoviruses in host cells at the single-particle level, which advances our understanding of the early steps of baculovirus entry.

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“…The constructs are normally used to visualize EEs (RAB5A) or LEs (RAB7). The expression of the proteins encoded by the plasmid requires the transport of baculovirus particles to the LE, 28 plasmid release and import into the nucleus. We compared the efficiency of BacMam 2.0 technology in HD and PT1 fibroblasts.…”

Section: Nfkb Does Not Translocate To the Nucleus In Response To Cpgmentioning

confidence: 99%

The Vici syndrome protein EPG5 regulates intracellular nucleic acid trafficking linking autophagy to innate and adaptive immunity

et al. 2018

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Vici syndrome is a human inherited multi-system disorder caused by recessive mutations in EPG5, encoding the EPG5 protein that mediates the fusion of autophagosomes with lysosomes. Immunodeficiency characterized by lack of memory B cells and increased susceptibility to infection is an integral part of the condition, but the role of EPG5 in the immune system remains unknown. Here we show that EPG5 is indispensable for the transport of the TLR9 ligand CpG to the late endosomal-lysosomal compartment, and for TLR9-initiated signaling, a step essential for the survival of human memory B cells and their ultimate differentiation into plasma cells. Moreover, the predicted structure of EPG5 includes a membrane remodeling domain and a karyopherin-like domain, thus explaining its function as a carrier between separate vesicular compartments. Our findings indicate that EPG5, by controlling nucleic acids intracellular trafficking, links macroautophagy/autophagy to innate and adaptive immunity.

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Visualization of intracellular pathways of engineered baculovirus in mammalian cells

Cited by 15 publications

References 54 publications

The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

The Major Hurdle for Effective Baculovirus Transduction into Mammalian Cells Is Passing Early Endosomes

Dissecting the Cell Entry Pathway of Baculovirus by Single-Particle Tracking and Quantitative Electron Microscopic Analysis

The Vici syndrome protein EPG5 regulates intracellular nucleic acid trafficking linking autophagy to innate and adaptive immunity

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