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Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells.

Laakkonen JP, Mäkelä AR, Kakkonen E, Turkki P, Kukkonen S, Peränen J, Ylä-Herttuala S, Airenne KJ, Oker-Blom C, Vihinen-Ranta M, Marjomäki V - PLoS ONE (2009)

Bottom Line: Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake.To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake.This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland.

ABSTRACT
The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants of RhoA, and of Arf6, a regulator of clathrin-independent entry. Furthermore, the entry of baculovirus induced ruffle formation and triggered the uptake of fluorescent E. coli bioparticles. To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake. This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells.

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Phagocytosis-like uptake of baculovirus.(A,B) Baculovirus (p24mCherry, MOI 200; red) internalized together with A488-labeled E. coli bioparticles (green) in HepG2 cells at 5 (A) and 10 min (B) p.t. Scale bars, 10 µm. (C) Induction of phagocytic uptake of E. coli during baculovirus transduction. Fluorescent E. coli were fed simultaneously with baculovirus (MOI 200; baculovirus +E. coli) into cells and fixed at 60 min p.t. As a control, E. coli particles were fed into cells without baculovirus (E. coli) or after virus transduction for 15 min (baculovirus 15′+E. coli). To separate the fluorescence of internalized and non-internalized particles, the cells were treated with trypan blue. Normalized mean fluorescence values (Ctrl = 1) and standard deviation from 250–300 cells are shown. Fluorescence intensity was measured from confocal microscopy images (see Materials and Methods). (D) Dynasore and filipin were tested for their effects on stimulated E. coli uptake during baculovirus transduction. Co-internalization of baculovirus and E. Coli for 1 h without drugs was set to 100%. Fluorescence intensitites were calculated from three separate experiments (30–40 cells) using segmentation tools embedded in the BioimageXD software. Dyn (dynasore, 80 µM) and Filip. (filipin, 1 µg/ml) were added 30 min before the experiment and they were present during the whole internalization assay. Statistical significance was determined by using the unpaired Student's t test with a two-tailed P value, ***P<0.001.
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pone-0005093-g006: Phagocytosis-like uptake of baculovirus.(A,B) Baculovirus (p24mCherry, MOI 200; red) internalized together with A488-labeled E. coli bioparticles (green) in HepG2 cells at 5 (A) and 10 min (B) p.t. Scale bars, 10 µm. (C) Induction of phagocytic uptake of E. coli during baculovirus transduction. Fluorescent E. coli were fed simultaneously with baculovirus (MOI 200; baculovirus +E. coli) into cells and fixed at 60 min p.t. As a control, E. coli particles were fed into cells without baculovirus (E. coli) or after virus transduction for 15 min (baculovirus 15′+E. coli). To separate the fluorescence of internalized and non-internalized particles, the cells were treated with trypan blue. Normalized mean fluorescence values (Ctrl = 1) and standard deviation from 250–300 cells are shown. Fluorescence intensity was measured from confocal microscopy images (see Materials and Methods). (D) Dynasore and filipin were tested for their effects on stimulated E. coli uptake during baculovirus transduction. Co-internalization of baculovirus and E. Coli for 1 h without drugs was set to 100%. Fluorescence intensitites were calculated from three separate experiments (30–40 cells) using segmentation tools embedded in the BioimageXD software. Dyn (dynasore, 80 µM) and Filip. (filipin, 1 µg/ml) were added 30 min before the experiment and they were present during the whole internalization assay. Statistical significance was determined by using the unpaired Student's t test with a two-tailed P value, ***P<0.001.

Mentions: Due to the large size of baculovirus, the induced ruffle formation, and the involvement of actin [34], Arf6, RhoA, as well as rafts in baculovirus transduction, the possible involvement of phagocytosis-like mechanisms in baculovirus entry was studied. For these experiments, we used heat-inactivated, Alexa-488-labelled E. coli (K12 strain, >1 µm) bioparticles, widely used as a marker of phagocytosis. First, we internalized E. coli particles and fluorescent baculovirus together for brief periods of time. The confocal results showed clear colocalization at 5 and 10 min p.t. in HepG2 cells (Figure 6A and 6B). Only a few E. coli particles were observed outside baculovirus-filled endosomes. We then monitored the intensity of fluorescent intracellular E. coli particles in baculovirus transduced cells by confocal microscopy. As a control, E. coli alone was fed to HepG2 cells for 1 h (Figure 6C). To separate the fluorescence of internalized and non-internalized particles, the cells were treated with trypan blue in order to quench the extracellular fluorescence. Untransduced control cells contained no apparent fluorescence after 1 h treatment with E. coli particles suggesting that bacteria did not enter the cells without baculovirus. Similarly, in 293 cells E.coli alone did not enter the cells and gave only low background fluorescence (data not shown). In contrast, virus-transduced HepG2 and 293 cells contained high amounts (49-fold and 10-fold more, respectively) of internalized E. coli particles (P<0.001), indicating that virus could induce entry of bacteria in non-phagocytic human cells. Interestingly, when baculovirus was first fed to HepG2 cells for 15 min and then E. coli for the next 60 min, bacteria could no longer enter cells efficiently. These results suggest that baculovirus is able to induce transient bacterial entry when administered simultaneously.


Clathrin-independent entry of baculovirus triggers uptake of E. coli in non-phagocytic human cells.

Laakkonen JP, Mäkelä AR, Kakkonen E, Turkki P, Kukkonen S, Peränen J, Ylä-Herttuala S, Airenne KJ, Oker-Blom C, Vihinen-Ranta M, Marjomäki V - PLoS ONE (2009)

Phagocytosis-like uptake of baculovirus.(A,B) Baculovirus (p24mCherry, MOI 200; red) internalized together with A488-labeled E. coli bioparticles (green) in HepG2 cells at 5 (A) and 10 min (B) p.t. Scale bars, 10 µm. (C) Induction of phagocytic uptake of E. coli during baculovirus transduction. Fluorescent E. coli were fed simultaneously with baculovirus (MOI 200; baculovirus +E. coli) into cells and fixed at 60 min p.t. As a control, E. coli particles were fed into cells without baculovirus (E. coli) or after virus transduction for 15 min (baculovirus 15′+E. coli). To separate the fluorescence of internalized and non-internalized particles, the cells were treated with trypan blue. Normalized mean fluorescence values (Ctrl = 1) and standard deviation from 250–300 cells are shown. Fluorescence intensity was measured from confocal microscopy images (see Materials and Methods). (D) Dynasore and filipin were tested for their effects on stimulated E. coli uptake during baculovirus transduction. Co-internalization of baculovirus and E. Coli for 1 h without drugs was set to 100%. Fluorescence intensitites were calculated from three separate experiments (30–40 cells) using segmentation tools embedded in the BioimageXD software. Dyn (dynasore, 80 µM) and Filip. (filipin, 1 µg/ml) were added 30 min before the experiment and they were present during the whole internalization assay. Statistical significance was determined by using the unpaired Student's t test with a two-tailed P value, ***P<0.001.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2662421&req=5

pone-0005093-g006: Phagocytosis-like uptake of baculovirus.(A,B) Baculovirus (p24mCherry, MOI 200; red) internalized together with A488-labeled E. coli bioparticles (green) in HepG2 cells at 5 (A) and 10 min (B) p.t. Scale bars, 10 µm. (C) Induction of phagocytic uptake of E. coli during baculovirus transduction. Fluorescent E. coli were fed simultaneously with baculovirus (MOI 200; baculovirus +E. coli) into cells and fixed at 60 min p.t. As a control, E. coli particles were fed into cells without baculovirus (E. coli) or after virus transduction for 15 min (baculovirus 15′+E. coli). To separate the fluorescence of internalized and non-internalized particles, the cells were treated with trypan blue. Normalized mean fluorescence values (Ctrl = 1) and standard deviation from 250–300 cells are shown. Fluorescence intensity was measured from confocal microscopy images (see Materials and Methods). (D) Dynasore and filipin were tested for their effects on stimulated E. coli uptake during baculovirus transduction. Co-internalization of baculovirus and E. Coli for 1 h without drugs was set to 100%. Fluorescence intensitites were calculated from three separate experiments (30–40 cells) using segmentation tools embedded in the BioimageXD software. Dyn (dynasore, 80 µM) and Filip. (filipin, 1 µg/ml) were added 30 min before the experiment and they were present during the whole internalization assay. Statistical significance was determined by using the unpaired Student's t test with a two-tailed P value, ***P<0.001.
Mentions: Due to the large size of baculovirus, the induced ruffle formation, and the involvement of actin [34], Arf6, RhoA, as well as rafts in baculovirus transduction, the possible involvement of phagocytosis-like mechanisms in baculovirus entry was studied. For these experiments, we used heat-inactivated, Alexa-488-labelled E. coli (K12 strain, >1 µm) bioparticles, widely used as a marker of phagocytosis. First, we internalized E. coli particles and fluorescent baculovirus together for brief periods of time. The confocal results showed clear colocalization at 5 and 10 min p.t. in HepG2 cells (Figure 6A and 6B). Only a few E. coli particles were observed outside baculovirus-filled endosomes. We then monitored the intensity of fluorescent intracellular E. coli particles in baculovirus transduced cells by confocal microscopy. As a control, E. coli alone was fed to HepG2 cells for 1 h (Figure 6C). To separate the fluorescence of internalized and non-internalized particles, the cells were treated with trypan blue in order to quench the extracellular fluorescence. Untransduced control cells contained no apparent fluorescence after 1 h treatment with E. coli particles suggesting that bacteria did not enter the cells without baculovirus. Similarly, in 293 cells E.coli alone did not enter the cells and gave only low background fluorescence (data not shown). In contrast, virus-transduced HepG2 and 293 cells contained high amounts (49-fold and 10-fold more, respectively) of internalized E. coli particles (P<0.001), indicating that virus could induce entry of bacteria in non-phagocytic human cells. Interestingly, when baculovirus was first fed to HepG2 cells for 15 min and then E. coli for the next 60 min, bacteria could no longer enter cells efficiently. These results suggest that baculovirus is able to induce transient bacterial entry when administered simultaneously.

Bottom Line: Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake.To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake.This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological and Environmental Science/Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland.

ABSTRACT
The prototype baculovirus, Autographa californica multiple nucleopolyhedrovirus, an insect pathogen, holds great potential as a gene therapy vector. To develop transductional targeting and gene delivery by baculovirus, we focused on characterizing the nature and regulation of its uptake in human cancer cells. Baculovirus entered the cells along fluid-phase markers from the raft areas into smooth-surfaced vesicles devoid of clathrin. Notably, regulators associated with macropinocytosis, namely EIPA, Pak1, Rab34, and Rac1, had no significant effect on viral transduction, and the virus did not induce fluid-phase uptake. The internalization and nuclear uptake was, however, affected by mutants of RhoA, and of Arf6, a regulator of clathrin-independent entry. Furthermore, the entry of baculovirus induced ruffle formation and triggered the uptake of fluorescent E. coli bioparticles. To conclude, baculovirus enters human cells via a clathrin-independent pathway, which is able to trigger bacterial uptake. This study increases our understanding of virus entry strategies and gives new insight into baculovirus-mediated gene delivery in human cells.

Show MeSH
Related in: MedlinePlus