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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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Related in: MedlinePlus

Baculovirus induces ruffling and internalizes along with fluid-phase markers.(A) Still images of baculovirus (p24mCherry, MOI 400) internalization in living 293 cells by confocal microscopy. Differential contrast image, baculovirus (red) and fixed time frames (0–245 s) are shown. Imaging was started at 5 min p.t. (0 s = 5 min). Some of the cellular protrusions guiding the baculovirus to the peripheral cytoplasm are within the rectangular box. Scale bar, 10 µm. (B) Cells positive for ruffles were calculated from control and baculovirus -treated cells 30 min p.t. (100 cells calculated). Mean values and standard deviations are shown. (C–D) Co-internalization of baculovirus (wt, MOI 500) with the fluid-phase marker HRP (10 mg/ml) was studied in 293 cells between 5 and 30 min p.t. HRP-negative (C) and positive (D) vesicles containing baculovirus (arrows) at 15 min p.t. are presented. Scale bar, 500 nm (C, D). (E) HRP (2 mg/ml) uptake after 30 min was measured with or without various amounts of baculovirus (200, 500 and 1000 MOI) co-internalized in cells. The amount of HRP is normalized to cellular protein content. Mean values and standard deviations are shown.
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pone-0005093-g002: Baculovirus induces ruffling and internalizes along with fluid-phase markers.(A) Still images of baculovirus (p24mCherry, MOI 400) internalization in living 293 cells by confocal microscopy. Differential contrast image, baculovirus (red) and fixed time frames (0–245 s) are shown. Imaging was started at 5 min p.t. (0 s = 5 min). Some of the cellular protrusions guiding the baculovirus to the peripheral cytoplasm are within the rectangular box. Scale bar, 10 µm. (B) Cells positive for ruffles were calculated from control and baculovirus -treated cells 30 min p.t. (100 cells calculated). Mean values and standard deviations are shown. (C–D) Co-internalization of baculovirus (wt, MOI 500) with the fluid-phase marker HRP (10 mg/ml) was studied in 293 cells between 5 and 30 min p.t. HRP-negative (C) and positive (D) vesicles containing baculovirus (arrows) at 15 min p.t. are presented. Scale bar, 500 nm (C, D). (E) HRP (2 mg/ml) uptake after 30 min was measured with or without various amounts of baculovirus (200, 500 and 1000 MOI) co-internalized in cells. The amount of HRP is normalized to cellular protein content. Mean values and standard deviations are shown.

Mentions: The binding and entry of the baculovirus (0–15 min p.t.) was next studied in live 293 and HepG2 cells by confocal microscopy. Interestingly, extensive ruffle formation on the cell surface of both cell types was detected early after administration of the virus (Figure 2A, Figure S2). The virions seemed to utilize the extended cellular protrusions for their attachment and further movement to the plasma membrane (Video S1). Additionally, engulfment of viruses from cellular ruffles was detected (Figure 2B). The cellular protrusions as well as cell surface areas, which were active in virus entry, were strongly positive for actin labeled with phalloidin rhodamine (Figure S2). Quantification of cells positive for ruffles showed that under control conditions 27% of the cells (±0.4% SD) showed ruffles, whereas at 30 min p.t. the number of cells showing ruffles was increased to 77% (±9% SD; n = 100).


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)

Baculovirus induces ruffling and internalizes along with fluid-phase markers.(A) Still images of baculovirus (p24mCherry, MOI 400) internalization in living 293 cells by confocal microscopy. Differential contrast image, baculovirus (red) and fixed time frames (0–245 s) are shown. Imaging was started at 5 min p.t. (0 s = 5 min). Some of the cellular protrusions guiding the baculovirus to the peripheral cytoplasm are within the rectangular box. Scale bar, 10 µm. (B) Cells positive for ruffles were calculated from control and baculovirus -treated cells 30 min p.t. (100 cells calculated). Mean values and standard deviations are shown. (C–D) Co-internalization of baculovirus (wt, MOI 500) with the fluid-phase marker HRP (10 mg/ml) was studied in 293 cells between 5 and 30 min p.t. HRP-negative (C) and positive (D) vesicles containing baculovirus (arrows) at 15 min p.t. are presented. Scale bar, 500 nm (C, D). (E) HRP (2 mg/ml) uptake after 30 min was measured with or without various amounts of baculovirus (200, 500 and 1000 MOI) co-internalized in cells. The amount of HRP is normalized to cellular protein content. Mean values and standard deviations are shown.
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Related In: Results  -  Collection

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pone-0005093-g002: Baculovirus induces ruffling and internalizes along with fluid-phase markers.(A) Still images of baculovirus (p24mCherry, MOI 400) internalization in living 293 cells by confocal microscopy. Differential contrast image, baculovirus (red) and fixed time frames (0–245 s) are shown. Imaging was started at 5 min p.t. (0 s = 5 min). Some of the cellular protrusions guiding the baculovirus to the peripheral cytoplasm are within the rectangular box. Scale bar, 10 µm. (B) Cells positive for ruffles were calculated from control and baculovirus -treated cells 30 min p.t. (100 cells calculated). Mean values and standard deviations are shown. (C–D) Co-internalization of baculovirus (wt, MOI 500) with the fluid-phase marker HRP (10 mg/ml) was studied in 293 cells between 5 and 30 min p.t. HRP-negative (C) and positive (D) vesicles containing baculovirus (arrows) at 15 min p.t. are presented. Scale bar, 500 nm (C, D). (E) HRP (2 mg/ml) uptake after 30 min was measured with or without various amounts of baculovirus (200, 500 and 1000 MOI) co-internalized in cells. The amount of HRP is normalized to cellular protein content. Mean values and standard deviations are shown.
Mentions: The binding and entry of the baculovirus (0–15 min p.t.) was next studied in live 293 and HepG2 cells by confocal microscopy. Interestingly, extensive ruffle formation on the cell surface of both cell types was detected early after administration of the virus (Figure 2A, Figure S2). The virions seemed to utilize the extended cellular protrusions for their attachment and further movement to the plasma membrane (Video S1). Additionally, engulfment of viruses from cellular ruffles was detected (Figure 2B). The cellular protrusions as well as cell surface areas, which were active in virus entry, were strongly positive for actin labeled with phalloidin rhodamine (Figure S2). Quantification of cells positive for ruffles showed that under control conditions 27% of the cells (±0.4% SD) showed ruffles, whereas at 30 min p.t. the number of cells showing ruffles was increased to 77% (±9% SD; n = 100).

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