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Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

Danthi P, Coffey CM, Parker JS, Abel TW, Dermody TS - PLoS Pathog. (2008)

Bottom Line: We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates.Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery.These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, USA. pdanthi@indiana.edu

ABSTRACT
Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

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K594D and I595K are apoptosis-defective.(A) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of apoptotic cells was determined after staining with acridine orange. Results are expressed as the mean percentage of apoptotic cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of cells with less than G1 DNA content (sub-G1) was quantified by staining with propidium iodide. Results are expressed as the mean percentage of sub-G1 cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) 293T cells were co-transfected with pNF-κB-Luc and pRenilla-Luc 24 h prior to adsorption with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Luciferase activity in cell lysates was determined at 24 h post-infection. Results are presented as the ratio of normalized luciferase activity from infected cell lysates to that from mock-infected lysates for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D.
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ppat-1000248-g004: K594D and I595K are apoptosis-defective.(A) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of apoptotic cells was determined after staining with acridine orange. Results are expressed as the mean percentage of apoptotic cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of cells with less than G1 DNA content (sub-G1) was quantified by staining with propidium iodide. Results are expressed as the mean percentage of sub-G1 cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) 293T cells were co-transfected with pNF-κB-Luc and pRenilla-Luc 24 h prior to adsorption with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Luciferase activity in cell lysates was determined at 24 h post-infection. Results are presented as the ratio of normalized luciferase activity from infected cell lysates to that from mock-infected lysates for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D.

Mentions: Residues Lys594 and Ile595 in μ1 ϕ modulate apoptosis induction following ectopic expression in cells (Figure 1). To determine whether these residues also are important for apoptosis induction during reovirus infection, we compared the apoptotic potential of wild-type and ϕ mutant viruses by acridine orange staining (Figure 4A). Following infection of HeLa cells with rsT3D, ∼30–35% of cells showed altered nuclear morphology characteristic of apoptosis. However, infection with the K594D or I595K elicited apoptosis in only ∼12% of cells. As a complementary approach, we used propidium iodide staining and flow cytometry to compare the capacity of these viruses to promote DNA fragmentation in infected cells (Figure 4B). Infection of HeLa cells with rsT3D resulted in ∼50% cells displaying hypodiploid (sub-G1) DNA staining, characteristic of apoptotic cells. In contrast, infection with either K594D or I595K resulted in a percentage of cells in sub-G1 that was comparable to mock infection. These results suggest that the apoptotic potential of reovirus is regulated by ϕ residues Lys594 and Ile595.


Independent regulation of reovirus membrane penetration and apoptosis by the mu1 phi domain.

Danthi P, Coffey CM, Parker JS, Abel TW, Dermody TS - PLoS Pathog. (2008)

K594D and I595K are apoptosis-defective.(A) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of apoptotic cells was determined after staining with acridine orange. Results are expressed as the mean percentage of apoptotic cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of cells with less than G1 DNA content (sub-G1) was quantified by staining with propidium iodide. Results are expressed as the mean percentage of sub-G1 cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) 293T cells were co-transfected with pNF-κB-Luc and pRenilla-Luc 24 h prior to adsorption with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Luciferase activity in cell lysates was determined at 24 h post-infection. Results are presented as the ratio of normalized luciferase activity from infected cell lysates to that from mock-infected lysates for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D.
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Related In: Results  -  Collection

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ppat-1000248-g004: K594D and I595K are apoptosis-defective.(A) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of apoptotic cells was determined after staining with acridine orange. Results are expressed as the mean percentage of apoptotic cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (B) HeLa cells were infected with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Following 48 h incubation, the percentage of cells with less than G1 DNA content (sub-G1) was quantified by staining with propidium iodide. Results are expressed as the mean percentage of sub-G1 cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D. (C) 293T cells were co-transfected with pNF-κB-Luc and pRenilla-Luc 24 h prior to adsorption with rsT3D or the indicated ϕ mutant at an MOI of 100 PFU/cell. Luciferase activity in cell lysates was determined at 24 h post-infection. Results are presented as the ratio of normalized luciferase activity from infected cell lysates to that from mock-infected lysates for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D.
Mentions: Residues Lys594 and Ile595 in μ1 ϕ modulate apoptosis induction following ectopic expression in cells (Figure 1). To determine whether these residues also are important for apoptosis induction during reovirus infection, we compared the apoptotic potential of wild-type and ϕ mutant viruses by acridine orange staining (Figure 4A). Following infection of HeLa cells with rsT3D, ∼30–35% of cells showed altered nuclear morphology characteristic of apoptosis. However, infection with the K594D or I595K elicited apoptosis in only ∼12% of cells. As a complementary approach, we used propidium iodide staining and flow cytometry to compare the capacity of these viruses to promote DNA fragmentation in infected cells (Figure 4B). Infection of HeLa cells with rsT3D resulted in ∼50% cells displaying hypodiploid (sub-G1) DNA staining, characteristic of apoptotic cells. In contrast, infection with either K594D or I595K resulted in a percentage of cells in sub-G1 that was comparable to mock infection. These results suggest that the apoptotic potential of reovirus is regulated by ϕ residues Lys594 and Ile595.

Bottom Line: We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates.Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery.These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

View Article: PubMed Central - PubMed

Affiliation: Department of Pediatrics, Vanderbilt University School of Medicine, Nashville, TN, USA. pdanthi@indiana.edu

ABSTRACT
Apoptosis plays an important role in the pathogenesis of reovirus encephalitis. Reovirus outer-capsid protein mu1, which functions to penetrate host cell membranes during viral entry, is the primary regulator of apoptosis following reovirus infection. Ectopic expression of full-length and truncated forms of mu1 indicates that the mu1 phi domain is sufficient to elicit a cell death response. To evaluate the contribution of the mu1 phi domain to the induction of apoptosis following reovirus infection, phi mutant viruses were generated by reverse genetics and analyzed for the capacity to penetrate cell membranes and elicit apoptosis. We found that mutations in phi diminish reovirus membrane penetration efficiency by preventing conformational changes that lead to generation of key reovirus entry intermediates. Independent of effects on membrane penetration, amino acid substitutions in phi affect the apoptotic potential of reovirus, suggesting that phi initiates apoptosis subsequent to cytosolic delivery. In comparison to wild-type virus, apoptosis-defective phi mutant viruses display diminished neurovirulence following intracranial inoculation of newborn mice. These results indicate that the phi domain of mu1 plays an important regulatory role in reovirus-induced apoptosis and disease.

Show MeSH
Related in: MedlinePlus