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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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Apoptosis-modulating ϕ mutations alter μ1 distribution in cells.(A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution 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-g005: Apoptosis-modulating ϕ mutations alter μ1 distribution in cells.(A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D.

Mentions: The capacity of ectopically expressed μ1 to induce apoptosis correlates with its distribution to intracellular membranous structures such as mitochondria, endoplasmic reticulum, and lipid droplets [33]. Moreover, a short loop and amphipathic helix within φ (residues 582 to 611) is necessary and sufficient for association of μ1 with intracellular membranous structures and induction of apoptosis [33] and contains residues Lys594 and Ile595. To determine whether the decrease in apoptotic potential of ϕ mutants K594D and I595K resulted from a diminished capacity of mutant μ1 to associate with intracellular membranes, we compared the distribution of wild-type and mutant μ1 proteins in infected CV-1 cells by immunofluorescence microscopy (Figure 5A). Consistent with previous findings [33], infection with rsT3D and each of the ϕ mutant viruses resulted in three discernable μ1 distribution patterns: (i) diffuse cytoplasmic distribution, (ii) incorporation within viral inclusions, or (iii) localization to intracellular membranes structures. Although μ1 was found in all three locations following infection with rsT3D, K594D, and I595K, a lower fraction of cells displayed diffuse μ1 localization following infection with either of the ϕ mutants (∼15%) in comparison to rsT3D (∼30%) (Figure 5B). Similarly, μ1 localized to intracellular membrane fractions in a significantly lower percentage of cells following infection with either K594D (∼35%) or I595K (∼20%) in comparison to rsT3D (∼50%). Concordant with the decrease in μ1 distribution throughout the cytoplasm and to intracellular membranes, a considerably higher fraction of cells showed μ1 within viral inclusions following infection with the ϕ mutants (∼80%) in comparison to wild-type virus (∼50%). Thus, mutations in ϕ that modulate apoptosis induction affect μ1 distribution in cells.


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)

Apoptosis-modulating ϕ mutations alter μ1 distribution in cells.(A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution 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-g005: Apoptosis-modulating ϕ mutations alter μ1 distribution in cells.(A) CV-1 cells were infected with 10 PFU/cell of rsT3D or the indicated ϕ mutant, fixed 48 h post-infection, permeabilized, and immunostained with anti-μ1 MAb 4A3 (green) and anti-μNS serum (red), followed by goat anti-mouse IgG conjugated to Alexa Fluor 488 (green) and goat anti-rabbit IgG conjugated to Alexa Fluor 594 (red). Scale bars, 10 µm. A representative image of the predominant μ1 distribution pattern following infection with each virus strain is shown. (B) The patterns of μ1 distribution in individual infected cells were scored for each time point as diffuse, associated with viral inclusions, or associated with intracellular membranes (marked by distinct ring-like distribution of μ1). Results are expressed as the mean percentage of cells showing the indicated μ1 distribution for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to rsT3D.
Mentions: The capacity of ectopically expressed μ1 to induce apoptosis correlates with its distribution to intracellular membranous structures such as mitochondria, endoplasmic reticulum, and lipid droplets [33]. Moreover, a short loop and amphipathic helix within φ (residues 582 to 611) is necessary and sufficient for association of μ1 with intracellular membranous structures and induction of apoptosis [33] and contains residues Lys594 and Ile595. To determine whether the decrease in apoptotic potential of ϕ mutants K594D and I595K resulted from a diminished capacity of mutant μ1 to associate with intracellular membranes, we compared the distribution of wild-type and mutant μ1 proteins in infected CV-1 cells by immunofluorescence microscopy (Figure 5A). Consistent with previous findings [33], infection with rsT3D and each of the ϕ mutant viruses resulted in three discernable μ1 distribution patterns: (i) diffuse cytoplasmic distribution, (ii) incorporation within viral inclusions, or (iii) localization to intracellular membranes structures. Although μ1 was found in all three locations following infection with rsT3D, K594D, and I595K, a lower fraction of cells displayed diffuse μ1 localization following infection with either of the ϕ mutants (∼15%) in comparison to rsT3D (∼30%) (Figure 5B). Similarly, μ1 localized to intracellular membrane fractions in a significantly lower percentage of cells following infection with either K594D (∼35%) or I595K (∼20%) in comparison to rsT3D (∼50%). Concordant with the decrease in μ1 distribution throughout the cytoplasm and to intracellular membranes, a considerably higher fraction of cells showed μ1 within viral inclusions following infection with the ϕ mutants (∼80%) in comparison to wild-type virus (∼50%). Thus, mutations in ϕ that modulate apoptosis induction affect μ1 distribution in cells.

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