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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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Charge substitutions of μ1 ϕ residues Lys594 and Ile595 diminish apoptosis-inducing capacity of ectopically expressed μ1 and ϕ.(A) Crystal structure of a μ1 monomer (PBD∶1JMU) [36]. The μ1N, δ, and ϕ domains are indicated in red, yellow, and blue, respectively. The location of a minimal functional region of ϕ (spanning amino acids 582–611) sufficient for apoptosis induction following expression in cells [33] is indicated in dark blue. A black box indicates the position of Lys594 and Ile595. (B) 293T cells were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (C) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for μ1 (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the PSTAIR peptide of Cdk1 (lower panel). (D) 293T cells were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (E) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for GFP (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the Cdk1 PSTAIR peptide (lower panel).
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ppat-1000248-g001: Charge substitutions of μ1 ϕ residues Lys594 and Ile595 diminish apoptosis-inducing capacity of ectopically expressed μ1 and ϕ.(A) Crystal structure of a μ1 monomer (PBD∶1JMU) [36]. The μ1N, δ, and ϕ domains are indicated in red, yellow, and blue, respectively. The location of a minimal functional region of ϕ (spanning amino acids 582–611) sufficient for apoptosis induction following expression in cells [33] is indicated in dark blue. A black box indicates the position of Lys594 and Ile595. (B) 293T cells were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (C) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for μ1 (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the PSTAIR peptide of Cdk1 (lower panel). (D) 293T cells were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (E) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for GFP (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the Cdk1 PSTAIR peptide (lower panel).

Mentions: In addition to its structural and proapoptotic functions, the μ1 protein plays an essential role in penetration of host cell membranes during reovirus cell entry. Following uptake into cellular endosomes, reovirus undergoes proteolytic disassembly resulting in removal of outer-capsid protein σ3 and cleavage of the μ1 protein to form the δ and ϕ fragments (Figure 1A), which remain associated with the newly generated infectious subvirion particles (ISVPs) [22]–[25]. Conformational changes within the ISVP-associated δ fragment lead to formation of ISVP*s and allow the autocatalytic cleavage and release of a small myristoylated N-terminal fragment of μ1, μ1N (Figure 1A) [26]–[30]. The released μ1N fragment is thought to function alone or act in concert with the lipophilic ISVP*s to form pores in host cell membranes that release the viral core into the cytoplasm to initiate infection [31],[32].


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)

Charge substitutions of μ1 ϕ residues Lys594 and Ile595 diminish apoptosis-inducing capacity of ectopically expressed μ1 and ϕ.(A) Crystal structure of a μ1 monomer (PBD∶1JMU) [36]. The μ1N, δ, and ϕ domains are indicated in red, yellow, and blue, respectively. The location of a minimal functional region of ϕ (spanning amino acids 582–611) sufficient for apoptosis induction following expression in cells [33] is indicated in dark blue. A black box indicates the position of Lys594 and Ile595. (B) 293T cells were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (C) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for μ1 (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the PSTAIR peptide of Cdk1 (lower panel). (D) 293T cells were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (E) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for GFP (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the Cdk1 PSTAIR peptide (lower panel).
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ppat-1000248-g001: Charge substitutions of μ1 ϕ residues Lys594 and Ile595 diminish apoptosis-inducing capacity of ectopically expressed μ1 and ϕ.(A) Crystal structure of a μ1 monomer (PBD∶1JMU) [36]. The μ1N, δ, and ϕ domains are indicated in red, yellow, and blue, respectively. The location of a minimal functional region of ϕ (spanning amino acids 582–611) sufficient for apoptosis induction following expression in cells [33] is indicated in dark blue. A black box indicates the position of Lys594 and Ile595. (B) 293T cells were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (C) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for μ1 (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the PSTAIR peptide of Cdk1 (lower panel). (D) 293T cells were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (E) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for GFP (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the Cdk1 PSTAIR peptide (lower panel).
Mentions: In addition to its structural and proapoptotic functions, the μ1 protein plays an essential role in penetration of host cell membranes during reovirus cell entry. Following uptake into cellular endosomes, reovirus undergoes proteolytic disassembly resulting in removal of outer-capsid protein σ3 and cleavage of the μ1 protein to form the δ and ϕ fragments (Figure 1A), which remain associated with the newly generated infectious subvirion particles (ISVPs) [22]–[25]. Conformational changes within the ISVP-associated δ fragment lead to formation of ISVP*s and allow the autocatalytic cleavage and release of a small myristoylated N-terminal fragment of μ1, μ1N (Figure 1A) [26]–[30]. The released μ1N fragment is thought to function alone or act in concert with the lipophilic ISVP*s to form pores in host cell membranes that release the viral core into the cytoplasm to initiate infection [31],[32].

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