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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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Infectivity and growth of μ1 ϕ mutants.(A) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 104 particles/cell. After incubation at 37°C for 18 h, cells were fixed using methanol and visualized by immunostaining. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (B) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 2 PFU/cell. Titers of virus in cell lysates at the indicated intervals post-infection were determined by plaque assay. Results are expressed as viral yield for triplicate samples. Error bars indicate SD.
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ppat-1000248-g002: Infectivity and growth of μ1 ϕ mutants.(A) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 104 particles/cell. After incubation at 37°C for 18 h, cells were fixed using methanol and visualized by immunostaining. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (B) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 2 PFU/cell. Titers of virus in cell lysates at the indicated intervals post-infection were determined by plaque assay. Results are expressed as viral yield for triplicate samples. Error bars indicate SD.

Mentions: To evaluate the function of residues 594 and 595 in apoptosis induction in the context of virus infection, we used reverse genetics [35] to rescue wild-type recombinant strain type 3 Dearing (rsT3D) and ϕ mutant viruses, K594D or I595K, which we hypothesized to be altered in prodeath signaling (Figure 1). Viruses with these substitutions in ϕ were rescued with equivalent efficiency (data not shown). To determine whether substitutions in ϕ affect reovirus infectivity, we quantified the number of L929 cells expressing nascent viral protein following infection with rsT3D and the mutant viruses (Figure 2A). We found that infection with either rsT3D or the ϕ mutants resulted in an approximately equal number of infected cells. These data suggest that mutations in ϕ do not affect the efficiency with which reovirus establishes infection of host cells. To evaluate whether these mutations affect the capacity of reovirus to efficiently replicate in host cells, we infected L929 cells with either rsT3D or the ϕ mutant viruses at a multiplicity of infection (MOI) of 2 plaque forming units (PFU)/cell and quantified viral titer at 0, 12, 24, and 48 h following infection (Figure 2B). At 24–48 h post-infection, each of the viruses produced ∼1000- to 10 000-fold increases in infectious progeny. In concordance with these findings, no differences were detected in the replication kinetics of rsT3D, K594D, and I595K when infection was initiated at an MOI of 0.01 PFU/cell (data not shown). These data indicate that mutations at ϕ residues 594 or 595 do not compromise viral replication kinetics or yield in cell culture.


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)

Infectivity and growth of μ1 ϕ mutants.(A) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 104 particles/cell. After incubation at 37°C for 18 h, cells were fixed using methanol and visualized by immunostaining. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (B) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 2 PFU/cell. Titers of virus in cell lysates at the indicated intervals post-infection were determined by plaque assay. Results are expressed as viral yield for triplicate samples. Error bars indicate SD.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2600812&req=5

ppat-1000248-g002: Infectivity and growth of μ1 ϕ mutants.(A) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 104 particles/cell. After incubation at 37°C for 18 h, cells were fixed using methanol and visualized by immunostaining. Reovirus-infected cells were quantified by counting fluorescent cells. Results are expressed as mean fluorescent focus units (FFU) per field for triplicate samples. Error bars indicate SD. (B) L929 cells were adsorbed with rsT3D or the indicated ϕ mutant at an MOI of 2 PFU/cell. Titers of virus in cell lysates at the indicated intervals post-infection were determined by plaque assay. Results are expressed as viral yield for triplicate samples. Error bars indicate SD.
Mentions: To evaluate the function of residues 594 and 595 in apoptosis induction in the context of virus infection, we used reverse genetics [35] to rescue wild-type recombinant strain type 3 Dearing (rsT3D) and ϕ mutant viruses, K594D or I595K, which we hypothesized to be altered in prodeath signaling (Figure 1). Viruses with these substitutions in ϕ were rescued with equivalent efficiency (data not shown). To determine whether substitutions in ϕ affect reovirus infectivity, we quantified the number of L929 cells expressing nascent viral protein following infection with rsT3D and the mutant viruses (Figure 2A). We found that infection with either rsT3D or the ϕ mutants resulted in an approximately equal number of infected cells. These data suggest that mutations in ϕ do not affect the efficiency with which reovirus establishes infection of host cells. To evaluate whether these mutations affect the capacity of reovirus to efficiently replicate in host cells, we infected L929 cells with either rsT3D or the ϕ mutant viruses at a multiplicity of infection (MOI) of 2 plaque forming units (PFU)/cell and quantified viral titer at 0, 12, 24, and 48 h following infection (Figure 2B). At 24–48 h post-infection, each of the viruses produced ∼1000- to 10 000-fold increases in infectious progeny. In concordance with these findings, no differences were detected in the replication kinetics of rsT3D, K594D, and I595K when infection was initiated at an MOI of 0.01 PFU/cell (data not shown). These data indicate that mutations at ϕ residues 594 or 595 do not compromise viral replication kinetics or yield in cell culture.

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