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MCMV-mediated inhibition of the pro-apoptotic Bak protein is required for optimal in vivo replication.

Fleming P, Kvansakul M, Voigt V, Kile BT, Kluck RM, Huang DC, Degli-Esposti MA, Andoniou CE - PLoS Pathog. (2013)

Bottom Line: Here we show that m41.1 is critical for optimal MCMV replication in vivo.Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak(-/-) mice.Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Western Australia, Australia.

ABSTRACT
Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity in vitro. Here we show that m41.1 is critical for optimal MCMV replication in vivo. Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak(-/-) mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins.

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Mutation of the m41 ORF inhibits viral replication.(A) Fibroblasts were infected with WT MCMV, the Δm41/m41.1 mutant or a revertant (Rev) virus at an MOI = 3. Total cell lysates were prepared at 24 hr pi and immunoblotted with anti-m41, anti-m41.1, or anti-IE1 antibodies. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 hr later treated with 100 µM etoposide (open columns) or vehicle (filled columns). Cell viability was quantified by Trypan Blue exclusion 24 hr later (n = 4) (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability determined 48 hr pi (n = 4) (D) BALB/c mice were infected with WT MCMV (filled square), Rev virus (filled circle), or Δm41/m41.1 (open diamond), organs were removed at the indicated times pi and viral load per organ determined by plaque assay. Viral titers were quantified in three separate experiments and the data pooled, mean ± S.D. of 6–9 mice per group is plotted. * P<0.05, ** P<0.005, *** P<0.0001. Dotted line indicates the limit of detection of the assay.
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ppat-1003192-g003: Mutation of the m41 ORF inhibits viral replication.(A) Fibroblasts were infected with WT MCMV, the Δm41/m41.1 mutant or a revertant (Rev) virus at an MOI = 3. Total cell lysates were prepared at 24 hr pi and immunoblotted with anti-m41, anti-m41.1, or anti-IE1 antibodies. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 hr later treated with 100 µM etoposide (open columns) or vehicle (filled columns). Cell viability was quantified by Trypan Blue exclusion 24 hr later (n = 4) (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability determined 48 hr pi (n = 4) (D) BALB/c mice were infected with WT MCMV (filled square), Rev virus (filled circle), or Δm41/m41.1 (open diamond), organs were removed at the indicated times pi and viral load per organ determined by plaque assay. Viral titers were quantified in three separate experiments and the data pooled, mean ± S.D. of 6–9 mice per group is plotted. * P<0.05, ** P<0.005, *** P<0.0001. Dotted line indicates the limit of detection of the assay.

Mentions: To evaluate the role of the m41 proteins during infection we constructed a mutant virus termed Δm41/41.1 that does not produce any of the m41 proteins. The Δm41/41.1 virus was generated by introducing a stop codon in-frame with the m41 coding sequence such that Asp4 of m41 was mutated to a stop. The mutation of the m41 sequence also results in the mutation of the m41.1 sequence such that Met1 is mutated to Leu. These changes were predicted to prevent the expression of the m41 proteins and m41.1. Western blot analysis of fibroblasts infected with the Δm41/41.1 virus confirmed that m41, m41L and m41.1 proteins were not expressed (Fig. 3A). A revertant (Rev) virus was produced by repairing the mutation within the m41 ORF. Comparing the revertant virus to the Δm41/41.1 mutant ensures that any growth defects detected in subsequent analysis are due to loss of the m41 proteins and not the result of mutations elsewhere in the viral genome. We have previously demonstrated that MCMV-infected cells are resistant to apoptosis initiated by stimuli such as growth factor withdrawal or cytotoxic drugs [17], [22]. As expected the viability of fibroblasts infected with either WT or Rev virus was not affected by etoposide treatment (Fig. 3B). By contrast, addition of etoposide to fibroblasts infected with Δm41/41.1 virus resulted in significant cell death (Fig. 3B). MCMV is capable of replicating in broad range of cell types, with cells such as macrophages and DCs more sensitive to the absence of anti-apoptotic proteins [17], [23]. We therefore, assessed the impact of infection of the Δm41/41.1 mutant on the viability of IC21 macrophages. Infection of macrophages with WT or Rev virus had no impact on cell viability, while a significant proportion of cells infected with the Δm41/41.1 virus had died by 48 h pi (Fig. 3C). Next, the capacity of the Δm41/41.1 virus to replicate in vivo was assessed by quantifying viral titers in BALB/c mice. Mice were infected with WT, Δm41/41.1 mutant, or Rev virus and viral titers in the target organs of spleen, liver, lungs and salivary glands determined. Replication of the Δm41/41.1 viral mutant was attenuated in all the organs assessed, with a pronounced defect in viral replication evident in the liver and lungs (Fig. 3D). Thus, proteins derived from the m41 ORF promote efficient in vivo replication of MCMV.


MCMV-mediated inhibition of the pro-apoptotic Bak protein is required for optimal in vivo replication.

Fleming P, Kvansakul M, Voigt V, Kile BT, Kluck RM, Huang DC, Degli-Esposti MA, Andoniou CE - PLoS Pathog. (2013)

Mutation of the m41 ORF inhibits viral replication.(A) Fibroblasts were infected with WT MCMV, the Δm41/m41.1 mutant or a revertant (Rev) virus at an MOI = 3. Total cell lysates were prepared at 24 hr pi and immunoblotted with anti-m41, anti-m41.1, or anti-IE1 antibodies. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 hr later treated with 100 µM etoposide (open columns) or vehicle (filled columns). Cell viability was quantified by Trypan Blue exclusion 24 hr later (n = 4) (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability determined 48 hr pi (n = 4) (D) BALB/c mice were infected with WT MCMV (filled square), Rev virus (filled circle), or Δm41/m41.1 (open diamond), organs were removed at the indicated times pi and viral load per organ determined by plaque assay. Viral titers were quantified in three separate experiments and the data pooled, mean ± S.D. of 6–9 mice per group is plotted. * P<0.05, ** P<0.005, *** P<0.0001. Dotted line indicates the limit of detection of the assay.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585157&req=5

ppat-1003192-g003: Mutation of the m41 ORF inhibits viral replication.(A) Fibroblasts were infected with WT MCMV, the Δm41/m41.1 mutant or a revertant (Rev) virus at an MOI = 3. Total cell lysates were prepared at 24 hr pi and immunoblotted with anti-m41, anti-m41.1, or anti-IE1 antibodies. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 hr later treated with 100 µM etoposide (open columns) or vehicle (filled columns). Cell viability was quantified by Trypan Blue exclusion 24 hr later (n = 4) (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability determined 48 hr pi (n = 4) (D) BALB/c mice were infected with WT MCMV (filled square), Rev virus (filled circle), or Δm41/m41.1 (open diamond), organs were removed at the indicated times pi and viral load per organ determined by plaque assay. Viral titers were quantified in three separate experiments and the data pooled, mean ± S.D. of 6–9 mice per group is plotted. * P<0.05, ** P<0.005, *** P<0.0001. Dotted line indicates the limit of detection of the assay.
Mentions: To evaluate the role of the m41 proteins during infection we constructed a mutant virus termed Δm41/41.1 that does not produce any of the m41 proteins. The Δm41/41.1 virus was generated by introducing a stop codon in-frame with the m41 coding sequence such that Asp4 of m41 was mutated to a stop. The mutation of the m41 sequence also results in the mutation of the m41.1 sequence such that Met1 is mutated to Leu. These changes were predicted to prevent the expression of the m41 proteins and m41.1. Western blot analysis of fibroblasts infected with the Δm41/41.1 virus confirmed that m41, m41L and m41.1 proteins were not expressed (Fig. 3A). A revertant (Rev) virus was produced by repairing the mutation within the m41 ORF. Comparing the revertant virus to the Δm41/41.1 mutant ensures that any growth defects detected in subsequent analysis are due to loss of the m41 proteins and not the result of mutations elsewhere in the viral genome. We have previously demonstrated that MCMV-infected cells are resistant to apoptosis initiated by stimuli such as growth factor withdrawal or cytotoxic drugs [17], [22]. As expected the viability of fibroblasts infected with either WT or Rev virus was not affected by etoposide treatment (Fig. 3B). By contrast, addition of etoposide to fibroblasts infected with Δm41/41.1 virus resulted in significant cell death (Fig. 3B). MCMV is capable of replicating in broad range of cell types, with cells such as macrophages and DCs more sensitive to the absence of anti-apoptotic proteins [17], [23]. We therefore, assessed the impact of infection of the Δm41/41.1 mutant on the viability of IC21 macrophages. Infection of macrophages with WT or Rev virus had no impact on cell viability, while a significant proportion of cells infected with the Δm41/41.1 virus had died by 48 h pi (Fig. 3C). Next, the capacity of the Δm41/41.1 virus to replicate in vivo was assessed by quantifying viral titers in BALB/c mice. Mice were infected with WT, Δm41/41.1 mutant, or Rev virus and viral titers in the target organs of spleen, liver, lungs and salivary glands determined. Replication of the Δm41/41.1 viral mutant was attenuated in all the organs assessed, with a pronounced defect in viral replication evident in the liver and lungs (Fig. 3D). Thus, proteins derived from the m41 ORF promote efficient in vivo replication of MCMV.

Bottom Line: Here we show that m41.1 is critical for optimal MCMV replication in vivo.Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak(-/-) mice.Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection.

View Article: PubMed Central - PubMed

Affiliation: Immunology and Virology Program, Centre for Ophthalmology and Visual Science, The University of Western Australia, Nedlands, Western Australia, Australia.

ABSTRACT
Successful replication and transmission of large DNA viruses such as the cytomegaloviruses (CMV) family of viruses depends on the ability to interfere with multiple aspects of the host immune response. Apoptosis functions as a host innate defence mechanism against viral infection, and the capacity to interfere with this process is essential for the replication of many viruses. The Bcl-2 family of proteins are the principle regulators of apoptosis, with two pro-apoptotic members, Bax and Bak, essential for apoptosis to proceed. The m38.5 protein encoded by murine CMV (MCMV) has been identified as Bax-specific inhibitor of apoptosis. Recently, m41.1, a protein product encoded by the m41 open reading frame (ORF) of MCMV, has been shown to inhibit Bak activity in vitro. Here we show that m41.1 is critical for optimal MCMV replication in vivo. Growth of a m41.1 mutant was attenuated in multiple organs, a defect that was not apparent in Bak(-/-) mice. Thus, m41.1 promotes MCMV replication by inhibiting Bak-dependent apoptosis during in vivo infection. The results show that Bax and Bak mediate non-redundant functions during MCMV infection and that the virus produces distinct inhibitors for each protein to counter the activity of these proteins.

Show MeSH
Related in: MedlinePlus