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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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In vitro analysis of m41 mutant viruses.(A) Fibroblasts were infected with WT, Rev or the indicated MCMV mutants and total cell lysates prepared 24 hr later. Immunoblot analysis was performed using antibodies specific for m41, m41.1 or IE1 as indicated. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 h later 100 µM etoposide added. Cell viability was quantified by Trypan Blue exclusion 24 hr after the addition of etoposide (n = 6). (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability assessed 48 hr later (n = 8). (D) Fibroblasts or (E) IC-21 macrophages were infected with WT MCMV (filled square), Rev (filled circle), Δm41 (open circle), Δm41.1 (cross) or Δm41/m41.1 (open diamond) (MOI = 0.05 for fibroblasts and MOI = 0.5 for IC-21) and viral replication measured at the indicated times pi (n = 6 for fibroblasts and macrophages). Dotted line indicates the limit of detection of the assay.
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ppat-1003192-g004: In vitro analysis of m41 mutant viruses.(A) Fibroblasts were infected with WT, Rev or the indicated MCMV mutants and total cell lysates prepared 24 hr later. Immunoblot analysis was performed using antibodies specific for m41, m41.1 or IE1 as indicated. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 h later 100 µM etoposide added. Cell viability was quantified by Trypan Blue exclusion 24 hr after the addition of etoposide (n = 6). (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability assessed 48 hr later (n = 8). (D) Fibroblasts or (E) IC-21 macrophages were infected with WT MCMV (filled square), Rev (filled circle), Δm41 (open circle), Δm41.1 (cross) or Δm41/m41.1 (open diamond) (MOI = 0.05 for fibroblasts and MOI = 0.5 for IC-21) and viral replication measured at the indicated times pi (n = 6 for fibroblasts and macrophages). Dotted line indicates the limit of detection of the assay.

Mentions: Having established that the m41 proteins enhance MCMV replication in vivo, additional mutants were constructed in order to assess the relative contribution of the m41 proteins, and m41.1, to viral pathogenesis. Mutants specifically lacking expression of m41.1 (termed Δm41.1) or both m41 and m41L (termed Δm41) were constructed using BAC mutagenesis. The Δm41 virus was produced by mutating the annotated m41 ATG to a stop codon, introducing a mutation at this position was predicted to have no impact on the expression of m41.1. Immunoblotting of lysates infected with the Δm41 virus confirmed that expression m41 and m41L was absent while expression of m41.1 was not affected (Fig. 4A). The Δm41.1 mutant was constructed by mutating the sequence coding for Leu21 of m41.1 to a stop codon. This mutation results in a silent mutation within the m41 sequence. Again immunoblotting of lysates from infected cells confirmed the specific deletion of m41.1 (Fig. 4A). The ability of the mutant viruses to inhibit apoptosis was then tested in fibroblasts. Cells infected with the Δm41 mutant were as resistant to etoposide-induced killing as those infected with WT or Rev MCMV (Fig. 4B). Conversely, etoposide treatment of cells infected with the m41.1 deletion virus resulted in cell death equivalent to that observed with the Δm41/41.1 mutant. Similarly, infection of macrophages with the Δm41.1 specific mutant virus resulted in cell death, while the Δm41 mutant had no impact on cell viability (Fig. 4C). Deletion of m41.1 or m41 and m41L did not affect viral replication in fibroblasts (Fig. 4D). However, in macrophages, deletion of m41.1 resulted in a significant attenuation of viral replication (Fig. 4E). These results confirm that m41.1 enhances viral replication in vitro by preventing the death of infected cells. Importantly, our data establishes that the ability of MCMV-infected cells to resist apoptosis in vitro is not dependent on m41 or m41L.


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)

In vitro analysis of m41 mutant viruses.(A) Fibroblasts were infected with WT, Rev or the indicated MCMV mutants and total cell lysates prepared 24 hr later. Immunoblot analysis was performed using antibodies specific for m41, m41.1 or IE1 as indicated. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 h later 100 µM etoposide added. Cell viability was quantified by Trypan Blue exclusion 24 hr after the addition of etoposide (n = 6). (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability assessed 48 hr later (n = 8). (D) Fibroblasts or (E) IC-21 macrophages were infected with WT MCMV (filled square), Rev (filled circle), Δm41 (open circle), Δm41.1 (cross) or Δm41/m41.1 (open diamond) (MOI = 0.05 for fibroblasts and MOI = 0.5 for IC-21) and viral replication measured at the indicated times pi (n = 6 for fibroblasts and macrophages). Dotted line indicates the limit of detection of the assay.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585157&req=5

ppat-1003192-g004: In vitro analysis of m41 mutant viruses.(A) Fibroblasts were infected with WT, Rev or the indicated MCMV mutants and total cell lysates prepared 24 hr later. Immunoblot analysis was performed using antibodies specific for m41, m41.1 or IE1 as indicated. (B) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 h later 100 µM etoposide added. Cell viability was quantified by Trypan Blue exclusion 24 hr after the addition of etoposide (n = 6). (C) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability assessed 48 hr later (n = 8). (D) Fibroblasts or (E) IC-21 macrophages were infected with WT MCMV (filled square), Rev (filled circle), Δm41 (open circle), Δm41.1 (cross) or Δm41/m41.1 (open diamond) (MOI = 0.05 for fibroblasts and MOI = 0.5 for IC-21) and viral replication measured at the indicated times pi (n = 6 for fibroblasts and macrophages). Dotted line indicates the limit of detection of the assay.
Mentions: Having established that the m41 proteins enhance MCMV replication in vivo, additional mutants were constructed in order to assess the relative contribution of the m41 proteins, and m41.1, to viral pathogenesis. Mutants specifically lacking expression of m41.1 (termed Δm41.1) or both m41 and m41L (termed Δm41) were constructed using BAC mutagenesis. The Δm41 virus was produced by mutating the annotated m41 ATG to a stop codon, introducing a mutation at this position was predicted to have no impact on the expression of m41.1. Immunoblotting of lysates infected with the Δm41 virus confirmed that expression m41 and m41L was absent while expression of m41.1 was not affected (Fig. 4A). The Δm41.1 mutant was constructed by mutating the sequence coding for Leu21 of m41.1 to a stop codon. This mutation results in a silent mutation within the m41 sequence. Again immunoblotting of lysates from infected cells confirmed the specific deletion of m41.1 (Fig. 4A). The ability of the mutant viruses to inhibit apoptosis was then tested in fibroblasts. Cells infected with the Δm41 mutant were as resistant to etoposide-induced killing as those infected with WT or Rev MCMV (Fig. 4B). Conversely, etoposide treatment of cells infected with the m41.1 deletion virus resulted in cell death equivalent to that observed with the Δm41/41.1 mutant. Similarly, infection of macrophages with the Δm41.1 specific mutant virus resulted in cell death, while the Δm41 mutant had no impact on cell viability (Fig. 4C). Deletion of m41.1 or m41 and m41L did not affect viral replication in fibroblasts (Fig. 4D). However, in macrophages, deletion of m41.1 resulted in a significant attenuation of viral replication (Fig. 4E). These results confirm that m41.1 enhances viral replication in vitro by preventing the death of infected cells. Importantly, our data establishes that the ability of MCMV-infected cells to resist apoptosis in vitro is not dependent on m41 or m41L.

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