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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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m41.1 enhances viral replication in leukocytes.(A) Whole blood was isolated from infected mice on day 5 pi., and red blood cells removed by isotonic lysis. The proportion of infected cells was determined by infectious centre assay. (B) Mice were infected with WT MCMV or the Δm41.1 mutant and spleens removed on day 4 pi. Viral burden in stromal cells (left panel), or leukocytes (right panel) were determined by plaque assay.
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ppat-1003192-g007: m41.1 enhances viral replication in leukocytes.(A) Whole blood was isolated from infected mice on day 5 pi., and red blood cells removed by isotonic lysis. The proportion of infected cells was determined by infectious centre assay. (B) Mice were infected with WT MCMV or the Δm41.1 mutant and spleens removed on day 4 pi. Viral burden in stromal cells (left panel), or leukocytes (right panel) were determined by plaque assay.

Mentions: In vitro, replication of the Δm41.1 mutant was significantly attenuated in macrophages while no defect was observed in fibroblasts (Fig. 4D and E). To determine if a similar effect occurs in vivo we measured virus levels in the blood of infected mice since myeloid progenitors are the predominant cell type infected by MCMV in the blood [25]–[27]. Blood was collected from mice infected with WT MCMV or the Δm41.1 mutant at day 5 pi and the number of infected cells in the circulation determined by infectious centre assay. Mice infected with the Δm41.1 mutant had significantly fewer leukocytes in the blood containing infectious virus (Fig. 7A). Thus, m41.1 protects leukocytes in the blood from apoptosis during MCMV infection. We extended this finding by determining if m41.1 enhanced the survival of virally-infected leukocytes in the spleen. In the spleen, MCMV replication occurs in several distinct cell types including cells of the myeloid lineage such as macrophages and dendritic cells (DC), and in splenic stromal cells such as endothelial cells [28]–[29]. Spleens were removed from mice infected with WT or the Δm41.1 mutant at day 4 pi and the viral load within the leukocytes or stromal cell fractions quantified. Viral replication within the leukocyte fraction was significantly reduced in mice infected with the Δm41.1 virus (Fig. 7B, right panel). However, viral titers of the Δm41.1 mutant were not significantly different from those of the WT virus when viral load within the stromal cell fraction was measured (Fig. 7B, left panel). Thus, m41.1-mediated inhibition of Bak is required for efficient MCMV replication in leukocytes, but is dispensable for MCMV replication in most permissive cells in the spleen.


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)

m41.1 enhances viral replication in leukocytes.(A) Whole blood was isolated from infected mice on day 5 pi., and red blood cells removed by isotonic lysis. The proportion of infected cells was determined by infectious centre assay. (B) Mice were infected with WT MCMV or the Δm41.1 mutant and spleens removed on day 4 pi. Viral burden in stromal cells (left panel), or leukocytes (right panel) were determined by plaque assay.
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Related In: Results  -  Collection

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

ppat-1003192-g007: m41.1 enhances viral replication in leukocytes.(A) Whole blood was isolated from infected mice on day 5 pi., and red blood cells removed by isotonic lysis. The proportion of infected cells was determined by infectious centre assay. (B) Mice were infected with WT MCMV or the Δm41.1 mutant and spleens removed on day 4 pi. Viral burden in stromal cells (left panel), or leukocytes (right panel) were determined by plaque assay.
Mentions: In vitro, replication of the Δm41.1 mutant was significantly attenuated in macrophages while no defect was observed in fibroblasts (Fig. 4D and E). To determine if a similar effect occurs in vivo we measured virus levels in the blood of infected mice since myeloid progenitors are the predominant cell type infected by MCMV in the blood [25]–[27]. Blood was collected from mice infected with WT MCMV or the Δm41.1 mutant at day 5 pi and the number of infected cells in the circulation determined by infectious centre assay. Mice infected with the Δm41.1 mutant had significantly fewer leukocytes in the blood containing infectious virus (Fig. 7A). Thus, m41.1 protects leukocytes in the blood from apoptosis during MCMV infection. We extended this finding by determining if m41.1 enhanced the survival of virally-infected leukocytes in the spleen. In the spleen, MCMV replication occurs in several distinct cell types including cells of the myeloid lineage such as macrophages and dendritic cells (DC), and in splenic stromal cells such as endothelial cells [28]–[29]. Spleens were removed from mice infected with WT or the Δm41.1 mutant at day 4 pi and the viral load within the leukocytes or stromal cell fractions quantified. Viral replication within the leukocyte fraction was significantly reduced in mice infected with the Δm41.1 virus (Fig. 7B, right panel). However, viral titers of the Δm41.1 mutant were not significantly different from those of the WT virus when viral load within the stromal cell fraction was measured (Fig. 7B, left panel). Thus, m41.1-mediated inhibition of Bak is required for efficient MCMV replication in leukocytes, but is dispensable for MCMV replication in most permissive cells in the spleen.

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