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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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Genomic arrangement and analysis of the m41 locus.RNA isolated from MCMV infected fibroblasts at IE, E and L times post-infection was subjected to (A) 5′ RACE or (B) 3′ RACE analysis. The resulting products were separated on 1% agarose gels. (C) The genomic region of MCMV encompassing the annotated m41 ORF (solid arrow), the newly identified 41 exon (solid box), and adjoining ORFs (open arrows) is shown. Direction of transcription is indicated by the orientation of the arrows. Location of the common polyadenylation site used by all genes in this region is denoted by the filled oval. (D) Sequence of the annotated m41 ORF is shown. The annotated m41 translation start site and the location of splice acceptor and donor sites are shown above the DNA sequence. Location of mutations within the m41 ORF are shown below the DNA sequence. (E) The indicated m41 constructs were transiently overexpressed in Cos-7 cells, total cell lysates prepared and expression of the m41 and m41L proteins detected by immunoblot using anti-flag antibodies. (F) Total cell lysates were prepared from fibroblasts infected with MCMV at the indicated time pi and the expression of m41, m41L, and IE1 proteins detected by immunoblot.
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ppat-1003192-g001: Genomic arrangement and analysis of the m41 locus.RNA isolated from MCMV infected fibroblasts at IE, E and L times post-infection was subjected to (A) 5′ RACE or (B) 3′ RACE analysis. The resulting products were separated on 1% agarose gels. (C) The genomic region of MCMV encompassing the annotated m41 ORF (solid arrow), the newly identified 41 exon (solid box), and adjoining ORFs (open arrows) is shown. Direction of transcription is indicated by the orientation of the arrows. Location of the common polyadenylation site used by all genes in this region is denoted by the filled oval. (D) Sequence of the annotated m41 ORF is shown. The annotated m41 translation start site and the location of splice acceptor and donor sites are shown above the DNA sequence. Location of mutations within the m41 ORF are shown below the DNA sequence. (E) The indicated m41 constructs were transiently overexpressed in Cos-7 cells, total cell lysates prepared and expression of the m41 and m41L proteins detected by immunoblot using anti-flag antibodies. (F) Total cell lysates were prepared from fibroblasts infected with MCMV at the indicated time pi and the expression of m41, m41L, and IE1 proteins detected by immunoblot.

Mentions: The m41 ORF encodes two protein products, m41 and m41.1, that are proposed to function by inhibiting apoptosis [19]–[20]. However, the relative contribution of these proteins to the pathogenesis of MCMV during in vivo infection has not been assessed. Moreover, previous work suggested that two distinct m41 protein products are produced from the m41 ORF, although their origin has not been defined [20]. We therefore utilized 5′ and 3′ rapid amplification of cDNA ends (RACE) to define transcripts produced from the m41 ORF (Fig. 1 A and B). Fibroblasts were infected with MCMV and RNA isolated at immediate early (IE), early (E) and late (L) times post-infection (pi). After two rounds of 5′ RACE two major transcripts were identified at E and L times pi (Fig. 1A). Sequencing of these transcripts determined that the shorter transcript encompasses the annotated m41 ORF sequence (Fig. 1C). The longer transcript is composed of two exons; the first exon is a short sequence originating upstream of the m42 ORF with a splice donor site at position 55,125. The first exon is spliced to the m41 sequence at position 54,216 which is in frame with the annotated m41 start codon (Fig. 1C and D). Sequencing of 3′ RACE products indicates that both transcripts terminate at the predicted m41 poly-A site (Fig. 1 B and C). Thus, two potential m41 protein products are produced during MCMV infection, the first from the annotated m41 ORF and the second is an N-terminally extended version of m41.


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)

Genomic arrangement and analysis of the m41 locus.RNA isolated from MCMV infected fibroblasts at IE, E and L times post-infection was subjected to (A) 5′ RACE or (B) 3′ RACE analysis. The resulting products were separated on 1% agarose gels. (C) The genomic region of MCMV encompassing the annotated m41 ORF (solid arrow), the newly identified 41 exon (solid box), and adjoining ORFs (open arrows) is shown. Direction of transcription is indicated by the orientation of the arrows. Location of the common polyadenylation site used by all genes in this region is denoted by the filled oval. (D) Sequence of the annotated m41 ORF is shown. The annotated m41 translation start site and the location of splice acceptor and donor sites are shown above the DNA sequence. Location of mutations within the m41 ORF are shown below the DNA sequence. (E) The indicated m41 constructs were transiently overexpressed in Cos-7 cells, total cell lysates prepared and expression of the m41 and m41L proteins detected by immunoblot using anti-flag antibodies. (F) Total cell lysates were prepared from fibroblasts infected with MCMV at the indicated time pi and the expression of m41, m41L, and IE1 proteins detected by immunoblot.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585157&req=5

ppat-1003192-g001: Genomic arrangement and analysis of the m41 locus.RNA isolated from MCMV infected fibroblasts at IE, E and L times post-infection was subjected to (A) 5′ RACE or (B) 3′ RACE analysis. The resulting products were separated on 1% agarose gels. (C) The genomic region of MCMV encompassing the annotated m41 ORF (solid arrow), the newly identified 41 exon (solid box), and adjoining ORFs (open arrows) is shown. Direction of transcription is indicated by the orientation of the arrows. Location of the common polyadenylation site used by all genes in this region is denoted by the filled oval. (D) Sequence of the annotated m41 ORF is shown. The annotated m41 translation start site and the location of splice acceptor and donor sites are shown above the DNA sequence. Location of mutations within the m41 ORF are shown below the DNA sequence. (E) The indicated m41 constructs were transiently overexpressed in Cos-7 cells, total cell lysates prepared and expression of the m41 and m41L proteins detected by immunoblot using anti-flag antibodies. (F) Total cell lysates were prepared from fibroblasts infected with MCMV at the indicated time pi and the expression of m41, m41L, and IE1 proteins detected by immunoblot.
Mentions: The m41 ORF encodes two protein products, m41 and m41.1, that are proposed to function by inhibiting apoptosis [19]–[20]. However, the relative contribution of these proteins to the pathogenesis of MCMV during in vivo infection has not been assessed. Moreover, previous work suggested that two distinct m41 protein products are produced from the m41 ORF, although their origin has not been defined [20]. We therefore utilized 5′ and 3′ rapid amplification of cDNA ends (RACE) to define transcripts produced from the m41 ORF (Fig. 1 A and B). Fibroblasts were infected with MCMV and RNA isolated at immediate early (IE), early (E) and late (L) times post-infection (pi). After two rounds of 5′ RACE two major transcripts were identified at E and L times pi (Fig. 1A). Sequencing of these transcripts determined that the shorter transcript encompasses the annotated m41 ORF sequence (Fig. 1C). The longer transcript is composed of two exons; the first exon is a short sequence originating upstream of the m42 ORF with a splice donor site at position 55,125. The first exon is spliced to the m41 sequence at position 54,216 which is in frame with the annotated m41 start codon (Fig. 1C and D). Sequencing of 3′ RACE products indicates that both transcripts terminate at the predicted m41 poly-A site (Fig. 1 B and C). Thus, two potential m41 protein products are produced during MCMV infection, the first from the annotated m41 ORF and the second is an N-terminally extended version of m41.

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