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A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence.

Theiss JM, Günther T, Alawi M, Neumann F, Tessmer U, Fischer N, Grundhoff A - PLoS Pathog. (2015)

Bottom Line: While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences.Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence.Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

View Article: PubMed Central - PubMed

Affiliation: Research Group Virus Genomics, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Merkel cell polyomavirus (MCPyV) is considered the etiological agent of Merkel cell carcinoma and persists asymptomatically in the majority of its healthy hosts. Largely due to the lack of appropriate model systems, the mechanisms of viral replication and MCPyV persistence remain poorly understood. Using a semi-permissive replication system, we here report a comprehensive analysis of the role of the MCPyV-encoded microRNA (miRNA) mcv-miR-M1 during short and long-term replication of authentic MCPyV episomes. We demonstrate that cells harboring intact episomes express high levels of the viral miRNA, and that expression of mcv-miR-M1 limits DNA replication. Furthermore, we present RACE, RNA-seq and ChIP-seq studies which allow insight in the viral transcription program and mechanisms of miRNA expression. While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences. We also report that MCPyV genomes can establish episomal persistence in a small number of cells for several months, a time period during which viral DNA as well as LT-Ag and viral miRNA expression can be detected via western blotting, FISH, qPCR and southern blot analyses. Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence. Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

No MeSH data available.


Related in: MedlinePlus

ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes.(A) Schematic depiction of the MCPyV genome with polyadenylation and transcriptional initiation sites as identified by RACE analyses. The position of the viral core origin of replication (ori) is marked by a grey box. (B, E) ChIP-seq profiles of LT-Ag (top panel, blue), H3K4me3 (center panel, green) or the negative IgG control (bottom panel, grey) along the MCPyV genome in PFSK-1 cells after 4 days of transfection with MCVSyn (B) or MCVSyn-pmt (E). Positions of early and late transcriptional initiation sites are marked by bent arrows pointing right and left, respectively. The putative promoter region mutated in MCVSyn-pmt is symbolized by a vertically hatched box in E. Graphs depict raw ChIP-seq read coverage; note that absolute read numbers depend on the efficiency of individual immunoprecipitations and thus only the relative coverage distribution along the viral genome is meaningful. (C) Mutations in the putative promoter region upstream of the mcv-miR-M1 locus. Substituted nucleotides are shown in red. The positions of the transcriptional initiation site TI-L2 is indicated by an arrow. The first four nucleotides of the mcv-miR-M1 locus are boxed in gray. (D) Quantitative stem-loop RT-PCR evaluation of mcv-miR-M1-5p expression in PFSK-1 cells after 4 days of transfection with MCVSyn (left), MCVSyn-pmt (center), or MCVSyn-hpko (negative control, right). Expression levels were normalized to the number of MCVSyn genomes per cell as determined by qPCR from genomic DNA. Mean values and standard deviations were calculated from three independent experiments. mcv-miR-M1 expression in MCVSyn-pmt is significantly decreased in comparison to MCVSyn (unpaired t-test).
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ppat.1004974.g006: ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes.(A) Schematic depiction of the MCPyV genome with polyadenylation and transcriptional initiation sites as identified by RACE analyses. The position of the viral core origin of replication (ori) is marked by a grey box. (B, E) ChIP-seq profiles of LT-Ag (top panel, blue), H3K4me3 (center panel, green) or the negative IgG control (bottom panel, grey) along the MCPyV genome in PFSK-1 cells after 4 days of transfection with MCVSyn (B) or MCVSyn-pmt (E). Positions of early and late transcriptional initiation sites are marked by bent arrows pointing right and left, respectively. The putative promoter region mutated in MCVSyn-pmt is symbolized by a vertically hatched box in E. Graphs depict raw ChIP-seq read coverage; note that absolute read numbers depend on the efficiency of individual immunoprecipitations and thus only the relative coverage distribution along the viral genome is meaningful. (C) Mutations in the putative promoter region upstream of the mcv-miR-M1 locus. Substituted nucleotides are shown in red. The positions of the transcriptional initiation site TI-L2 is indicated by an arrow. The first four nucleotides of the mcv-miR-M1 locus are boxed in gray. (D) Quantitative stem-loop RT-PCR evaluation of mcv-miR-M1-5p expression in PFSK-1 cells after 4 days of transfection with MCVSyn (left), MCVSyn-pmt (center), or MCVSyn-hpko (negative control, right). Expression levels were normalized to the number of MCVSyn genomes per cell as determined by qPCR from genomic DNA. Mean values and standard deviations were calculated from three independent experiments. mcv-miR-M1 expression in MCVSyn-pmt is significantly decreased in comparison to MCVSyn (unpaired t-test).

Mentions: If the region upstream of the mcv-miR-M1 locus exhibits promoter activity, then intact episome should exhibit an open chromatin conformation that permits transcriptional initiation at this position. To investigate this notion, we performed ChIP-seq experiments to evaluate patterns of the postranslational histone modification H3K4me3, a mark which is strongly enriched at transcriptional start sites. Additionally, we performed ChIP-seq to elucidate binding patterns of LT-Ag binding across the viral episome. For this purpose, we transfected PFSK-1 cells with MCVSyn and, 48 hours later, performed chromatin immunoprecipitation with antibodies specific for H3K4me3, or with the LT-Ag antibody CM2B4. An immunoprecipitation with IgG served as a negative control. The complete coverage data is given in S5 Dataset. As shown in the top panel of Fig 6B, the CM2B4 antibody produced a marked peak centered at the core origin of replication, consistent with the previously observed binding of LT-Ag to an array of GRGCC pentamers located in this region [41, 42]. No additional peaks were observed, indicating that, at least under the conditions used here, LT-Ag appears not to stringently bind to other loci on the viral episome. As expected, the NCCR of MCPyV was also highly enriched in the activation-associated histone mark H3K4me3 (Fig 6B, center panel). The H3K4me3 profile in this region presented as a broad peak which extended from the late to the early transcription initiation sites mapped during our 5’-RACE analysis. Indeed, consistent with our previous experiments that had suggested promoter activity of the region upstream of the viral miRNA locus, a second prominent H3K4me3-enriched zone was located within the T-Ag coding region. The summit of this peak mapped precisely to the transcriptional start site upstream of the viral miRNA locus identified during our 5’-RACE analysis. We next sought to determine whether mutation of upstream sequences would negatively affect miRNA expression. Given that the H3K4me3 enriched region is located in the early coding region, deletion of the putative promoter would also disrupt LT-Ag expression and thus abrogate the replication ability of MCVSyn episomes. However, we hypothesized that introduction of synonymous triplet mutations which preserve the LT-Ag coding capacity may be sufficient to ablate or reduce promoter activity. Accordingly, we generated a MCVSyn mutant (termed MCVSyn-pmt in the following) with a total of 68 triplet mutations in a ~200 bp region located 28 nt upstream of the miRNA locus (Fig 6C). Introduction of the same set of mutations in the context of early region-only construct pER (see Fig 5A) and subsequent transfection of the resulting plasmid pER-pmt into PFSK-1 cells confirmed that the mutations led to a significant reduction of NCCR-independent miRNA expression (S3 Fig).


A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence.

Theiss JM, Günther T, Alawi M, Neumann F, Tessmer U, Fischer N, Grundhoff A - PLoS Pathog. (2015)

ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes.(A) Schematic depiction of the MCPyV genome with polyadenylation and transcriptional initiation sites as identified by RACE analyses. The position of the viral core origin of replication (ori) is marked by a grey box. (B, E) ChIP-seq profiles of LT-Ag (top panel, blue), H3K4me3 (center panel, green) or the negative IgG control (bottom panel, grey) along the MCPyV genome in PFSK-1 cells after 4 days of transfection with MCVSyn (B) or MCVSyn-pmt (E). Positions of early and late transcriptional initiation sites are marked by bent arrows pointing right and left, respectively. The putative promoter region mutated in MCVSyn-pmt is symbolized by a vertically hatched box in E. Graphs depict raw ChIP-seq read coverage; note that absolute read numbers depend on the efficiency of individual immunoprecipitations and thus only the relative coverage distribution along the viral genome is meaningful. (C) Mutations in the putative promoter region upstream of the mcv-miR-M1 locus. Substituted nucleotides are shown in red. The positions of the transcriptional initiation site TI-L2 is indicated by an arrow. The first four nucleotides of the mcv-miR-M1 locus are boxed in gray. (D) Quantitative stem-loop RT-PCR evaluation of mcv-miR-M1-5p expression in PFSK-1 cells after 4 days of transfection with MCVSyn (left), MCVSyn-pmt (center), or MCVSyn-hpko (negative control, right). Expression levels were normalized to the number of MCVSyn genomes per cell as determined by qPCR from genomic DNA. Mean values and standard deviations were calculated from three independent experiments. mcv-miR-M1 expression in MCVSyn-pmt is significantly decreased in comparison to MCVSyn (unpaired t-test).
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Related In: Results  -  Collection

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ppat.1004974.g006: ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes.(A) Schematic depiction of the MCPyV genome with polyadenylation and transcriptional initiation sites as identified by RACE analyses. The position of the viral core origin of replication (ori) is marked by a grey box. (B, E) ChIP-seq profiles of LT-Ag (top panel, blue), H3K4me3 (center panel, green) or the negative IgG control (bottom panel, grey) along the MCPyV genome in PFSK-1 cells after 4 days of transfection with MCVSyn (B) or MCVSyn-pmt (E). Positions of early and late transcriptional initiation sites are marked by bent arrows pointing right and left, respectively. The putative promoter region mutated in MCVSyn-pmt is symbolized by a vertically hatched box in E. Graphs depict raw ChIP-seq read coverage; note that absolute read numbers depend on the efficiency of individual immunoprecipitations and thus only the relative coverage distribution along the viral genome is meaningful. (C) Mutations in the putative promoter region upstream of the mcv-miR-M1 locus. Substituted nucleotides are shown in red. The positions of the transcriptional initiation site TI-L2 is indicated by an arrow. The first four nucleotides of the mcv-miR-M1 locus are boxed in gray. (D) Quantitative stem-loop RT-PCR evaluation of mcv-miR-M1-5p expression in PFSK-1 cells after 4 days of transfection with MCVSyn (left), MCVSyn-pmt (center), or MCVSyn-hpko (negative control, right). Expression levels were normalized to the number of MCVSyn genomes per cell as determined by qPCR from genomic DNA. Mean values and standard deviations were calculated from three independent experiments. mcv-miR-M1 expression in MCVSyn-pmt is significantly decreased in comparison to MCVSyn (unpaired t-test).
Mentions: If the region upstream of the mcv-miR-M1 locus exhibits promoter activity, then intact episome should exhibit an open chromatin conformation that permits transcriptional initiation at this position. To investigate this notion, we performed ChIP-seq experiments to evaluate patterns of the postranslational histone modification H3K4me3, a mark which is strongly enriched at transcriptional start sites. Additionally, we performed ChIP-seq to elucidate binding patterns of LT-Ag binding across the viral episome. For this purpose, we transfected PFSK-1 cells with MCVSyn and, 48 hours later, performed chromatin immunoprecipitation with antibodies specific for H3K4me3, or with the LT-Ag antibody CM2B4. An immunoprecipitation with IgG served as a negative control. The complete coverage data is given in S5 Dataset. As shown in the top panel of Fig 6B, the CM2B4 antibody produced a marked peak centered at the core origin of replication, consistent with the previously observed binding of LT-Ag to an array of GRGCC pentamers located in this region [41, 42]. No additional peaks were observed, indicating that, at least under the conditions used here, LT-Ag appears not to stringently bind to other loci on the viral episome. As expected, the NCCR of MCPyV was also highly enriched in the activation-associated histone mark H3K4me3 (Fig 6B, center panel). The H3K4me3 profile in this region presented as a broad peak which extended from the late to the early transcription initiation sites mapped during our 5’-RACE analysis. Indeed, consistent with our previous experiments that had suggested promoter activity of the region upstream of the viral miRNA locus, a second prominent H3K4me3-enriched zone was located within the T-Ag coding region. The summit of this peak mapped precisely to the transcriptional start site upstream of the viral miRNA locus identified during our 5’-RACE analysis. We next sought to determine whether mutation of upstream sequences would negatively affect miRNA expression. Given that the H3K4me3 enriched region is located in the early coding region, deletion of the putative promoter would also disrupt LT-Ag expression and thus abrogate the replication ability of MCVSyn episomes. However, we hypothesized that introduction of synonymous triplet mutations which preserve the LT-Ag coding capacity may be sufficient to ablate or reduce promoter activity. Accordingly, we generated a MCVSyn mutant (termed MCVSyn-pmt in the following) with a total of 68 triplet mutations in a ~200 bp region located 28 nt upstream of the miRNA locus (Fig 6C). Introduction of the same set of mutations in the context of early region-only construct pER (see Fig 5A) and subsequent transfection of the resulting plasmid pER-pmt into PFSK-1 cells confirmed that the mutations led to a significant reduction of NCCR-independent miRNA expression (S3 Fig).

Bottom Line: While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences.Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence.Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

View Article: PubMed Central - PubMed

Affiliation: Research Group Virus Genomics, Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany; Institute for Medical Microbiology, Virology and Hygiene, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.

ABSTRACT
Merkel cell polyomavirus (MCPyV) is considered the etiological agent of Merkel cell carcinoma and persists asymptomatically in the majority of its healthy hosts. Largely due to the lack of appropriate model systems, the mechanisms of viral replication and MCPyV persistence remain poorly understood. Using a semi-permissive replication system, we here report a comprehensive analysis of the role of the MCPyV-encoded microRNA (miRNA) mcv-miR-M1 during short and long-term replication of authentic MCPyV episomes. We demonstrate that cells harboring intact episomes express high levels of the viral miRNA, and that expression of mcv-miR-M1 limits DNA replication. Furthermore, we present RACE, RNA-seq and ChIP-seq studies which allow insight in the viral transcription program and mechanisms of miRNA expression. While our data suggest that mcv-miR-M1 can be expressed from canonical late strand transcripts, we also present evidence for the existence of an independent miRNA promoter that is embedded within early strand coding sequences. We also report that MCPyV genomes can establish episomal persistence in a small number of cells for several months, a time period during which viral DNA as well as LT-Ag and viral miRNA expression can be detected via western blotting, FISH, qPCR and southern blot analyses. Strikingly, despite enhanced replication in short term DNA replication assays, a mutant unable to express the viral miRNA was severely limited in its ability to establish long-term persistence. Our data suggest that MCPyV may have evolved strategies to enter a non- or low level vegetative stage of infection which could aid the virus in establishing and maintaining a lifelong persistence.

No MeSH data available.


Related in: MedlinePlus