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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

A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication.(A) Predicted secondary structures of the wt (left) and mutated (right) mcv-miR-M1 pre-miRNA sequences. Regions encoding mature miRNAs are shown in bold, mutated nucleotides are highlighted in red. (B) Small RNA Northern Blot analysis of mcv-miR-M1 expression 24 and 48 hours after transfection of MCVSyn (left panel) or MCVSyn-hpko (right panel) genomes. Mock transfected cells or cells transfected with a plasmid containing the mcv-miR-M1 pre-miRNA cloned behind a CMV promoter served as negative and positive controls, respectively. Positions of pre- and mature miRNAs are marked by arrows. (C) Western Blot analysis of LT antigen expression in PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko at 4d post transfection. The position of LT-Ag is marked by an arrow. (D) Analysis of de novo replicated viral DNA by Southern Blot analysis of HIRT extracts prepared from PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko genomes 4d post transfection. The position of DpnI-resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI-sensitive input DNA are visible near the bottom of the blot.
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ppat.1004974.g002: A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication.(A) Predicted secondary structures of the wt (left) and mutated (right) mcv-miR-M1 pre-miRNA sequences. Regions encoding mature miRNAs are shown in bold, mutated nucleotides are highlighted in red. (B) Small RNA Northern Blot analysis of mcv-miR-M1 expression 24 and 48 hours after transfection of MCVSyn (left panel) or MCVSyn-hpko (right panel) genomes. Mock transfected cells or cells transfected with a plasmid containing the mcv-miR-M1 pre-miRNA cloned behind a CMV promoter served as negative and positive controls, respectively. Positions of pre- and mature miRNAs are marked by arrows. (C) Western Blot analysis of LT antigen expression in PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko at 4d post transfection. The position of LT-Ag is marked by an arrow. (D) Analysis of de novo replicated viral DNA by Southern Blot analysis of HIRT extracts prepared from PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko genomes 4d post transfection. The position of DpnI-resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI-sensitive input DNA are visible near the bottom of the blot.

Mentions: Regardless of their exact seed sequence, all mature miRNA species are perfectly complementary to transcripts originating from the opposite strand of the MCPyV genome. Consequently, provided they are efficiently incorporated into RISC, all species should be able to negatively regulate early transcripts. Indeed, a number of previous studies have suggested that the ability to autoregulate LT-Ag expression may represent an evolutionary conserved function of polyomavirus miRNAs [11, 15–17, 19, 20, 22–25]. In support of this notion, Seo and colleagues have formally demonstrated that ectopically expressed mcv-miR-M1 can negatively regulate luciferase expression of a chimeric reporter construct containing the mcv-mir-M1 complementary region and flanking sequences [11]. To determine whether mcv-mir-M1 also can suppress LT-Ag expression in the context of transcription from intact episomes, we generated a mutant MCPyV genome unable to express the viral miRNA. To this end, we introduced a total of 14 mutations designed to disrupt the mcv-mir-M1 pre-miRNA hairpin structure (Fig 2A) which is required for the processing of pre- and mature miRNAs by Drosha and Dicer, respectively. All nucleotide substitutions were designed such that the coding capacity of LT-Ag encoded on the opposite strand remained unaltered (S1 Fig). The resulting viral genome (referred to as MCVSyn-hpko in the following) or the parental MCVSyn genome was transfected into PFSK-1 cells. As shown in Fig 2B, parental MCVSyn genomes expressed viral miRNA moieties that were readily detectable by northern blotting at 4 days post infection. As expected, no mcv-mir-M1 pre- or mature miRNAs were produced in cells transfected with MCVSyn-hpko mutants. As shown in Fig 2C, the absence of viral miRNA expression resulted in substantially higher expression of LT-Ag on the protein level. To investigate whether elevated LT-Ag expression also affected the efficacy of viral DNA replication, we performed a DpnI resistance assay on HIRT extracts. As shown in the Southern Blots of Fig 2D, MCVSyn-hpko genomes indeed replicated to appreciably higher levels when compared to the wt genome. Collectively, the above data thus suggest that i) complementary target sites in full length LT-Ag transcripts are accessible to mcv-mir-M1 binding, ii) levels of mcv-mir-M1 expressed by replicating MCPyV genomes are sufficient to induce substantial downregulation of LT-Ag expression and iii) the extent of LT-Ag downregulation mediated by mcv-mir-M1 is sufficient to limit the replication of transfected MCPyV genomes.


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)

A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication.(A) Predicted secondary structures of the wt (left) and mutated (right) mcv-miR-M1 pre-miRNA sequences. Regions encoding mature miRNAs are shown in bold, mutated nucleotides are highlighted in red. (B) Small RNA Northern Blot analysis of mcv-miR-M1 expression 24 and 48 hours after transfection of MCVSyn (left panel) or MCVSyn-hpko (right panel) genomes. Mock transfected cells or cells transfected with a plasmid containing the mcv-miR-M1 pre-miRNA cloned behind a CMV promoter served as negative and positive controls, respectively. Positions of pre- and mature miRNAs are marked by arrows. (C) Western Blot analysis of LT antigen expression in PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko at 4d post transfection. The position of LT-Ag is marked by an arrow. (D) Analysis of de novo replicated viral DNA by Southern Blot analysis of HIRT extracts prepared from PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko genomes 4d post transfection. The position of DpnI-resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI-sensitive input DNA are visible near the bottom of the blot.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4517807&req=5

ppat.1004974.g002: A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication.(A) Predicted secondary structures of the wt (left) and mutated (right) mcv-miR-M1 pre-miRNA sequences. Regions encoding mature miRNAs are shown in bold, mutated nucleotides are highlighted in red. (B) Small RNA Northern Blot analysis of mcv-miR-M1 expression 24 and 48 hours after transfection of MCVSyn (left panel) or MCVSyn-hpko (right panel) genomes. Mock transfected cells or cells transfected with a plasmid containing the mcv-miR-M1 pre-miRNA cloned behind a CMV promoter served as negative and positive controls, respectively. Positions of pre- and mature miRNAs are marked by arrows. (C) Western Blot analysis of LT antigen expression in PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko at 4d post transfection. The position of LT-Ag is marked by an arrow. (D) Analysis of de novo replicated viral DNA by Southern Blot analysis of HIRT extracts prepared from PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko genomes 4d post transfection. The position of DpnI-resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI-sensitive input DNA are visible near the bottom of the blot.
Mentions: Regardless of their exact seed sequence, all mature miRNA species are perfectly complementary to transcripts originating from the opposite strand of the MCPyV genome. Consequently, provided they are efficiently incorporated into RISC, all species should be able to negatively regulate early transcripts. Indeed, a number of previous studies have suggested that the ability to autoregulate LT-Ag expression may represent an evolutionary conserved function of polyomavirus miRNAs [11, 15–17, 19, 20, 22–25]. In support of this notion, Seo and colleagues have formally demonstrated that ectopically expressed mcv-miR-M1 can negatively regulate luciferase expression of a chimeric reporter construct containing the mcv-mir-M1 complementary region and flanking sequences [11]. To determine whether mcv-mir-M1 also can suppress LT-Ag expression in the context of transcription from intact episomes, we generated a mutant MCPyV genome unable to express the viral miRNA. To this end, we introduced a total of 14 mutations designed to disrupt the mcv-mir-M1 pre-miRNA hairpin structure (Fig 2A) which is required for the processing of pre- and mature miRNAs by Drosha and Dicer, respectively. All nucleotide substitutions were designed such that the coding capacity of LT-Ag encoded on the opposite strand remained unaltered (S1 Fig). The resulting viral genome (referred to as MCVSyn-hpko in the following) or the parental MCVSyn genome was transfected into PFSK-1 cells. As shown in Fig 2B, parental MCVSyn genomes expressed viral miRNA moieties that were readily detectable by northern blotting at 4 days post infection. As expected, no mcv-mir-M1 pre- or mature miRNAs were produced in cells transfected with MCVSyn-hpko mutants. As shown in Fig 2C, the absence of viral miRNA expression resulted in substantially higher expression of LT-Ag on the protein level. To investigate whether elevated LT-Ag expression also affected the efficacy of viral DNA replication, we performed a DpnI resistance assay on HIRT extracts. As shown in the Southern Blots of Fig 2D, MCVSyn-hpko genomes indeed replicated to appreciably higher levels when compared to the wt genome. Collectively, the above data thus suggest that i) complementary target sites in full length LT-Ag transcripts are accessible to mcv-mir-M1 binding, ii) levels of mcv-mir-M1 expressed by replicating MCPyV genomes are sufficient to induce substantial downregulation of LT-Ag expression and iii) the extent of LT-Ag downregulation mediated by mcv-mir-M1 is sufficient to limit the replication of transfected MCPyV genomes.

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