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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

Mapping of early and late strand polyadenylation sites.(A) Schematic representation of early (blue) and late (red) strand polyadenylation sites as identified via 3’-RACE analysis. The location of nested gene specific primers is indicated by double arrows. Dashed arrows indicate individual clones recovered during 3’-RACE analysis for each of the strands. (B, C): Sequence representation of the identified polyadenylation on the early (B) and late (C) strands. The nucleotide after which cleavage occurs is marked by an arrow. Canonical polyadenlyation signals upstream of the cleavage sites are boxed. GU- or U-rich regions downstream of the cleavage sites are underlined with solid or dashed lines, respectively.
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ppat.1004974.g003: Mapping of early and late strand polyadenylation sites.(A) Schematic representation of early (blue) and late (red) strand polyadenylation sites as identified via 3’-RACE analysis. The location of nested gene specific primers is indicated by double arrows. Dashed arrows indicate individual clones recovered during 3’-RACE analysis for each of the strands. (B, C): Sequence representation of the identified polyadenylation on the early (B) and late (C) strands. The nucleotide after which cleavage occurs is marked by an arrow. Canonical polyadenlyation signals upstream of the cleavage sites are boxed. GU- or U-rich regions downstream of the cleavage sites are underlined with solid or dashed lines, respectively.

Mentions: Polyadenylation sites were determined with a conventional 3’RACE protocol, using gene specific 5'-primers for the distal coding regions of early and late transcripts together with anchored oligo dT 3'-primers (Fig 3A). Amplification products were subcloned in bulk, and between 16 and 26 (for early and late transcripts, respectively) randomly picked clones were subjected to Sanger sequencing. As shown in Fig 3A and 3B, 100% (16 of 16) clones derived from early transcripts terminated at position 3094, 14 nucleotides downstream of a canonical polyadenylation signal (AAUAAA) which overlaps with the T-Ag stop codon, and 9 nucleotides upstream of a GU-rich element (Fig 3B). The 3’-RACE products from late transcripts were more diverse: As shown in Fig 3A and 3C, 14 (53%) of the 26 clones terminated at position 2842 (pA site L1), 317 nucleotides downstream of the VP1 stop codon. Another 8 clones (31%) terminated in a distance of 451 from the VP1 stop codon at position 2708 (pA site L2). Canonical polyadenylation signals were observed immediately upstream of both cleavage sites (Fig 3C). Although U-rich regions are present 13 or 31 nucleotides downstream of pA sites L1 and L2, respectively, neither site exhibits a clearly discernible GU-rich element. The four remaining clones from late transcripts were predominantly found at A-rich regions of the viral genome, suggesting they had resulted from mispriming of the oligo dT primers to internal regions of viral transcripts extending into the early region. Together, these results suggested highly efficient termination of early transcripts, but relatively weak late polyadenylation signals that allow at least some transcriptional read-through.


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)

Mapping of early and late strand polyadenylation sites.(A) Schematic representation of early (blue) and late (red) strand polyadenylation sites as identified via 3’-RACE analysis. The location of nested gene specific primers is indicated by double arrows. Dashed arrows indicate individual clones recovered during 3’-RACE analysis for each of the strands. (B, C): Sequence representation of the identified polyadenylation on the early (B) and late (C) strands. The nucleotide after which cleavage occurs is marked by an arrow. Canonical polyadenlyation signals upstream of the cleavage sites are boxed. GU- or U-rich regions downstream of the cleavage sites are underlined with solid or dashed lines, respectively.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004974.g003: Mapping of early and late strand polyadenylation sites.(A) Schematic representation of early (blue) and late (red) strand polyadenylation sites as identified via 3’-RACE analysis. The location of nested gene specific primers is indicated by double arrows. Dashed arrows indicate individual clones recovered during 3’-RACE analysis for each of the strands. (B, C): Sequence representation of the identified polyadenylation on the early (B) and late (C) strands. The nucleotide after which cleavage occurs is marked by an arrow. Canonical polyadenlyation signals upstream of the cleavage sites are boxed. GU- or U-rich regions downstream of the cleavage sites are underlined with solid or dashed lines, respectively.
Mentions: Polyadenylation sites were determined with a conventional 3’RACE protocol, using gene specific 5'-primers for the distal coding regions of early and late transcripts together with anchored oligo dT 3'-primers (Fig 3A). Amplification products were subcloned in bulk, and between 16 and 26 (for early and late transcripts, respectively) randomly picked clones were subjected to Sanger sequencing. As shown in Fig 3A and 3B, 100% (16 of 16) clones derived from early transcripts terminated at position 3094, 14 nucleotides downstream of a canonical polyadenylation signal (AAUAAA) which overlaps with the T-Ag stop codon, and 9 nucleotides upstream of a GU-rich element (Fig 3B). The 3’-RACE products from late transcripts were more diverse: As shown in Fig 3A and 3C, 14 (53%) of the 26 clones terminated at position 2842 (pA site L1), 317 nucleotides downstream of the VP1 stop codon. Another 8 clones (31%) terminated in a distance of 451 from the VP1 stop codon at position 2708 (pA site L2). Canonical polyadenylation signals were observed immediately upstream of both cleavage sites (Fig 3C). Although U-rich regions are present 13 or 31 nucleotides downstream of pA sites L1 and L2, respectively, neither site exhibits a clearly discernible GU-rich element. The four remaining clones from late transcripts were predominantly found at A-rich regions of the viral genome, suggesting they had resulted from mispriming of the oligo dT primers to internal regions of viral transcripts extending into the early region. Together, these results suggested highly efficient termination of early transcripts, but relatively weak late polyadenylation signals that allow at least some transcriptional read-through.

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