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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

LT antigen expression in long term cultures of MCVSyn or MCVSyn-hpko transfected PFSK-1 cells.(A) Western blot analysis and (B) confocal laser scanning immunofluorescence microscopy of PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko and analyzed at the indicated time points. Material was derived from the same cultures shown in Fig 8A to 8C. The asterisk in (A) denotes the position of an unspecific background band. Antibody CM2B4 recognizes LT-Ag as well as the alternative splice product 57k T. The positions of both protein bands are indicated by arrowheads.
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ppat.1004974.g009: LT antigen expression in long term cultures of MCVSyn or MCVSyn-hpko transfected PFSK-1 cells.(A) Western blot analysis and (B) confocal laser scanning immunofluorescence microscopy of PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko and analyzed at the indicated time points. Material was derived from the same cultures shown in Fig 8A to 8C. The asterisk in (A) denotes the position of an unspecific background band. Antibody CM2B4 recognizes LT-Ag as well as the alternative splice product 57k T. The positions of both protein bands are indicated by arrowheads.

Mentions: Western Blot analysis of the bulk cultures confirmed the absence of LT antigen in MCVSyn-hpko transfected cells after the loss of genomic DNA (Fig 9A, lane 12). In contrast, LT antigen expression could be readily observed in MCVSyn transfected cultures even after more than 160 days (lane 11). To also analyze LT-Ag expression on the single cell level, we performed immunofluorescence analyses using the CM2B4 antibody. Fig 9B shows representative images from an early (4d) and several late time points of MCVSyn transfected cells. LT-Ag staining presented as distinct, strictly nuclear dots that are likely to represent foci of viral DNA replication. At 4 days post-transfection, we estimated the percentage of LT-Ag positive cells in both MCVSyn and MCVSyn-hpko transfected cultures to be approximately 2–3%. In accord with our qPCR and southern blot experiments, LT-Ag positive cells became approximately 100fold less frequent, but cells with multiple foci (albeit smaller than those observed after 4 days) remained clearly detectable for several months in MCVSyn cultures. Apart from the fact that LT-Ag positive cells were absent from late time points of MCVSyn-hpko-transfected cultures, we did not detect fundamental differences in the LT-Ag staining patterns between MCVSyn or MCVSyn-hpko transfected cells.


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)

LT antigen expression in long term cultures of MCVSyn or MCVSyn-hpko transfected PFSK-1 cells.(A) Western blot analysis and (B) confocal laser scanning immunofluorescence microscopy of PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko and analyzed at the indicated time points. Material was derived from the same cultures shown in Fig 8A to 8C. The asterisk in (A) denotes the position of an unspecific background band. Antibody CM2B4 recognizes LT-Ag as well as the alternative splice product 57k T. The positions of both protein bands are indicated by arrowheads.
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004974.g009: LT antigen expression in long term cultures of MCVSyn or MCVSyn-hpko transfected PFSK-1 cells.(A) Western blot analysis and (B) confocal laser scanning immunofluorescence microscopy of PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko and analyzed at the indicated time points. Material was derived from the same cultures shown in Fig 8A to 8C. The asterisk in (A) denotes the position of an unspecific background band. Antibody CM2B4 recognizes LT-Ag as well as the alternative splice product 57k T. The positions of both protein bands are indicated by arrowheads.
Mentions: Western Blot analysis of the bulk cultures confirmed the absence of LT antigen in MCVSyn-hpko transfected cells after the loss of genomic DNA (Fig 9A, lane 12). In contrast, LT antigen expression could be readily observed in MCVSyn transfected cultures even after more than 160 days (lane 11). To also analyze LT-Ag expression on the single cell level, we performed immunofluorescence analyses using the CM2B4 antibody. Fig 9B shows representative images from an early (4d) and several late time points of MCVSyn transfected cells. LT-Ag staining presented as distinct, strictly nuclear dots that are likely to represent foci of viral DNA replication. At 4 days post-transfection, we estimated the percentage of LT-Ag positive cells in both MCVSyn and MCVSyn-hpko transfected cultures to be approximately 2–3%. In accord with our qPCR and southern blot experiments, LT-Ag positive cells became approximately 100fold less frequent, but cells with multiple foci (albeit smaller than those observed after 4 days) remained clearly detectable for several months in MCVSyn cultures. Apart from the fact that LT-Ag positive cells were absent from late time points of MCVSyn-hpko-transfected cultures, we did not detect fundamental differences in the LT-Ag staining patterns between MCVSyn or MCVSyn-hpko transfected cells.

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