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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

mcv-miR-M1 can be expressed independently of NCCR-initiated late gene expression.(A) Schematic illustration of heterologous mcv-miR-M1 constructs. pCMV:ER-AS and-S contain the entire early coding region in antisense (ER-AS) or sense orientation (ER-S) relative to the CMV promoter. pER contains the entire early coding region without an heterologous promoter. (B) Small RNA Northern Blot analysis of PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. Blots from cells transfected with CMV constructs were exposed overnight. The blot from pER transfected cells was exposed for five days to facilitate visualization of miRNA signals. (C) Quantitative stem-loop RT-PCR for mcv-miR-M1-5p expression in PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. (D) Quantitative RT-PCR for mcv-miR-M1-5p (right columns), GAPDH mRNA (center columns) or tRNA meth expression (left columns) in PFSK-1 cells after 2 days of transfection with construct pER. Expression of the indicated transcripts in the presence of α-amanitin (light grey bars) is displayed as mean values from three independent experiments relative to the expression in untreated control cells (black bars).
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ppat.1004974.g005: mcv-miR-M1 can be expressed independently of NCCR-initiated late gene expression.(A) Schematic illustration of heterologous mcv-miR-M1 constructs. pCMV:ER-AS and-S contain the entire early coding region in antisense (ER-AS) or sense orientation (ER-S) relative to the CMV promoter. pER contains the entire early coding region without an heterologous promoter. (B) Small RNA Northern Blot analysis of PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. Blots from cells transfected with CMV constructs were exposed overnight. The blot from pER transfected cells was exposed for five days to facilitate visualization of miRNA signals. (C) Quantitative stem-loop RT-PCR for mcv-miR-M1-5p expression in PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. (D) Quantitative RT-PCR for mcv-miR-M1-5p (right columns), GAPDH mRNA (center columns) or tRNA meth expression (left columns) in PFSK-1 cells after 2 days of transfection with construct pER. Expression of the indicated transcripts in the presence of α-amanitin (light grey bars) is displayed as mean values from three independent experiments relative to the expression in untreated control cells (black bars).

Mentions: Given the observation of transcripts initiating upstream of the viral miRNA we sought to investigate whether mcv-miR-M1 could be expressed independently of NCCR-initiated transcription. For this purpose, we sub-cloned the entire early T-Ag coding region in either sense or antisense orientation downstream of an heterologous CMV promoter (pCMV:ER-S and –AS, respectively; see Fig 5A). As expected, forced transcription of the early region antisense strand gave rise to readily detectable pre- and mature miRNA moieties of mcv-miR-M1 (Fig 5B, left panel). However, similar levels of miRNA expression were observed when the CMV-promoter initiated transcription traversed the early region in the sense (i.e. T-Ag coding) orientation (Fig 5B, center panel). Indeed, a promoterless construct harboring the entire early region (pER) was likewise able to express the viral miRNA (Fig 5B, right panel), albeit at considerably (approx. 10 fold) lower levels than either CMV promoter-driven construct (see GAPDH-normalized stem-loop RT-qPCR data in Fig 5C; note that the Northern Blot in the right panel Fig 5B was exposed for longer time period than those shown for the pCMV constructs). While we presently cannot explain the seemingly disparate observation that strong miRNA expression was observed independent of the CMV promoter’s orientation relative to mcv-miR-M1, we suspect that CMV-promoter driven transcription through the locus may activate an intrinsic promoter. The fact that we had observed a transcriptional initiation site approx. 100 nt. upstream of the miRNA using a 5’-CAP dependent RACE protocol suggested that such transcripts are likely produced by RNA polymerase II. To investigate this assumption, we treated pER-transfected PFSK-1 cells with α-amanitin, a potent inhibitor of RNA-polymerase (RNA-pol) II, and investigated mcv-miR-M1 expression 24 hours later. As controls for RNA pol II and III transcribed RNAs, we additionally measured levels of GAPDH mRNA and tRNA-meth, respectively. As shown in Fig 5D, α-amanitin treatment strongly reduced expression of GAPDH and mcv-miR-M1, but not that of tRNA-meth. Hence, an intrinsic promoter activity within in the early region of MCPyV can lead to RNA pol II-dependent transcription of mcv-miR-M1.


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)

mcv-miR-M1 can be expressed independently of NCCR-initiated late gene expression.(A) Schematic illustration of heterologous mcv-miR-M1 constructs. pCMV:ER-AS and-S contain the entire early coding region in antisense (ER-AS) or sense orientation (ER-S) relative to the CMV promoter. pER contains the entire early coding region without an heterologous promoter. (B) Small RNA Northern Blot analysis of PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. Blots from cells transfected with CMV constructs were exposed overnight. The blot from pER transfected cells was exposed for five days to facilitate visualization of miRNA signals. (C) Quantitative stem-loop RT-PCR for mcv-miR-M1-5p expression in PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. (D) Quantitative RT-PCR for mcv-miR-M1-5p (right columns), GAPDH mRNA (center columns) or tRNA meth expression (left columns) in PFSK-1 cells after 2 days of transfection with construct pER. Expression of the indicated transcripts in the presence of α-amanitin (light grey bars) is displayed as mean values from three independent experiments relative to the expression in untreated control cells (black bars).
© Copyright Policy
Related In: Results  -  Collection

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

ppat.1004974.g005: mcv-miR-M1 can be expressed independently of NCCR-initiated late gene expression.(A) Schematic illustration of heterologous mcv-miR-M1 constructs. pCMV:ER-AS and-S contain the entire early coding region in antisense (ER-AS) or sense orientation (ER-S) relative to the CMV promoter. pER contains the entire early coding region without an heterologous promoter. (B) Small RNA Northern Blot analysis of PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. Blots from cells transfected with CMV constructs were exposed overnight. The blot from pER transfected cells was exposed for five days to facilitate visualization of miRNA signals. (C) Quantitative stem-loop RT-PCR for mcv-miR-M1-5p expression in PFSK-1 cells after 2 days of transfection with the mcv-miR-M1 constructs shown in A. (D) Quantitative RT-PCR for mcv-miR-M1-5p (right columns), GAPDH mRNA (center columns) or tRNA meth expression (left columns) in PFSK-1 cells after 2 days of transfection with construct pER. Expression of the indicated transcripts in the presence of α-amanitin (light grey bars) is displayed as mean values from three independent experiments relative to the expression in untreated control cells (black bars).
Mentions: Given the observation of transcripts initiating upstream of the viral miRNA we sought to investigate whether mcv-miR-M1 could be expressed independently of NCCR-initiated transcription. For this purpose, we sub-cloned the entire early T-Ag coding region in either sense or antisense orientation downstream of an heterologous CMV promoter (pCMV:ER-S and –AS, respectively; see Fig 5A). As expected, forced transcription of the early region antisense strand gave rise to readily detectable pre- and mature miRNA moieties of mcv-miR-M1 (Fig 5B, left panel). However, similar levels of miRNA expression were observed when the CMV-promoter initiated transcription traversed the early region in the sense (i.e. T-Ag coding) orientation (Fig 5B, center panel). Indeed, a promoterless construct harboring the entire early region (pER) was likewise able to express the viral miRNA (Fig 5B, right panel), albeit at considerably (approx. 10 fold) lower levels than either CMV promoter-driven construct (see GAPDH-normalized stem-loop RT-qPCR data in Fig 5C; note that the Northern Blot in the right panel Fig 5B was exposed for longer time period than those shown for the pCMV constructs). While we presently cannot explain the seemingly disparate observation that strong miRNA expression was observed independent of the CMV promoter’s orientation relative to mcv-miR-M1, we suspect that CMV-promoter driven transcription through the locus may activate an intrinsic promoter. The fact that we had observed a transcriptional initiation site approx. 100 nt. upstream of the miRNA using a 5’-CAP dependent RACE protocol suggested that such transcripts are likely produced by RNA polymerase II. To investigate this assumption, we treated pER-transfected PFSK-1 cells with α-amanitin, a potent inhibitor of RNA-polymerase (RNA-pol) II, and investigated mcv-miR-M1 expression 24 hours later. As controls for RNA pol II and III transcribed RNAs, we additionally measured levels of GAPDH mRNA and tRNA-meth, respectively. As shown in Fig 5D, α-amanitin treatment strongly reduced expression of GAPDH and mcv-miR-M1, but not that of tRNA-meth. Hence, an intrinsic promoter activity within in the early region of MCPyV can lead to RNA pol II-dependent transcription of mcv-miR-M1.

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