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

<div><p>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.</p></div

LT antigen expression in long term cultures of MCVSyn or MCVSyn-hpko transfected PFSK-1 cells.

<p><b>(A)</b> Western blot analysis and <b>(B)</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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004974#ppat.1004974.g008" target="_blank">Fig 8A to 8C</a>. 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.</p

RNA-seq analysis of replicating MCPyV genomes.

<p>(<b>A</b>) Schematic depiction of the MCPyV genome. <b>(B, C)</b> RNA-seq coverage of MCVSyn (B) or MCVSyn-hpko (C) genomes in PFSK-1 cells after 4 days of transfection. Read coverage on the early (positive axis; blue) or late strand (negative axis; red) is shown relative to the maximally observed nucleotide coverage (set to 100%). Note that the negative/late strand axis is shown at a lower scale in C to facilitate comparison of late strand read coverage profiles. <b>(D, E)</b> Structure of known or predicted early (D) or late (E) transcripts. The position of donor or acceptor sites is indicated by vertically drawn hatched lines. Previously identified donors or acceptors are marked with asterisks. Known or predicted protein products and estimated abundance values are given next to all transcripts that map to the major early or late transcription cassettes (T1-T’7 and L1-L3, respectively). We did not estimate abundance for transcripts that contain splice junctions with donors or acceptors located outside of the major transcriptional units (T’8, T’9, LL, L’4). For simplicity, regions of transcripts which are of unknown structure, or that may display splice patterns which are of no immediate consequence for coding potential are symbolized by horizontal hatched lines. Coding regions (first AUG-initiated ORF) in each of the transcripts are shown by boxes that are colored according to reading frame relative to the genomic position of major early or late transcriptional start positions (149 and 5245, respectively).</p

A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication.

<p><b>(A)</b> 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>(B)</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. <b>(C)</b> 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. <b>(D)</b> Analysis of <i>de novo</i> 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 <i>DpnI</i>-resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the <i>DpnI</i>-sensitive input DNA are visible near the bottom of the blot.</p

ChIP-seq analysis and miRNA expression in wt and mutated MCVSyn genomes.

<p>(<b>A</b>) 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>B, E</b>) 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>B</b>) or MCVSyn-pmt (<b>E</b>). 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. (<b>C</b>) 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. <b>(D)</b> 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).</p

Expression of mcv-miR-M1 by replicating MCPyV genomes.

<p><b>(A)</b><i>Top</i>: Schematic illustration of the MCPyV genome. Early (light blue) and late (red) strand open reading frames as well as the mcv-miR-M1 locus are shown as solid block arrows. The 57K T-Ag ORF is not shown for simplicity. <i>Bottom</i>: Small RNA read coverage on early (positive axis) or late (negative axis) strands as observed in MCVSyn transfected PFSK-1 cells (NEBNext library preparation protocol). <b>(B)</b> Enlarged depiction of late strand coverage at the genomic mcv-miR-M1 locus as shown in (A). The regions producing the major 5p and 3p mature miRNAs are shown in bold and their seed sequences are underlined. The asterisk denotes a single nucleotide polymorphism that is frequently observed in MCPyV field strains. <b>(C, D)</b> Relative frequency of mature 5p (C) or 3p (D) mcv-miR-M1 reads containing the indicated seed sequences in small RNA libraries prepared with the NEBNext (left panels) or TruSeq (right panels) library preparation protocols.</p

mcv-miR-M1-5p/3p ranks.

<p>Numbers indicate ranks of mcv-miR-M1-5p and -3p relative to human miRNAs according to expression values.</p><p>mcv-miR-M1-5p/3p ranks.</p

Mapping of early and late strand transcriptional initiation sites.

<p>(<b>A</b>) Schematic depiction of the MCPyV genome. The binding sites of nested gene specific 5’-RACE primers for early and late strand are indicated by blue or red double arrows, respectively. The identified transcriptional initiation zones TI-E1 (early strand) or TI-L1 and –L2 (late strand) are shown as black arrows drawn towards the top or bottom, respectively. (<b>B</b>) Relative frequency of transcriptional initiation site (TIS) reads on the early (positive axis, blue) or late (negative axis, red) strands. (<b>C</b>) Detailed depiction of TIS coverage near the major early initiation zone TI-E1. A TATA box located upstream of TI-E1 is underlined. A minor initiation site upstream of the TATA box is marked with an asterisk. The T-Ag start codon is boxed and highlighted in yellow. (<b>D</b>) Depiction of TIS read coverage near the major late initiation zone TI-L1 (top panel), and a second initiation site outside of the NCCR (TI-L2, lower panel). The VP2 start codon is boxed and the first 9 nucleotides of the genomic mcv-miR-M1 locus are boxed and highlighted in yellow or grey, respectively.</p

RNA-seq summary statistics.

<p><b><i>a</i>, <i>b</i></b>: number of reads mapped to regions delineated by transcriptional start and polyadenylation sites identified in this study (nts. 149 to 3094 (+) and 2708 to 5245 (-), respectively).</p><p>RNA-seq summary statistics.</p

Mapping of early and late strand polyadenylation sites.

<p>(<b>A</b>) 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>B, C</b>): Sequence representation of the identified polyadenylation on the early (<b>B</b>) and late (<b>C</b>) 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.</p