Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: The illustration is based on a multiple sequence alignment using the Clustal W algorithm (see for accession numbers used in the alignment) of MCVSyn and all full length MCPyV sequences deposited in the NCBI Database as of August 2011. Aligned genomes were compared to the consensus sequence (which is identical to MCVSyn as well as the isolates 17b, 18b and 20b). Nucleotide substitutions/mismatches relative to this sequence are shown as vertical black bars, whereas deletions are shown in red. Nucleotide insertions in a given sequence are shown as blue bars, and register as gaps in the backbone of the remaining genomes. Genomes that were isolated from MCC or MCC-derived cell lines are marked by an asterisk; the mutations which lead to the truncation of LT-Ag sequences in these genomes are likewise marked.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Sequencing, Isolation, Derivative Assay

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Cell lines used to study MCVSyn replication, early and late transcription as well as particle formation.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques:

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Expressing, SDS Page, Western Blot, Staining, Molecular Weight, Isolation, Extraction, Agarose Gel Electrophoresis, Southern Blot, Labeling, Software

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: 5×10 4 H1299, PSFK-1 or 293 cells were transfected with 100 ng re-circularized MCVSyn DNA or equivalent amounts of pUC18 DNA (Mock control). At the indicated time points, cells were lysed and analyzed by immunoblotting for MCPyV LT-Ag expression using the monoclonal LT-Ag antibody Cm2B4. Equal protein loading was confirmed by re-incubating the membrane with an anti-actin antibody. An LT-Ag expression control (pos. control; transient transfection with a CMV-promoter driven LT-Ag expression construct for 48 h) was loaded as an internal control.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Control, Western Blot, Expressing, Membrane, Construct

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: RNA was isolated at the indicated time points after transfection, DNAse I digested and used for cDNA synthesis followed by real time PCR using a LT-Ag or VP1 specific primer set. Results were normalized against GAPDH transcript levels.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Isolation, Transfection, cDNA Synthesis, Real-time Polymerase Chain Reaction

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: (A) Double staining of CV-1 cells transfected with SV40 viral DNA. 4d p.t. the cells were fixed, and VP1 was detected with a polyclonal anti-VP1 antibody. LT-Ag was visualized with the monoclonal anti-LT antibody Pab419. Z-stack pictures were taken using confocal microscopy. Each picture represents an individual Z-stack. VP1 staining was observed primarily in speckles close to or at the nuclear membrane. LT-Ag staining was observed throughout the nucleoplasm with the nucleoli excluded. In some cells granular LT-Ag staining was observed. The panel on the lower right represents a 3× zoomed picture of a CV1 transfected cell with the two channels merged. Double staining of Merkel cell polyomavirus VP1 and LT-Ag in H1299 cells (B) and PFSK-1 cells (C) 4d p.t. reveals inner peripheral nuclear localization of MCVSyn VP1 protein. VP1 was visualized with a polyclonal anti-VP1 serum and anti-rabbit FITC, while LT-Ag was visualized with the monoclonal antibody Cm2B4 specifically recognizing MCPyV LT-Ag. 40 Z-stack pictures were taken scanning through the cells using a 63× magnification and 2fold zoom on a confocal microscope. The picture shown represents an individual image from the center of a Z-stack.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Double Staining, Transfection, Confocal Microscopy, Staining, Membrane, Microscopy

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Optiprep™ gradient centrifugation was performed with cell lysates from CV1 (A) and H1299 (B) cells 4d after transfection with viral DNA. 15×250 µl fractions were collected (fraction 1 represents the fraction with the highest density and fraction 15 represents the lowest density fraction). (A) Left panel: Real time PCR of micrococcal nuclease treated fractions was performed using SV40 VP1 primer sequences. 20 µl of each gradient fraction was loaded on a 10% SDS-page followed immunoblotting using anti-VP1 serum. Right panel: Negative EM staining of SV40 particles identified in fraction 9. (B) Left panel: Real time PCR results of H1299 MCVSyn gradient fractions after micrococcal nuclease treatment using MCPyV VP1-specific primers. Right panel: Negative EM staining of particles identified in fractions 10 and 6.

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Gradient Centrifugation, Transfection, Real-time Polymerase Chain Reaction, SDS Page, Western Blot, Staining

Journal: PLoS ONE

Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

doi: 10.1371/journal.pone.0029112

Figure Lengend Snippet: Images were prepared from PFSK-1 cultures at 8 days post transfection with MCVSyn DNA. (A and B) ∼40 µm electron dense particles were observed in approximately 1 out of 50 cells with the particles localizing in the nucleus close to membrane structures (additional particles in B that are located outside of the enlarged inset are marked by arrows). (C) Membrane-attached MCPyV particles, reminiscent of the structures observed in SV40 infected cells as shown in .

Article Snippet: Plasmid pwM expresses a codon optimized MCPyV VP1 ORF (GenBank accession FJ548568) and was obtained from Addgene (plasmid #22515).

Techniques: Transfection, Membrane, Infection