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
Fig 2
A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication.
(A) 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) 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. (C) 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. (D) Analysis of de novo 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 DpnI-resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI-sensitive input DNA are visible near the bottom of the blot.