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 5
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).