Journal: Journal of Virology
Article Title: Relationship between Vaccinia Virus Intracellular Cores, Early mRNAs, and DNA Replication Sites
Figure Lengend Snippet: Double transfection of BrUTP and the antisense H5R oligonucleotide at different times of infection reveals two different structures that accumulate viral mRNAs in infected cells. HeLa cells were infected and transfected with BrUTP for 2 h in the presence of 2 mM HU. HU was washed out, and cells were transfected with the antisense oligonucleotide corresponding to H5R mRNA. Double-transfected cells were fixed 2 h after HU washout and triple labeled with anti-BrU (FITC) (yellow channel), streptavidin-rhodamine (H5R) (red channel), and DAPI (bluechannel). The solid arrows in all four panels indicate DAPI-positive replication sites, the labeling of which entirely colocalizes with the pattern of the H5R oligonucleotide. The BrU pattern, indicated by hatched arrows (BrU and merge panels), does not colocalize with either of the two labels. It accumulates in granular structures that appear to have no relationship with the replication sites. This is most dramatically shown in the upper left cell, in which the replication sites all seem to collect at the upper left side of the cell, while the BrU-positive structures accumulate at the lower right side of the same cell. Bar, 10 μm.
Article Snippet: Fixed cells were then either double labeled with anti-BrU and DAPI or triple labeled with anti-BrU (followed by an anti-rat antibody coupled to rhodamine), DAPI, and streptavidin-FITC.
Techniques: Transfection, Infection, Labeling