FIG 5.

Export of ATI mRNA from factory region is translation dependent. (A to D) HeLa cells were transfected with a plasmid expressing the uninterrupted or mutated ATI ORF with a C-terminal HA tag and after 4 h were infected with VACV vΔA25ΔΑ26, which lacks either the CPXV ATI ORF or the truncated VACV ATI ORF, for an additional 6 h. ATI RNA was detected by FISH using fluorescently labeled deoxyoligonucleotide antisense ATI RNA probes, ATI protein with anti-HA antibody, followed by fluorescent secondary antibody and DNA with DAPI. Diagrams of the expressed ATI proteins are shown on the right of each row. Repeat sequences are indicated by black bars, and coiled-coil domains are underlined. Arrows point to cytoplasmic factories. (E) Localization of ATI protein and mRNA expressed by VACV with the naturally truncated A25 ATI ORF. After 16 h, the cells were fixed and analyzed with fluorescent-labeled deoxyoligonucleotide antisense ATI RNA probes and antibody to the ATI protein followed by a fluorescent secondary antibody and DAPI. Arrows point to cytoplasmic factories. Maximum projections of typical cells are shown.