Fig. 1

Truncated ORF1 proteins require the RRM domain for their nuclear localization. a (top) ORF1 domains are indicated as an N-terminal domain (N), a coiled-coil domain (CCD), and RNA recognition motif (RRM) and a C-terminal domain (CTD). Positions of domains shown are approximate. The antibody symbols (name above) denote approximate location of antibodies on the ORF1p. Putative bipartite nuclear localization signal (Bipartite) is shown in green and putative monopartite nuclear localization signal (monopartite) is shown in brown. (bottom) Schematic of the ORF1 truncation approach. The expected molecular weight of each construct is listed to the right as well as detection of the protein generated from the construct (Detected column). The numbers listed on the left represent the position of the site of truncation based on a full-length ORF1p. The 53–157 construct has a T7 tag. b Western blot analysis of truncated human ORF1 transiently transfected in HeLa cells. Proteins are separated into nuclear (N) and cytoplasmic (C) fractions. Human ORF1 protein was detected with human-specific ORF1 polyclonal antibodies (hORF1). Calregulin (cytoplasmic marker) and Lamin A/C (nuclear marker) are used as loading and cell fractionation controls and eIF3 is used to control for cytoplasmic stress granules. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid. b Western blot analysis of truncated human ORF1 transiently transfected in HeLa cells. Proteins are separated into nuclear (N) and cytoplasmic (C) fractions. Human ORF1 protein was detected with human-specific ORF1 polyclonal antibodies (hORF1 201). Calregulin (cytoplasmic marker) and Lamin A/C (nuclear marker) are used as loading and cell fractionation controls. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid. d Western blot analysis of truncated human ORF1 transiently transfected in HeLa cells. Proteins are separated into nuclear (N) and cytoplasmic (C) fractions. Human ORF1 protein was detected with T7 tag polyclonal antibodies (T7). GAPDH (cytoplasmic marker) and Lamin A/C (nuclear marker) are used as loading and cell fractionation controls. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid