Fig. 3

Mutation of the putative nuclear localization signal results in a shift of the truncated ORF1p into the cytoplasmic fraction. 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 symbol (name above) denotes approximate location of the antibody on the ORF1p. Putative bipartite nuclear localization signal (Bipartite) is shown in green and putative monopartite nuclear localization signal (monopartite) is shown in brown. The red “X” (X) denotes the mutation of the ORF1 R206/R210/R211 amino acid positions into alanine residues. (bottom) Schematic of the truncated and full-length ORF1 constructs. The expected molecular weight of each construct is listed to the right. The numbers listed on the left represent the position of the site of truncation based on a full-length ORF1p. b Western blot analysis of truncated and full-length 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. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid. c Western blot analysis of full-length 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. Positions of molecular markers are indicated on the right in kDa. Control indicates cells transfected with empty plasmid