Journal: Journal of Virology
Article Title: Signals That Dictate Nuclear Localization of Human Papillomavirus Type 16 Oncoprotein E6 in Living Cells
Figure Lengend Snippet: Cellular localizations of low-risk and high-risk HPV E6 in COS-1 cells. COS-1 cells were transfected with plasmids pZMZ66 (6E6E7, nt 85 to 939), pZMZ67 (11E6E7, nt 85 to 924), pZMZ69 (18E6E7, nt 103 to 967), pZMZ70 (16E6E7, nt 81 to 880), and pZMZ81 (16E6E7, nt 81 to 880 with a mutant nt 226 5′ splice site [GU to GG]) (A) and pZMZ82 (16E6, nt 81 to 559 with a mutant nt 226 5′ splice site [GU to GG]), pZMZ71 (16E6*I cDNA, nt 81 to 559), pZMZ72 (16E6*II cDNA, nt 81 to 559), pZMZ73 (16E6, nt 81 to 559), and pZMZ74 (16E7, nt 560 to 880) (B). 16E6*I and 16E6*II cDNA constructs have no coding sequences in the E6 intron region because of pre-mRNA splicing. Diagrams at the top of each panel show the insertion and position of the “E6” or “E7” coding region in mammalian expression vector pEGFP-C1. The individual images were collected at 6 (6E6 and 11E6) or 24 h after transfection. Plasmid pEGFP-C1 was used as a GFP control in each transfection. A fluorescence image, a differential interference contrast (DIC) image, and a merged fluorescence-DIC image for each fusion protein arepresented. (A) Localization of 6E6, 11E6, 16E6, and 18E6 translated from the inserted E6E7 coding region in COS-1 cells. 16E6 mt 5′ ss indicates the plasmid containing the 16E6E7 coding region but having a mutant 5′ splice site at nt 226 position (GU to GG), used as a transfection control for full-length 16E6, since this mutation blocks the splicing of the E6E7 pre-mRNA. The single nucleotide mutation in the 5′ splice site of 16E6E7 converts a GUA codon for valine in 16E6 to a GGA codon for glycine. (B) Localization of various 16E6 proteins translated from the inserted E6 coding region and E6*I or E6*II cDNA. 16E7 protein translated from plasmid pZMZ74 was used for comparison. + or − indicates the presence or absence of cytoplasmic (C), nuclear (N), or nucleolar (No) localization of GFP fusions. Scale bars, ∼8 μm. (C) Western blotting analysis of GFP-E6 or GFP-E7 fusions expressed from pEGFP-C1 vectors. As shown in panels A and B, all GFP fusions except for mutant E6 were included for comparison of the expected full-length E6 or E7 protein, even through the 6E6 and 11E6 coding regions do not have an intron. GFP-16E6 in lane 5, encoded from a plasmid with the 16E6E7 coding region, was included for comparison with GFP-16E6 in lane 8, encoded from a plasmid with the 16E6 coding region. Protein samples prepared from COS-1 cells transfected with the various plasmids were resolved on an SDS-12% polyacrylamide gel and blotted with anti-GFP antibody. GFP and its derived fusions are indicated above the lanes. Prestained protein markers (Bio-Rad) in kilodaltons are shown at the left.
Article Snippet: Mammalian expression vectors pEGFP-C1 and pEGFP-N1 were purchased from Clontech (Palo Alto, Calif.).
Techniques: Transfection, Mutagenesis, Construct, Expressing, Plasmid Preparation, Fluorescence, Western Blot, Derivative Assay