Journal: Journal of Virology

Article Title: Membrane Association and Dimerization of a Cysteine-Rich, 16-Kilodalton Polypeptide Released from the C-Terminal Region of the Coronavirus Infectious Bronchitis Virus 1a Polyprotein

doi: 10.1128/JVI.76.12.6257-6267.2002

Figure Lengend Snippet: (a) Subcellular localization of the 16-kDa protein. (A) Cos-7 cells overexpressing pEGFP-16k at 16 h posttransfection showing perinuclear staining. (B) Cos-7 cells overexpressing the EGFP control vector (pEGFP) showing diffuse staining. (C) Immunofluorescent staining of IBV-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection showing distinct punctate perinuclear staining. (D) Immunofluorescent staining of mock-infected Vero cells with anti-16-kDa protein serum (1:20) at 13 h postinfection. (E) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining profile of anti-16-kDa protein serum. (F) Double labeling of IBV-infected Vero cells that were transfected with BrUTP at 10 h postinfection showing the staining pattern of anti-BrUTP serum. (G) Superimposition of images E and F. All images were taken from a Zeiss Axioplan confocal microscope. (b) Membrane association of the 16-kDa protein. Cos-7 cells expressing the 16-kDa protein were labeled with [ 35 S]methionine-cysteine for 4 h and harvested. Cells were lysed with a Dounce homogenizer and fractionated into membrane (M) and cytosol (C) fractions at pH 7 (lanes 1 and 2) by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography. Membrane (M) pellets were resuspended in hypotonic buffer; treated with 1% Triton X-100 (TX-100), 100 mM Na 2 CO 3 , or 1 M KCl; and further fractionated into supernatant (S) and pellet (P) fractions by ultracentrifugation. Polypeptides were immunoprecipitated with anti-16-kDa protein serum, separated on an SDS-15% polyacrylamide gel, and detected by fluorography.

Article Snippet: To investigate this possibility, IBV-infected Vero cells were BrUTP labeled at 10 h postinfection for 4 h and double labeled with an anti-BrdU monoclonal antibody (Roche) and anti-16-kDa protein serum, showing perinuclear punctate vesicular staining patterns (Fig. , parts E and F).

Techniques: Staining, Plasmid Preparation, Infection, Labeling, Transfection, Microscopy, Expressing, Immunoprecipitation