Article Figures & Data
Figures
- Fig. 1.
ORF 57 increases cytoplasmic transport of viral mRNA. Cos-7 cells transfected with pUCgB (a) and pEcoB (b) in the absence or presence of pRSVORF57. Total (lanes 1 and 2), nuclear (lanes 3 and 4), and cytoplasmic (lanes 5 and 6) RNA was then isolated and separated by elecrophoresis on a 1% denaturing formaldehyde agarose gel. The RNA was transferred to Hybond-N membranes and hybridized with32P-radiolabelled randomly primed probes specific for the HVS gB and the capsid coding sequence.
- Fig. 2.
The ORF 57 protein binds viral mRNA. (a) Coomassie blue-stained gel of GST (lane 1) and GST-ORF 57 (lane 2) proteins expressed in E. coli DH5α and purified from the crude lysate by incubation with glutathione-Sepharose 4B beads according to manufacturer’s specifications. (b) RNA-binding assay of the ORF 57 protein. GST and GST-ORF 57 proteins were separated on an SDS–12% polyacrylamide gel and transferred onto nitrocellulose (Amersham). The nitrocellulose bound proteins were denatured by using 6 M guanidine-HCl and then renatured by using six 50% stepwise dilutions of the guanidine-HCl in binding buffer. The filter-bound ORF 57 protein was then hybridized with 32P-radiolabelled RNA probes specific for HVS gB (i) and actin (ii).
- Fig. 3.
(a) Hoechst dye staining of monkey and mouse cell nuclei. Hoechst dye allowed differentiation between monkey (i) and mouse (ii) nuclei. Monkey cells stained diffusely throughout the nuclei, whereas mouse nuclei stained with a distinctive speckled pattern. (b) The ORF 57 protein shuttles between the nucleus and cytoplasm. Cos-7 cells seeded at 2 × 105 cells per 35-mm-diameter petri dish were transiently transfected with 2 μg of pRSVORF57. After 18 h, mouse 3T3 cells (5 × 105cells/well) were plated onto the Cos-7 cells in medium containing 50 μg of cycloheximide per ml. Four hours later the cells were washed in PBS and fused by the addition of 2 ml of 50% polyethylene glycol (wt/wt) in PBS. After being washed, the cells were returned to medium containing 50 μg of cycloheximide per ml for 60 min. Cells were incubated with a 1:100 dilution of ORF 57 antibody and then costained with 0.5 μg of Hoechst dye (i) and fluorescein-conjugated anti-mouse immunoglobulin (ii) per ml.
- Fig. 4.
The ORF 57 gene product expresses a nuclear export signal. Cos-7 cell monolayers were transfected with 2 μg of either pEGFP-C1 (i) or pEGFP-57NES (ii). After 24 h, the subcellular localization of GFP was observed by using fluorescence microscopy.
- Fig. 5.
Mutation of the ORF 57 NES abrogated the ability of the ORF 57 protein to shuttle between the nucleus and cytoplasm. Cos-7 cells seeded at 2 × 105 cells per 35-mm-diameter petri dish were transiently transfected with 2 μg of p57-NES. After 18 h, mouse 3T3 cells (5 × 105 cells/well) were plated onto the Cos-7 cells in medium containing 50 μg of cycloheximide per ml. Four hours later the cells were washed in PBS and fused by the addition of 2 ml of 50% polyethylene glycol (wt/wt) in PBS. After being washed, the cells were returned to medium containing 50 μg of cycloheximide per ml for 60 min. Cells were then incubated with a 1:100 dilution of ORF 57 antibody and costained with 0.5 μg of Hoechst dye (i) and fluorescein-conjugated anti-mouse immunoglobulin (ii) per ml.
