Article Figures & Data
Figures
- Fig. 1.
UL11-Gag chimeras. The wild-type RSV Gag (unshaded) and UL11 (hatched) proteins are aligned at their N termini. The positions of the assembly domains are marked along the top of Gag. Sites cleaved by the viral protease are marked by vertical lines through Gag. N-terminal myristylation is indicated by a squiggled line. Numbers indicate the amino acids included in each construct. The p6 sequence of HIV, the first 10 amino acids of the Src oncoprotein, and the GFP sequences are indicated by black boxes.
- Fig. 2.
Expression of HMG. COS-1 cells transfected with the indicated constructs were labeled for 2.5 h withl-[35S]methionine, and the Gag proteins from the media and cell lysates were immunoprecipitated with anti-RSV antibodies, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. The numbers to the left are the positions (in kilodaltons) of molecular mass markers. The positions of the CA and PR Gag cleavage products are also indicated. Gag products in the medium are indicative of budding.
- Fig. 3.
Release properties of modified forms of HMG. The indicated constructs were transfected into duplicate plates of COS-1 cells. The right panel (media) was from one set of plates that were treated as described in the legend to Fig. 2. Gag proteins in the left panel (lysates) were collected and visualized as for Fig. 2, but were from the second set of plates that had been labeled for only 5 min.
- Fig. 4.
Microscopic analyses of HMG-expressing cells. COS-1 cells were transfected with the indicated constructs. Cells in panels A to F were analyzed by immunofluorescence. In panels A to C, antibodies specific for RSV Gag were used. Cells in panels D to F were double labeled with a mixture of rabbit antibodies against Gag and mouse antibodies against the Golgi 58K protein, which were detected by using a mixture of goat anti-rabbit antibodies conjugated to TRITC and goat anti-mouse antibodies conjugated to FITC. Panels D and E are the same field viewed by confocal microscopy with the appropriate wavelength to excite TRITC (D) or FITC (E) and were digitally combined to provide the image in panel F. Cells transfected with GFP constructs in panels G and H were viewed by light microscopy without fixing or staining. Cells in panels I and J were analyzed by standard electron microscopy.
- Fig. 5.
Deletion analysis of UL11. (A) The level of particle release of the indicated mutants was measured by labeling the transfected cells as in Fig. 3. (B) COS-1 cells were transfected, fixed, and stained with RSV Gag-specific antibodies and visualized by confocal microscopy.
- Fig. 6.
Expression of HMG in avian cells. The HMG and RSV Gag genes were transferred into the BHRCAN (12) vector for expression in avian cells. (A) Plasmids were transfected by the calcium-phosphate precipitation method into QT6 cells, and the proteins were labeled and collected as described in the legend to Fig. 2. (B) COS-1 or QT6 cells were transfected as for panel A with either GFP or UL11.GFP and viewed by confocal microscopy.
- Fig. 7.
Alignment of UL11 homologs. The amino acid sequences of the UL11 homologs from several herpesviruses are shown. HHV, human herpesvirus. Identical amino acids are indicated by an asterisk, while similar amino acids are marked by a colon. A conserved acidic cluster motif is underlined.
