Skip to main content
. 2013 Jan;87(2):1083–1097. doi: 10.1128/JVI.02529-12

Fig 7.

Fig 7

Structure function analysis of the role of the A17 N terminus in virion morphogenesis. (A and B) BSC-40 cells were infected with vindA17-IPTG and transfected with empty vector (V) or plasmids encoding WT or mutant alleles of A17. Cells were harvested at 18 to 24 hpi and processed for conventional electron microscopy. Representative images of the most advanced and most predominant phenotypes are shown in panel A. A total of 20 cells were examined and scored as described for Fig. 3C. The scoring categories were vesicles and virosomes, crescents and virosomes, RIF-like membranes, IV and IVN, MV, and aberrant MV; data representing averages of the results of two independent experiments are shown graphically in panel B. In panel A, the arrows indicate the RIF-like membranes (flaccid sheets of membranes surrounding virosomes) seen in the presence of the PRECLV N or Y3,6,7F mutants, and the triangles indicate aberrant MV (spherical MV with internal core) seen in the presence of the UNCLV N mutant. (C) To complement the conventional EM analysis, cells infected with vindA17-IPTG and transfected with plasmids encoding the PRECLV N or UNCLV N mutants were also examined by immunoelectron microscopy using antisera to the D13 or A17 proteins. The star marks D13-containing inclusion bodies. (D) Cells were infected in the presence of araC and transfected with plasmids encoding WT A17 or the PRECLV N or Y3,6,7F mutants, alone or with V5-D13; cells transfected with V5-D13 alone or with empty vector (V) were also included as controls. At 24 hpi, cell lysates were prepared. An aliquot was removed for analysis as the input, and the remainder of the D13 protein was purified on anti-V5 beads along with any interacting proteins. The V5-D13 (arrowhead) and A17 (triangle) in the input and pulldown samples were assessed by immunoblot analysis. (The IgG heavy chain [diamond] was also seen in the pulldown samples.) The dashed lines indicate where the immunoblots were cut prior to being probed with different antibodies; the numbers at the left of each panel indicate the MW (× 10−3) of the protein standards.