Figure 2

Characterization of the endogenous I5V5 protein. (A). I5 is expressed as a late protein. Cells were left uninfected (lane 1) or infected (MOI 5) with wt virus (lane 2) or with vI5V5 in the absence (lane 3) or presence (lane 4) of AraC (cytosine arabinoside, 20 μM). Cells were harvested at 8 hpi and lysates were probed with either an anti-G7 or anti-V5 antibody. The molecular masses (in kDa) of protein standards are shown at the left. (B) I5 is not phosphorylated in vivo. BSC40 cells were infected with wt virus (lanes 1,2,5,6) or vI5V5 (lanes 3,4,7,8) (MOI 5) in the presence of either ³⁵S-met (lanes 1–4) or ³²PPi (lanes 5–8) and then harvested for immunoprecipitation analysis. Immunoprecipitation was performed with antisera specific for the F18 protein (odd numbered lanes) or the V5 epitope (even numbered lanes); immune complexes were resolved electrophoretically and visualized by audioradiography. The molecular masses (in kDa) of protein standards are shown at the right. (C) I5 shows a punctate distribution throughout the cytoplasm. BSC40 cells were infected with wt virus or vI5V5 for 8 hpi; fixed cells were stained with the anti-V5 antibody and a secondary antibody conjugated to Alexafluor 594 and DAPI. (D) I5 localizes to crescents, immature and mature virions. Cells were infected with V5I5 (MOI 2) and harvested at 17 hpi for post-embedding immunoelectron microscopy. Sections were incubated with anti-V5 antibody and secondary antibodies conjugated to 5 nm (panels B, C, E) or 10 nm (panels A, D, F) gold particles.