FIG. 2.

2Nt phosphorylation is important for nuclear exit of infectious virus in transformed cells. (A) Subcellular localization of MVMp particles in a single round of infection of transformed 324K cells synchronized by growth to confluence. Shown are representative fields of cells visualized by confocal microscopy and stained with the MVM-capsid MAb and the α-2Nt antibody specific for DNA-filled virions. +LMB, wt particles at 24 hpi in cells treated with LMB (100 ng/ml) from 14 hpi. Insets, intranuclear punctate phenotype of S/G particles. (B) Subcellular distribution of newly formed MVM particles. Infected synchronous cells were labeled with ³⁵S since 6 hpi, and wt viral particles harvested from fractionated cells at the indicated times were sedimented through a sucrose cushion and centrifuged to equilibrium in a CsCl gradient. A representative result of ³⁵S cpm distribution in the banding positions of the empty capsids (c) and DNA-filled virions (v) is shown. (C) The S/G phosphorylation mutant shows a defect in nuclear egress. (Left) Infectious units (IU) of wt and S/G viruses in the culture medium (extra) and inside the cells (intra) at the indicated postinfection times for synchronized 324K cells. DL, detection limit of the assay. (Right) Percentage of subcellular distribution of infectious viruses in fractionated cells. Data are means and standard errors from three independent experiments.