Figure 4

Repression or deletion of the I5 locus does not have a deleterious effect on virus replication in tissue culture. (A) The vΔindI5V5 virus allows tight repression of the I5 protein. BSC40 cells were infected (MOI 2) with vΔind I5V5 in the presence (lanes 1,3) or absence (lanes 2,4) of TET. Cells were harvested at 17 hpi and lysates were subjected to immunoblot analysis with the anti-V5 serum (lower panel) or antibodies specific for the intermediate and late viral proteins I3 and L4, respectively (top panel). The molecular masses (in kDa) of protein standards are shown at the right. (B) Repression of I5 does not affect the viral yield produced in a single infectious cycle in BSC40 cells or primary human fibroblasts. BSC40 cells or primary human fibroblasts were infected with vΔind I5V5 (MOI 3) in the presence or absence of TET and harvested at 24 hpi. Viral yield (pfu/ml) was determined by titration on BSC40 cells; experiments were preformed in duplicate and titrated in duplicate. Error bars represent standard deviation. (C) The vΔI5 virus is deleted for the I5 locus. Cells were infected with the parental vI5V5 virus (lanes 2,4) or with two isolates of the vΔI5 virus (lanes 1,3) at an MOI 2 and harvested at 18 hpi. Lysates were examined by immunoblot analysis with the anti-V5 antibody. The molecular masses (in kDa) of protein standards are shown at the right. (D) Deletion of I5 does not affect the viral yield produced in a single infectious cycle in BSC40 cells or primary human fibroblasts. BSC40 cells or human diploid fibroblasts were infected with wt virus, vI5V5 or vΔI5 (MOI 3) for 24 h, and the viral yield was determined as described for panel B. (E) Repression or deletion of I5 does not affect viral plaque size. BSC40 cells were infected with 50–75 PFU of vI5V5, vΔind I5V5 + TET, vΔind I5V5 – TET, or vΔI5; plates were fixed and stained with crystal violet at 48 hpi.