rVSV-EnvG₄-G₆ characterization.

<p>(a) After a 24 hr infection, total infected Vero cell lysates were collected and proteins were separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE). Blot was probed with anti-VSV-N and anti-VSV-G_IN CT, which does not recognize G_NJ. IN: rVSV-EnvG₄-G₆^IN; NJ: rVSV-EnvG₄-G₆^NJ. (b-d) Sucrose-gradient purified rVSV-EnvG₄-G₆ particles were separated by SDS-PAGE. (b) Blots of 10⁶ pfu of rVSV-EnvG₄-G₆^IN and 2.5×10⁶ pfu rVSV-EnvG₄-G₆^NJ vectors were probed separately for EnvG using anti-gp120. (c) Blot of 10⁶ pfu rVSV-EnvG₄-G₆^IN was probed with anti-VSV-G^IN CT. (d) Blot of 10⁶ pfu rVSV-EnvG₄-G₆^IN and rVSV-EnvG₄-G₆^NJ was probed with anti-VSV-G. (e) Replication kinetics of rVSV-EnvG₄-G₆ and rVSV-G₄ viruses in Vero cells. Vero cells were infected in 6-well plates at an MOI of 0.1. At various intervals post infection, supernatant was collected from duplicate wells and virus was titrated. Plaques were counted manually after cell fixation.