FIG 8.

The dynein regulators p50 and NudEL are required for a post-reverse transcription step in MLV infection. (A) Control, p50, or NudEL KD cells were infected with Mo-MLV. Low-molecular-weight DNA was isolated 20 h after infection, and the viral DNA synthesized in the infected cells was detected by PCR using primer pairs specific for the different forms of the linear DNA (primers directed against the CA region) or the LTR-LTR junction nuclear entry marker. The amplification of mitochondrial DNA was used as a control. (B) The correct localization of the ecotropic receptor mCAT1 in p50 and NudEL KD cells was determined. Control (nonsilenced), p50, and NudEL KD cells were transfected with a plasmid encoding an HA-tagged version of mCAT1. The subcellular localization of the overexpressed receptor was detected by indirect immunofluorescence using an α-HA antibody. The arrowheads indicate membrane-localized receptors. (C) Stable TE671 cells in which p50 and NudEL were silenced were generated and challenged with amphotropic HIV-1. (Bottom) NudEL and p50 mRNA levels in cells engineered by KD of NudEL or p50 were determined by quantitative PCR. The values were normalized to GAPDH mRNA and expressed as fold reduction over the nonsilenced control (**, P < 0.001). (Top) Relative infection by amphotropic HIV-1–luciferase reporter in the KD cell compared to the nonsilenced control. The error bars represent the standard deviations among the 3 independent experiments (n.s., nonsignificant). (D) Relative infection of ecotropic HIV-1–luciferase reporter in NIH 3T3 p50 and NudEL KD cells compared to the nonsilenced control. The error bars represent the standard deviations among the 3 independent experiments (*, P < 0.05).