Rab14 interaction with FIP1C is required for HIV-1 Env incorporation.

<p>(A) HeLa cells were transfected with proviral plasmid pNL4-3 or NL4-3 CT144 and either a dominant negative form of Rab14 (Rab14S25N) or a constitutively active form of Rab14 (Rab14Q70L). 48 hours after transfection, cellular and particle content of HIV-1 Env was examined by Western blot. Numbers above Env blots represent densitometry values with leftmost lanes set to 100. (B) shRNA-mediated depletion of Rab11 or Rab14 was performed in HeLa cells. Following selection in puromycin, cells were transfected with either pNL4-3 or pNL4-3 CT144 plasmid. Cellular and particle-associated Env was detected by immunoblotting 48 hours following transfection. (C) shRNA-resistant GFP-FIP1C* WT and GFP-FIP1C* (S580N/S582L) were transfected together with pNL4-3 in control HeLa cells or FIP1C-depleted HeLa cells as in the repletion experiment described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003278#ppat-1003278-g002" target="_blank">Figure 2</a>. Input FIP1C* plasmid levels were 2 µg in lanes 3, 5, 7, and 8 and increased to 4 µg in lanes 4 and 6. In a separate experiment the ability of Rab11 binding mutant GFP-FIP1C* I62E and GFP-FIP1C* D622N to rescue Env particle incorporation in FIP1C-depleted HeLa cells (lanes 7 and 8). Note that gp160 and gp120 were detected with polyclonal antiserum; gp41 particle blots were probed with a monoclonal specific for gp41.