Fig 2.

The NC domain is required for PTAP-mediated budding of SIVsmmE543. (A) Schematic representation of the SIVsmmE543 NC domain (amino acids 1 to 50). Lysine (K) and arginine (R) residues replaced with alanine in the smRKI and smRKII mutants are circled and underlined, respectively. (B) Comparison of virus release among WT SIVsmmE543, the L domain mutant L1L2/YL (lacking all known L domains), and the Alix binding site mutant YL (relying only on PTAP for release) in the context of a WT or mutated NC. 293T cells were transfected with either WT SIVsmmE543 or the indicated mutant. Cell lysates and viral fractions were analyzed by Western blotting using the indicated antibodies. Relative virus release efficiencies are shown at the bottom (± the standard deviations; n = 3). (C) Release defects of SIVsmmE543/YL NC mutant viruses are rescued by ectopic expression of Nedd4.2s. 293T cells were transfected with the indicated NC mutant alone (lanes 1 and 2) or in the presence of increasing amounts of Nedd4.2s (lanes 3, 4, 6, and 7). Pelleted virion and cell lysate fractions were analyzed by Western blotting using the indicated antibodies. Quantification of the stimulatory effect of Nedd4.2s on viral release is shown at the bottom. The n-fold increase in viral release upon cotransfection with Nedd4.2s for each mutant was calculated as described in the legend to Fig. 1.