The incorporation of Env onto HIV-1 particles is saturable in a cytoplasmic tail-dependent fashion.

<p>(A) Increasing amount of wildtype and the tailless mutant (CT144) of NL4-3 Env protein expressing plasmids were co-transfected with fixed amount of pNL4.3Env- proviral plasmid into HeLa cells. Progeny viral particles were harvested two days after transfection through a 20% sucrose cushion and the particles were lysed with SDS loading buffer. Proteins were analyzed by immunoblot using Env and CA specific antibodies. (B) The intensity of each Env band and Gag band in the particle immunoblot shown in A was quantified by Licor software. Background-subtracted pixel intensity values were plotted on the graph as Env band intensity versus CA band intensity. (C) The absolute quantity of Env and CA/P24 on particles was measured by gp120 ELISA and p24 ELISA. The number of Env molecules per particle was calculated as an approximation, assuming that 5000 CA molecules are present in one HIV-1 virion. (D) The infectivity of the progeny viral particles was measured using TZM-bl reporter cells. Infectivity is plotted as number of blue cells per nanogram of p24 antigen used in the infection assay.