FIG. 3.

Sucrose density gradient fractionation of virus-associated HIV-1 RT. 293T cells were cotransfected with HA-tagged R560 (hR560) and pGAG. At 48 h posttransfection, culture supernatant was collected, filtered, and pelleted through 20% sucrose cushions. Viral pellets were resuspended and centrifuged through 30 to 70% sucrose gradients containing a layer of 1% Triton X-100 as described in Materials and Methods. Ten fractions of equal size were collected from the top of each gradient after 16 h of centrifugation. In addition to measuring the density of each fraction, we used Western immunoblotting to analyze Gag and RT protein levels. Fraction densities (in g/ml) are indicated at the top.