Production and Purification of HIV-1 Virions

Replication deficient VSV-G pseudotyped HIV-1 virions were produced in HEK293T cells using pCRV1-GagPol, pCSGW and pMD2.G as described previously (Price et al., 2014 (link)). At 24–48 hr post transfection, the supernatants were harvested and passed through 0.22 μm nitrocellulose filter. The virions were concentrated by ultracentrifugation through a 20% (w/v) sucrose cushion (2 hr at 28,000 rpm in a SW32 rotor [Beckman Coulter Life Sciences]). The pellet was resuspended in PBS, snap-frozen and stored at –80 °C. LEN-treated virions were incubated in presence of 700 nM LEN for 1.5 hr at room temperature prior to plunge-freezing for cryo-ET.