FIG. 4.

(A) Purification of chimerical particles. Particles were purified using a sucrose gradient. Supernatant from 293T cells transfected with pMN gag-pol or pLentiopt, pCMV-Ampho, and CMV 26Sm2 was deposited on a continuous sucrose gradient, 20% to 60%, in 35-ml tubes. Tubes were centrifuged at 100,000 × g for 2 h using an SW 28 swinging rotor. Fractions were collected using a Pharmacia collector equipped with a 260-nm UV detector for protein detection (optical density [OD]). Each fraction was checked for RT activity using rA/dT oligonucleotides in the presence of αdATP³². Black lozenges and squares represent RT activity for MLV and lentivirus vectors, respectively. For HIV-based vectors, we also measured the CA p24 concentration in each fraction (gray dots on the right panel). In the indicated fractions, we detected the presence of SFV genome by RT-PCR. Primer localization on vectors was chosen to detect full-length RNA by targeting the nsP1. MW, molecular weight marker. The lower panel gives a densitometric measurement of PCR fragment signal in arbitrary units. (B) Western blot detection of nsP1 in vectors. Vector proteins were extracted after ultracentrifugation on a sucrose cushion of supernatant harvested from producing tritransfected BHK-21 cells. Proteins were separated on sodium dodecyl sulfate-polyacrylamide gels. nsP1-specific antibody recognized the two forms of the protein, the uncleaved precursor and the free cleaved protein. Lane 1, supernatant from pSFV1/AMenv-transfected BHK-21 cells; lane 2, supernatant from pSFV1/AMenv- and pSFV-C/gag-pol-transfected BHK-21 cells; lane 3, supernatant from pSFV1/AMenv-, pSFV-C/gag-pol-, and 26Sm2-transfected BHK-21 cells; lane 4, negative control, supernatant from 293T cells transfected with pMN gag-pol, pCMV-Ampho, and pBullet. Only vectors produced using the SFV system contained both the uncleaved and cleaved forms of nsP1, as shown by the detection in lanes 1 to 3 of 250-kDa and 61-kDa bands, respectively.