TABLE 1.
Titers of infectious LZRNL virus preparations prepared by exposure of noninfectious gag-pol RNA particles to VSV-G in vitro by mixing of conditioned media, mixing of media followed by pelleting, or mixing of pelleted mediaa
| Source of VSV-G | Virus titer (CFU/ml)b
|
||
|---|---|---|---|
| Conditioned media mixed | Media mixed before pelleting | Media pelleted, then mixed | |
| 293/pCMV-G | |||
| Conditioned medium | 6.0 × 102 | 8.4 × 103 | 3.6 × 103 |
| Conditioned medium + I1 antibody | <1 | <1 | <1 |
| Cell lysate | ND | 4.4 × 103 | ND |
| 293GP/pCMV-G | |||
| Conditioned medium | 5.8 × 102 | 1.1 × 104 | 4.3 × 103 |
| Conditioned medium + I1 antibody | <1 | <1 | <1 |
| Cell lysate | ND | 6.1 × 103 | ND |
| Detergent-purified VSV-G | |||
| β-OG | ND | ND | <1 |
| C12H8 | ND | ND | 2.0 × 102 |
| 293/pCMV-Ampho | |||
| Conditioned medium | <1 | <1 | <1 |
| 293/pCMV-Eco | |||
| Conditioned medium | <1 | <1 | <1 |
| Cell lysate | <1 | <1 | <1 |
Sources of VSV-G included 293 or 293GP cells transfected with pCMV-G as described in Materials and Methods. For samples mixed after centrifugation, 1 ml of each medium was centrifuged, the pellets were resuspended in 10 μl of PBS and mixed, and serial dilutions were assayed for infection on rat 208F cells. For assays involving mixing prior to pelleting, 0.5-ml aliquots of the media were mixed and centrifuged as above. The pellets were resuspended in 50 μl of PBS, and serial dilutions were assayed for infectivity on rat 208F cells. Prior to infection of 208F cells, an aliquot of each sample was incubated with VSV-G neutralizing antibody I1 or with anti-VSV-G polyclonal antibody for 15 min at room temperature. Results of antibody I1-treated samples are shown. The virus titer is indicated as the total infectious virus in 1 ml of the starting 293GP/LZRNL conditioned medium. The titer of a conventionally prepared pseudotyped LZRNL virus produced under identical conditions was 2.3 × 105 CFU/ml. Solubilized and partially purified VSV-G samples were prepared as described in Materials and Methods. One microgram of purified VSV-G was mixed with the resuspended pellet from 293GP/LZRNL conditioned medium and assayed for infectivity as described above. Conditioned media and cell lysates of 293 cells transfected with pCMV-Ampho or pCMV-Eco were also examined by the same procedure as those of pCMV-G-transfected cells.
Total CFU of virus produced from 1 ml of conditioned medium from 293GP/LZRNL cells. ND, not determined.