CELL LINE-DEPENDENT RELEASE OF HIV-LIKE GAG PARTICLES AFTER INFECTIONOF MAMMALIAN-CELLS WITH RECOMBINANT VACCINIA VIRUSES

We investigated the production of Gag particles by Vero, CV-1, or 1D c ells infected with different vaccinia virus recombinants expressing HI V gag or gag-pol genes. Immunoblots of(centrifuged) culture media from 1D cells infected with vMM5, a vaccinia virus recombinant expressing the HIV-2 gag-pol genes, revealed the presence of abundant particles t hat contained (mostly processed) Gag antigens. In contrast, Gag partic les were found only in low amounts in the culture medium from Vero cel ls infected with the same HIV gag-pol vaccinia virus recombinant; the Gag precursor remained associated with the infected Vero cells and was efficiently processed. This low excretion of Gag particles after infe ction of Vero cells with vMM5 was also demonstrated by assays of rever se transcriptase activity in the pellet of centrifuged culture medium. Cell fractionation showed that Gag proteins were predominantly found in the membrane fraction from both 1D and Vero cells. Electron microsc opy observations of 1D or of Vero cells infected with vMM5 vaccinia vi rus recombinant revealed in both cases the presence of particles buddi ng at the plasma membrane. However, the shape of the budding particles was different in the two cell lines, with immature forms present in t he membrane from the infected Vero cells. An inefficient excretion of Gag particles was also observed after infection of Vero cells with dif ferent vaccinia virus recombinants expressing either an uncleaved HIV- 2 Gag protein or the HIV-I gag-pol genes, as judged both by immunoblot and reverse transcriptase activity assays. Radioactive myristic acid was incorporated to similar levels into the p55(gag) precursor express ed in 1D or in Vero cells, revealing that the low excretion of Gag par ticles from the Vero cells did not result from a defect in myristylati on of the Gag precursor in these cells. These results suggest that Ver o cells are lacking an (unidentified) cellular factor involved in the last stage of budding particle formation which mediates the separation from the cells of the HIV-like particles formed in the cell membrane.