STABLE TRANSFECTION OF PROVIRUS OF HUMAN-IMMUNODEFICIENCY-VIRUS INTO A MURINE PACKAGING CELL-LINE

In order to generate HIV (murine leukemia virus (MuLV)) pseudotypes, H IV genome was transfected into the ecotropic murine packaging cell lin e (GP+E86) and four of the nine transfected clones were extensively ch aracterized. One clone (801), harbouring a full copy of integrated HIV sequences, exhibited a detectable level of intracellular HIV p24 anti gen expression. Northern blot analysis revealed that clone 801 express ed all three classes of HIV mRNAs. Multispliced 2kb mRNAs were detecte d in another clone (8.14). Two other clones (1.31 and 1.32) also exhib ited a complete HIV provirus, but did not show any viral expression, a s evaluated by Northern blot analysis or HIV p24 ELISA. Reverse transc ription-polymerase chain reaction (RT-PCR) experiments revealed the pr esence of full length genomic RNA in four transfected clones, which we re extensively characterized. A co-cultivation of clone 801 with human CD4' cells resulted in syncytia formation. By electron microscopy, ma ture HIV particles were observed after co-cultivation of uninfected C8 166 cells with 801 cells. These results demonstrated that the murine c lone was stably transfected with the complete HIV genome and was capab le of shuttling infectious HIV to human cells. Clone 801 was cocultiva ted with murine NIH-3T3 fibroblasts. In several experiments, HIV infec tion of NIH-3T3 cells was revealed by PCR technique. Thus, 801 cells a ppear to produce low levels of HIV (MuLV) pseudotypes capable of trans ferring the HIV genome into mouse cells.