A macrophage fusion assay for rapid screening of cloned HIV-1 Env using dual recombinant vaccinia viruses expressing distinct RNA polymerases

HIV-1 cell tropism is determined initially at the level of fusion mediated by the viral envelope glycoprotein (Env). Cell-cell fusion assays are emplo yed widely to study Env-mediated fusion, and generally require transfection of target cells with a reporter plasmid that is activated upon fusion with Env-expressing effector cells. Macrophages are an important target for HIV -1, but fusion studies using primary macrophages are limited by their resis tance to transfection. An assay described previously used recombinant vacci nia virus to express T7 polymerase in macrophages, and effector ells transf ected with a T7-driven reporter plasmid and infected with recombinant vacci nia virus expressing Env. However, this requires a recombinant vaccinia vir us for each Env. We developed a method to study fusion using primary macrop hages and HIV-1 env plasmid clones under control of the T7 promoter. Macrop hages were infected with a recombinant vaccinia virus expressing the SP6 RN A polymerase. Effector 293T cells were infected with a recombinant vaccinia virus expressing T7 polymerase, and co-transfected with T7-driven env plas mids and an SP6-driven reporter gene plasmid. Cell-cell fusion mediated by T7-driven Env results in SP6-driven reporter gene transactivation. This app roach is suitable for rapid analysis of multiple primary isolate, chimeric, or mutant env genes cloned into plasmid vectors. (C) 1999 Elsevier Science B.V. All rights reserved.