title: A macrophage fusion assay for rapid screening of cloned HIV-1 Env using dual recombinant vaccinia viruses expressing distinct RNA polymerases.
abstract: HIV-1 cell tropism is determined initially at the level of fusion mediated by the viral envelope glycoprotein (Env). Cell-cell fusion assays are employed 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 resistance to transfection. An assay described previously used recombinant vaccinia virus to express T7 polymerase in macrophages, and effector cells transfected with a T7-driven reporter plasmid and infected with recombinant vaccinia virus expressing Env. However, this requires a recombinant vaccinia virus for each Env. We developed a method to study fusion using primary macrophages and HIV-1 env plasmid clones under control of the T7 promoter. Macrophages were infected with a recombinant vaccinia virus expressing the SP6 RNA polymerase. Effector 293T cells were infected with a recombinant vaccinia virus expressing T7 polymerase, and co-transfected with T7-driven env plasmids and an SP6-driven reporter gene plasmid. Cell-cell fusion mediated by T7-driven Env results in SP6-driven reporter gene transactivation. This approach is suitable for rapid analysis of multiple primary isolate, chimeric, or mutant env genes cloned into plasmid vectors.
authors.affiliation: Division of Infectious Diseases, University of Pennsylvania School of Medicine, Philadelphia 19104-6060, USA. isaacs@mail.med.upenn.edu
authors.forename: S N
authors.initials: SN
authors.lastname: Isaacs
authors.forename: Y
authors.initials: Y
authors.lastname: Yi
authors.forename: A
authors.initials: A
authors.lastname: Singh
authors.forename: R G
authors.initials: RG
authors.lastname: Collman
mesh_terms: D002459:Cell Fusion / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000235:genetics; D005814:Genes, Viral; D015700:HIV Envelope Protein gp41 / Q000235:genetics / Q000302:isolation & purification*; D015497:HIV-1 / Q000235:genetics* / Q000302:isolation & purification; D006801:Humans; D008264:Macrophages / Q000737:chemistry / Q000821:virology*; D012313:RNA / Q000235:genetics*; D012326:RNA Splicing / Q000235:genetics; D014616:Vaccinia virus / Q000201:enzymology* / Q000235:genetics*; D015678:Viral Structural Proteins / Q000235:genetics
chemical_list: D015700:HIV Envelope Protein gp41; C039919:RNA, recombinant; D015678:Viral Structural Proteins; D012313:RNA; D012321:DNA-Directed RNA Polymerases