title: Modification of a viral envelope glycoprotein cell-cell fusion assay by utilizing plasmid encoded bacteriophage RNA polymerase.
abstract: Many viruses enter cells via an interaction of the viral envelope glycoprotein (Env) with receptor inducing fusion of viral and cellular membranes. These interactions are often evaluated in cell-cell fusion, gene-reporting systems with effector cells expressing Env and target cells expressing receptors. A common system utilizes vaccinia virus encoding T7 RNA polymerase (RNAP) in effector cells and a T7 promoted reporter plasmid in target cells. Fusion is quantified with expression of the reporter plasmid. However, direct activation of reporter plasmid from vaccinia virus can occur increasing background activity. We report here a modification of this assay in which T7 RNAP is expressed from a plasmid rather than vaccinia. This modification increased sensitivity with a ten-fold reduction in background. A novel dual T7/SP6 RNAP fusion assay was also developed to allow rapid screening for functional Env clones. Using these assays, we show that Envs from two CD4-independent HIV-2 isolates (VCP and ROD/B), which are able to fuse with chemokine receptor CXCR4 in a CD4-independent manner, are also able to fuse with alternative coreceptors GPR1 and GPR15 in the absence of CD4. The assay could also detect fusion of murine leukemia virus on target cells expressing the ecotropic MCAT-1 receptor showing its broad utility in other viral systems.
authors.affiliation: Department of Medicine, Hematology-Oncology Division, University of Pennsylvania, Philadelphia, PA 19104, USA. lin@bmi-md.com
authors.forename: George
authors.initials: G
authors.lastname: Lin
authors.forename: Samuel L
authors.initials: SL
authors.lastname: Murphy
authors.forename: Glen N
authors.initials: GN
authors.lastname: Gaulton
authors.forename: James A
authors.initials: JA
authors.lastname: Hoxie
mesh_terms: D000818:Animals; D015704:CD4 Antigens / Q000378:metabolism; D002459:Cell Fusion / Q000379:methods*; D002460:Cell Line; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism*; D015686:Gene Products, env / Q000235:genetics / Q000378:metabolism*; D015498:HIV-2 / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D006801:Humans; D009052:Leukemia Virus, Murine / Q000378:metabolism / Q000472:pathogenicity; D051379:Mice; D010957:Plasmids / Q000235:genetics; D019718:Receptors, CXCR4 / Q000378:metabolism; D015728:Receptors, HIV / Q000378:metabolism; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D015704:CD4 Antigens; D015686:Gene Products, env; D019718:Receptors, CXCR4; D015728:Receptors, HIV; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases