title: HIV-1 virion fusion assay: uncoating not required and no effect of Nef on fusion.
abstract: We recently described a sensitive and specific assay that detects the fusion of HIV-1 virions to a broad range of target cells, including primary CD4 cells. This assay involves the use of virions containing beta-lactamase-Vpr (BlaM-Vpr) and the loading of target cells with CCF2, a fluorogenic substrate of beta-lactamase. Since Vpr strongly associates with the viral core, uncoating of the viral particle might be required for effective cleavage of CCF2 by BlaM-Vpr. Here, we show that BlaM-Vpr within mature viral cores effectively cleaves CCF2, indicating that this assay measures virion fusion independently of uncoating. We also show that wildtype and Nef-deficient HIV-1 virions fuse with equivalent efficiency to HeLa-CD4 cells, SupT1 T cells, and primary CD4 T cells. Since Nef enhances cytoplasmic delivery of viral cores and increases viral infectivity, these findings indicate that Nef enhances an early post-fusion event in the multistep process of viral entry. Possible sites of Nef action include enlargement of the fusion pore, enhanced uncoating of viral particles, and more efficient passage of viral cores through the dense cortical actin network located immediately beneath the plasma membrane.
authors.affiliation: Gladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94141-9100, United States.
authors.forename: Marielle
authors.initials: M
authors.lastname: Cavrois
authors.forename: Jason
authors.initials: J
authors.lastname: Neidleman
authors.forename: Wes
authors.initials: W
authors.lastname: Yonemoto
authors.forename: David
authors.initials: D
authors.lastname: Fenard
authors.forename: Warner C
authors.initials: WC
authors.lastname: Greene
mesh_terms: D002460:Cell Line; D003593:Cytoplasm / Q000821:virology; D005452:Fluoresceins; D015693:Gene Products, nef / Q000172:deficiency / Q000502:physiology*; D016336:Gene Products, vpr / Q000378:metabolism; D016655:HIV Core Protein p24 / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism / Q000472:pathogenicity / Q000502:physiology*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D007769:Lactams; D008561:Membrane Fusion; D013379:Substrate Specificity; D014774:Virulence; D014779:Virus Replication; D001618:beta-Lactamases; D047090:beta-Lactams; D054311:nef Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus
chemical_list: C109764:CCF 2; D005452:Fluoresceins; D015693:Gene Products, nef; D016336:Gene Products, vpr; D016655:HIV Core Protein p24; D007769:Lactams; D047090:beta-Lactams; D054311:nef Gene Products, Human Immunodeficiency Virus; D054325:vpr Gene Products, Human Immunodeficiency Virus; D001618:beta-Lactamases