Document 0425
 DOCN  M9490425
 TI    Fusogenic mechanisms of enveloped-virus glycoproteins analyzed by a
       novel recombinant vaccinia virus-based assay quantitating cell
       fusion-dependent reporter gene activation.
 DT    9411
 AU    Nussbaum O; Broder CC; Berger EA; Laboratory of Viral Diseases, National
       Institute of Allergy and; Infectious Diseases, National Institutes of
       Health, Bethesda, MD; 20892.
 SO    J Virol. 1994 Sep;68(9):5411-22. Unique Identifier : AIDSLINE
       MED/94335053
 AB    The fusogenic activities of enveloped-virus glycoproteins were analyzed
       by using a quantitative, sensitive, rapid, and highly versatile
       recombinant vaccinia virus-based assay measuring activation of a
       reporter gene upon fusion of two distinct cell populations. One
       population uniformly expressed vaccinia virus-encoded viral
       glycoproteins mediating specific binding and fusion activities; the
       other expressed the corresponding cellular receptor(s). The cytoplasm of
       one population also contained vaccinia virus-encoded bacteriophage T7
       RNA polymerase; the cytoplasm of the other contained a transfected
       plasmid with the Escherichia coli lacZ gene linked to the T7 promoter.
       When the two populations were mixed, cell fusion resulted in activation
       of the LacZ gene in the cytoplasm of the fused cells; beta-galactosidase
       activity was assessed by colorimetric assay of detergent cell lysates or
       by in situ staining. We applied this approach to study the human
       immunodeficiency virus type 1 envelope glycoprotein (Env)-CD4
       interaction. Beta-Galactosidase was detected within 1 h after cell
       mixing and accumulated over the next several hours. Cell fusion
       dependence was demonstrated by the strict requirement for both CD4 and
       functional Env expression and by the inhibitory effects of known
       fusion-blocking monoclonal antibodies and pharmacological agents.
       Quantitative measurements indicated much higher sensitivity compared
       with analysis of syncytium formation. The assay was used to probe
       mechanisms of the cell type specificity for Env-CD4-mediated fusion. In
       agreement with known restrictions, cell fusion occurred only when CD4
       was expressed on a human cell type. Membrane vesicle transfer
       experiments indicated that CD4 initially produced in either human or
       nonhuman cells was functional when delivered to human cells, suggesting
       that the fusion deficiency with nonhuman cells was not associated with
       irreversible defects in CD4. We also demonstrated that the infectivity
       specificities of different human immunodeficiency virus type 1 isolates
       for peripheral blood lymphocytes versus continuous CD4+ cell lines were
       associated with corresponding fusion selectivities of the respective
       recombinant Env proteins. The assay enabled analysis of the fusogenic
       activity of the fusion glycoprotein/hemagglutinin-neuraminidase of the
       paramyxovirus simian virus 5. This system provides a powerful tool to
       study fusion mechanisms mediated by enveloped-virus glycoproteins, as
       well as to screen fusion-blocking antibodies and pharmacological agents.
 DE    Antigens, CD4/METABOLISM  *Cell Fusion  Cell Line  HIV Envelope Protein
       gp120/*PHYSIOLOGY  HIV-1/PHYSIOLOGY  HN Protein/PHYSIOLOGY
       Paramyxovirus/*PHYSIOLOGY  Recombinant Proteins  Support, U.S. Gov't,
       P.H.S.  Vaccinia Virus  Viral Envelope Proteins/PHYSIOLOGY  Viral Fusion
       Proteins/*PHYSIOLOGY  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).