title: Immunologic and Ultrastructural Characterization of HIV Pseudovirions Containing Gag and Env Precursor Proteins Engineered in Insect Cells
abstract: Expression of human immunodeficiency virus (HIV) Gag precursor protein (Pr55) by recombinant baculoviruses in insect cells results in the assembly and budding of Pr55 as virus-like particles, or Gag pseudovirions. The ultrastructural morphology, size, and sucrose sedimentation rate of Gag pseudovirions are indistinguishable from immature lentivirus particles produced by HIV-infected human cells. Recombinant baculoviruses were engineered to express individually Pr55 and HIV Env glycoprotein precursor (gp160). These recombinant baculoviruses were used to co-infect insect cells to produce chimeric HIV Gag pseudovirions containing gp160 in experiments to develop methodologies for producing complex noninfectious particulate vaccines for HIV. Coexpression of HIV Pr55 and gp160 resulted in the apparent incorporation of gp160 into Gag pseudovirions as determined by immunoblotting with envelope-specific monoclonal antibodies. Furthermore, results from indirect immunogold electron microscopy using monoclonal antibodies to HIV gp120, a component of the Env glycoprotein precursor, suggested that HIV gp160 was specifically incorporated during the budding process into the outer surface of chimeric Gag pseudovirions. Parallel labeling experiments to localize gp120 and Pr55 epitopes on HIV-infected H9 lymphocytes provided results similar to those obtained with chimeric Gag pseudovirions producing recombinant baculovirus-infected insect cells. Parameters influencing immunoelectron microscopy results in cell-surface and postembedding labeling experiments are discussed.
authors.affiliation: Laboratory of Cell and Molecular Structure, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, Maryland, 21702-1201
authors.initials: GJ
authors.lastname: Tobin
authors.initials: K
authors.lastname: Nagashima
authors.initials: MA
authors.lastname: Gonda