Budding and secretion of HIV Gag–Env virus-like particles from recombinant human adenovirus infected cells

Abstract

We have characterized the assembly, budding and extra-cellular release of human immunodeficiency virus (HIV) Gag–Env virus-like particles (VLPs) from human embryonic kidney cells (293 cells expressing the E1a protein of adenovirus) infected with recombinant replication-defective human adenovirus type 5. Recombinant human adenovirus vectors expressing the chimeric Gag–Env protein were constructed by inserting the gag– env fusion gene into the E1a region of the human adenovirus type 5. Biochemical and immunological analyses of VLPs recovered from the culture supernatant revealed that these particles contain the HIV-2 Gag protein and segments of Env protein from the HIV-1 gp120. This chimeric Gag–Env protein interacted with HIV-1 positive patient sera and with HIV-2 Gag monoclonal antibody. Immunoelectron microscopy of the 293 cells infected with the recombinant adenoviruses showed that the HIV Gag–Env antigen is present in the VLPs. Thin-section electron microscopy (EM) revealed that the Gag–Env VLPs bud through the cytoplasmic membrane, as well as through membranes of intracellular vacuoles. The thin-section EM showed that the Gag–Env VLPs have a spherical morphology with an electron-dense ring. The size of VLPs range from 110 to 140 nm in diameter, which is slightly larger than that of the Gag particles without Env protein fusion. Mice immunized with recombinant adenoviruses generated antibodies that specifically reacted with Gag–Env chimeric proteins. Our results support the idea that the replication-defective adenovirus could be used to induce immune responses that might be useful in a vaccine against HIV/AIDS.