EXPRESSION OF Gag AND Gag-Pol OF BOVINE IMMUNODEFICIENCY VIRUS

Abstract

The gag and gag-pol genes of bovine immunodeficiency virus (BIV) were expressed and characterized in the baculovirus expression vector system. It was known that retrovirus gag gene products alone were capable of assembly and production of virus-like particles (VLPs) in mammalian cells. To determine if this was true for the BIV Gag precursor (Pr53) in the baculovirus system and to produce recombinant protein for the characterization of the BIV gag gene products, the gag gene of BIV was expressed in the baculovirus expression vector system. Since gag and pol are expressed from the same message through a frameshifting mechanism which permits production of Gag Pr53 or Gag,-Pol Pr174, the entire gag-pol gene segment of the BIV genome was also expressed in the baculovirus expression vector system to (1) determine if the frameshifting mechanism was functional in insect cells expressing the BIV gag-pol gene, (2) determine if mature VLPs would be produced by the inclusion of the pol gene products, and (3) characterize the pol gene products. The coding regions of BIV gag and gag-pol were excised from the full-length infectious molecular clone, BIV 106, and cloned into the baculovirus transfer vector, pVL941. The clones pVL941-BIVᶢᵃᶢ and pVL941-BIVᶢᵃᶢ˗ᵖᵒᶥ were used to produce recombinant baculoviruses AcNPV-BIVᶢᵃᶢ and AcNPV-BIVᶢᵃᶢ˗ᵖᵒᶥ, respectively. These constructs were used to infect Sf-9 cells for the production and characterization of BIV Gag and Gag-Pol proteins. Cells infected with AcNPV-BIVᶢᵃᶢ produced large quantities of Pr53 protein. In addition the expressed protein self-assembled into VLPs. The VLPs budded from the cell membrane into the media where they exhibited virus-like properties. The VLPs could be precipitated by polyethylene glycol and purified on a 20- to 60-percent (w/w) sucrose gradient. The baculovirus-produced VLPs and authentic BIV virus both band at the same sucrose density, 1.16 g/ml. The easily purified VLPs provided large amounts of relatively pure Pr53 protein, and in the absence of the viral protease, Pr53 was not processed. The purified Pr53 was used as an antigen in radioimmunoprecipitation, western blot, and enzyme-linked immunosorbent assays. Pr53 was also used to immunize animals for the production of Gag specific antiserum and for monoclonal antibody production. In contrast, cells infected with AcNPV-BIVᶢᵃᶢ˗ᵖᵒᶥ virus did not make VLPs, but did produce Pr53 and Pr174 and exhibited significant reverse transcriptase activity. This demonstrated that the frameshifting mechanism employed by BIV in eukaryotic cells was functional in Sf-9 insect cells and that the expressed Pol enzymatic proteins were biologically active. It is likely that the high concentration of BIV protease produced in the AcNPV-BIVᶢᵃᶢ˗ᵖᵒᶥ-infected insect cells contributed to the rapid processing of Gag and Gag-Pol into the mature viral proteins, thus inhibiting VLP formation.