12th International AIDS Conference Geneva, Switzerland - June 28-July 3, 1998

Int Conf AIDS 1998 Jun 28-Jul 3; 12:9 (abstract no. 11136)

Saman E, Ottevaere I, Vanden Haesevelde M, Cornelissen M, Devos K;;; Innogenetics, Gent, Belgium.

OBJECTIVE: Expression of the HIV-1 gag gene in a Baculovirus insect system to generate particles which resemble the native viral core structure. Physical and immunochemical characterization of these structures.

METHODS: The gag gene of the HIV-1 isolate Ant166 was introduced in the baculovirus expression vector pAcUW51 under the control of the polyhedrin promoter. This construct was introduced into the baculovirus genome via homologous recombination and the recombinant virus was used to infect Sf9 cells in different culture media. The culture supernatant and the cell lysate were analysed for the presence of high molecular weight particles by sucrose gradient centrifugation, western blot and p24 capturing ELISA. Budding of the particles was visualized by transmission electron microscopy of infected cells. The stability of the particles was investigated by treatment with different detergents.

RESULTS: Sucrose gradient (20-60% sucrose) analysis of the Sf9 cell culture supernatant revealed the presence of high density material (1.15 g/cm3) reactive in a p24 capturing ELISA. When cells were grown in serum containing medium, a broad distribution of the p24 containing material was seen over the gradient whereas in serum free medium a sharp and symmetrical antigen profile was found at 1.15 g/cm3. Treatment of the high density material with SDS or nonionic detergents (e.g. NP40) resulted in disappearance of the high density antigen peak, indicating the disruption of the particulate structure of the antigen. Electron microscopy confirmed the presence of budding particles at the surface of the infected Sf9 cells, giving additional evidence for the presence of highly ordered CLP.

CONCLUSIONS: Production of the HIV-1 gag precursor in Sf9 insect cells generates CLP which bud from the cell membrane into the culture medium. Treatment of this material with NP40 detergent disrupts these structures, which is in contrast to published findings. The relative lower stability of our CLP may be attributed to a point mutation in the N-terminal part of the matrix protein. This possibility is being investigated.

Copyright © 1998 - International AIDS Society (IAS). Reproduction of this abstract (other than one copy for personal reference) must be cleared through the IAS.