Construction and characterization of a recombinant adenovirus vector carrying the human preproinsulin gene under the control of the metallothionein gene promoter.

A new adenovirus vector carrying human-preproinsulin (h-PPI) genomic DNA, which was placed under the control of the mouse metallothionein gene promoter, was constructed. In the recombinant virus-infected cells, h-PPI gene expression increased as a function of ZnSO4 concentration. Reversed-phase high-performance liquid chromatography analysis revealed that the recombinant adenovirus-infected cells secreted immature insulin containing proinsulin and incorrectly processed insulin. Tyrosyl phosphorylation of human insulin receptor substrate 1 occurred when HepG2 cells were treated with the cultured medium, indicating that the h-PPI gene product was functionally active in vitro. We also examined the biological activity of the product using diabetic severe combined immunodeficient mice and confirmed that the h-PPI gene product reduced the blood glucose concentration in vivo. This study suggests that the adenovirus vector can be used to express a foreign gene under the control of an external promoter in various human cells.

Purification and characterization of virus-like particles and pentamers produced by the expression of SV40 capsid proteins in insect cells.

Three capsid proteins of SV40 (VP1, VP2, and VP3) were expressed in insect cells using recombinant baculoviruses. When the VP1 capsid protein was expressed alone or co-expressed with VP2 and VP3, virus-like particles (VLP) were produced. In the latter case, the minor capsid proteins, VP2 and VP3, were incorporated into the VLP. VLPs with and without VP2 and VP3, and the wild type SV40 virions were indistinguishable under electron microscope. The sedimentation coefficient, S20,w' obtained for the VLP consisting of VP1 alone (VP1-VLP) was 170 S, and that for the VLP consisting of all of the capsid proteins (VP1/2/3-VLP) was 174 S. Treatment of the VP1-VLP with a calcium ion chelating agent and a reducing agent caused dissociation of the VP1-VLP. The dissociated and purified VP1 proteins were identified as pentamers of VP1 based on the molecular weight determination by sedimentation equilibrium. The pentamers were shown to possess the ability to re-assemble into VLP which had the S20,w of 141S. The results are discussed in relation to the morphogenesis of SV40.