Use of persistent infections with vaccinia virus recombinants to introduce alterations in foreign proteins: An application to HIV-1 env protein

With the aim of generating a virus-cell system to introduce alterations in proteins of interest—which may be of use in studies of their biological functions—we established a persistent infection on a B-lymphoma cell line (A20.2J) with vaccinia virus (VV) recombinants. As a model, we used a vaccinia virus recombinant expressing the human immunodeficiency virus HIV-1 env gene. In this unique virus-cell system, we found that it is possible to introduce several structural and functional alterations in the env protein with passage numbers. From passage 10–20, two new env products emerged: an uncleaved gp160 and a glycoprotein fragment of 110 kDa. The uncleaved gp160 exhibit interesting properties as an immunogen. This protein forms stable oligomers, is not released from the cells, cannot fuse CD4+ presenting HeLa cells and activates a stronger cellular immune response than the parental cleaved env. In contrast, the 110 kDa product is a poor immunogen, since it lacks the gp41 domains, cannot form oligomers, accumulates intracellularly and cannot fuse CD4+ cells. In the persistently infected cells we have also found alterations in another heterologous protein—β-galactosidase—a gene inserted in the same locus of VV as the env gene. This alteration resulted in a truncation of the (β-galactosidase protein from 125 kDa to about 70 kDa. A similar size truncation of env and of β-galactosidase was observed in many of the isolated VV recombinants.