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 alteration s in proteins of interest-which may be of use in studies of their biol ogical 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 immunodefic iency virus HIV-1 env gene. In this unique virus-cell system, we found that it is possible to introduce several structural and functional al terations in the enu protein with passage numbers. From passage 10-20, two new enu products emerged: an uncleaved gp160 and a glycoprotein f ragment 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 activate s a stronger cellular immune response than the parental cleaved env. I n contrast, the 110 kDa product is a poor immunogen, since it lacks th e gp41 domain, cannot form oligomers, accumulates intracellularly and cannot fuse CD4(+) cells. In the persistently infected cells we have a lso found alterations in another heterologous protein-beta-galactosida se-a gene inserted in the same locus of VV as the env gene. This alter ation resulted in a truncation of the (beta-galactosidase protein from 125 kDa to about 70 kDa. A similar size truncation of env and of beta -galactosidase was observed in many of the isolated VV recombinants. C opyright (C) 1996 Elsevier Science B.V.