title: Use of persistent infections with vaccinia virus recombinants to introduce alterations in foreign proteins: an application to HIV-1 env protein.
abstract: 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 domain, cannot form oligomers, accumulates intracellularly and cannot fuse CD4+ cells. In the persistently infected cells we have also found alterations in another heterologous protein-beta-galactosidase-a gene inserted in the same locus of VV as the env gene. This alteration 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.
authors.affiliation: Department of Cell and Molecular Biology, CSIC, Madrid, Spain. mesteban@samba.cmb.uam.es
authors.forename: M
authors.initials: M
authors.lastname: Esteban
authors.forename: J
authors.initials: J
authors.lastname: Ramón Rodríguez
authors.forename: V
authors.initials: V
authors.lastname: Jimenez
authors.forename: D
authors.initials: D
authors.lastname: Rodríguez
mesh_terms: D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis; D015967:Gene Expression Regulation, Viral / Q000276:immunology; D015686:Gene Products, env / Q000737:chemistry / Q000235:genetics*; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics*; D006801:Humans; D008213:Lymphocyte Activation; D016393:Lymphoma, B-Cell / Q000235:genetics / Q000821:virology; D051379:Mice; D011995:Recombination, Genetic; D012692:Serial Passage; D014407:Tumor Cells, Cultured; D014615:Vaccinia / Q000235:genetics* / Q000378:metabolism; D014616:Vaccinia virus / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
chemical_list: D000914:Antibodies, Viral; D015686:Gene Products, env