Clonal isolates of mouse 3T3 cells and primary rat embryo cells, recovered nonselectively after infection by simian virus 40 (SV40), have been tested for tumorigenicity in the immune-deficient nude mice in order to determine the cellular growth properties in vitro specifically correlated with neoplastic growth in vivo. In addition, mouse 3T3 cells transformed by murine sarcoma virus (MuSV, Kirsten strain), and revertants isolated from cells fully transformed by either SV40 or MuSV were also studied. Results suggest that the single cellular property consistently associated with tumorigenicity in nude mice is the acquisition by virus-transformed cells of the ability to proliferate in vitro in the absence of anchorage. Other cellular parameters of virusinduced transformation, such as lack of sensitivity to high cell density and the capacity to grow in low serum concentration, are dissociable from cellular tumorigenicity. This conclusion is supported further by the demonstration that specific selection in vivo for tumorigenic cells from anchorage-dependent cells results in the isolation of anchorage-independent cells. Conversely, a single-step selection in vitro for anchorage-independent cells from nontumorigenic cells results in a simultaneous selection of highly tumorigenic subclones. Infection of susceptible animal cells in vitro by tumor viruses usually results in a spectrum of stable alterations in cellular growth properties, as well as in the appearance of virus-specific antigens in the transformed cells (1). In particular, division in populations of untransformed cells is inhibited by any of the following three environmental constraints: extensive cell-cell contact (2), reduction of serum concentration (3, 4), or deprivation of a solid substrate for cell anchorage (5, 6).Recent results have demonstrated that cellular responses to the experimental parameters which differentiate the normal cell from its transformed counterpart are not coordinately controlled (7,8). Each constraint is the source of a selective assay that yields a different class of transformed cell line. Nonselective transformations of 3T3 mouse cells and of primary rat embryo cells by simian virus 40 (SV40) yielded lines displaying many different transformed phenotypes. While some lines were fully insensitive to each of the three constraints, most transformed lines lost only one or two of these constraints and remained normal for the others. Negative selection of revertant cell lines from a fully transformed 3T3 cell also dissociated these three parameters of growth control (9, 10).These observations suggested to us that not all of the altered cellular growth properties commonly associated with Abbreviations: SV40, simian virus 40; MuSV, murine sarcoma virus, Kirsten strain; RE, rat embryo; ME, mouse embryo. t Present address:
The human immunodeficiency virus transmembrane glycoprotein gp4l has at its amino terminus a strongly hydrophobic stretch of 28 amino acids flanked by a highly conserved series of polar amino acids. To investigate the role in syncytium formation of the hydrophobic amino terminus of gp4l and the polar border of this hydrophobic region, we introduced eight single-amino acid substitutions and one double-amino acid substitution in the amino-terminal 31 amino acids of gp4l. The mutant envelope glycoproteins were expressed from two distinct human immunodeficiency virus type 1 envelope glycoprotein expression vectors; the effects of the mutations on syncytium formation, envelope glycoprotein transport, secretion, and CD4 receptor-binding were analyzed. Results showed that polar substitutions throughout the hydrophobic amino terminus of gp4l greatly reduced or blocked syncytium formation mediated by the human immunodeficiency virus type 1 envelope glycoproteins, as did nonconservative mutations in the polar border of the hydrophobic amino terminus. Mutations at gp4l amino acids 15, 26, and 29 also significantly increased the extent of gpl20 secretion into the extracellular medium. None of the mutations detectably affected envelope glycoprotein processing or envelope glycoprotein binding to CD4.Enveloped viruses, under appropriate conditions, induce cell fusion, or syncytium formation, upon infection of permissive cell types. Early descriptions of such virus-induced cell fusion involved the orthomyxoviruses and paramyxoviruses (1-3), and one of the early retroviral plaque assays depended upon the ability of murine leukemia viruses to induce syncytium formation in the XC cell line (4). Later work indicated that expression of the orthomyxovirus envelope proteins was necessary and sufficient to induce cell fusion (5). More recently, syncytium formation has been described as a characteristic of human immunodeficiency virus (HIV) infection of CD4+ cells, and, as demonstrated for the orthomyxoviruses and paramyxoviruses (3), the envelope glycoproteins have been shown to be responsible for this cell-fusion property (6, 7). The syncytium formation induced by HIV leads to cell death in culture and may contribute to the depletion of CD4+ helper T cells in AIDS patients (6-10). Membrane fusion is also an essential step in the infection process (11).HIV envelope glycoproteins, like those of other retroviruses, are synthesized as a polyprotein precursor that is cleaved to generate the surface and transmembrane (TM) envelope proteins (12,13 (17,18). Because of their role in fusion, the hydrophobic amino-terminal regions of the orthomyxovirus and paramyxovirus TM proteins were termed "fusion peptides." Several studies suggested that the analogous hydrophobic regions at the amino termini of retroviral TM envelope proteins were also involved in the cell-fusion properties of these proteins. Linker insertion mutations introduced after AA 10 of the HIV-1 gp41 glycoprotein blocked cell fusion (19), as did point mutations at positions...
The envelope glycoproteins of the human immunodeficiency virus (HIV) type 1 are synthesized as a precursor molecule, gpl60, which is cleaved to generate the two mature envelope glycoproteins, gp120 and gp4l. The cleavage reaction, which is mediated by a host protease, occurs at a sequence highly conserved in retroviral envelope glycoprotein precursors. We have investigated the sequence requirements for this cleavage reaction by introducing four single-amino-acid changes into the glutamic acid-lysine-arginine sequence immediately amino terminal to the site of cleavage. We have also examined the effects of these mutations on the syncytium formation induced by HIV envelope glycoproteins. Our results indicate that a glutamic acid to glycine change at gpl20 amino acid 516, a lysine to isoleucine change at amino acid 517, and an arginine to lysine change at amino acid 518 affect neither gpl60 cleavage nor syncytium formation. The results obtained with the arginine to lysine change at amino acid 518 differ significantly from the results obtained with the same mutation at the envelope precursor cleavage site of a murine leukemia virus (E. 0. Freed, and R. Risser, J. Virol. 61:2852-2856, 1987). An arginine to threonine mutation at gpl20 amino acid 518, the terminal residue of gpl20, abolishes both gpl60 cleavage and syncytium formation. These findings demonstrate that despite its highly conserved nature, the basic pair of amino acids at the site of gpl60 cleavage is not absolutely required for proper envelope glycoprotein processing. This report also supports the idea that cleavage of gpl60 is required for activation of the HIV envelope fusion function.
The V3 loop, located near the middle of the surface envelope glycoprotein gpl20, is the major neutralizing domain of human immunodeficiency virus type 1 (HIV-1). Although the majority of the V3 loop is highly variable between different strains of HIV-1, a Gly-Pro-Gly-Arg motif at the tip of the loop is highly conserved. To determine whether this region plays a role in fusion mediated by the HIV-1 envelope glycoproteins, we introduced seven single-amino-acid changes in the V3 loop. The mutant envelope glycoproteins were expressed from an HIV-1 envelope expression vector and analyzed for their ability to induce cell fusion in the absence of virus replication. Our results indicated that single-amino-acid changes in the V3 loop were capable of completely abolishing or greatly reducing the ability of the HIV-1 envelope glycoproteins to induce cell fusion, thereby identifying the V3 loop as a fusion domain of HIV-1. Mutations in the highly conserved tip of the loop or in a nonconserved region flanking the highly conserved tip had no effect on envelope glycoprotein synthesis, processing, transport, or binding to the CD4 receptor molecule. Mutation of the putative disulfide bridgeforming Cys at residue 336 blocked gpl60 cleavage and CD4 binding.
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