Recognition of adenovirus E1A gene products on immortalized cell surfaces by cytotoxic T lymphocytes.

The experiments described in this report were designed to examine whether target cells transfected with the adenovirus E1A gene and exhibiting increased susceptibility to lysis by natural killer cells and activated macrophages (J. L. Cook, T. A. Walker, A. M. Lewis, Jr., H. E. Ruley, F. L. Graham, and S. H. Pilder, Proc. Natl. Acad. Sci. USA 83:6965-6969, 1986) also express E1A proteins on their surfaces. MT1A, 12S, and 13S are strain Fischer baby rat kidney (BRK) cell lines immortalized by transfection with plasmids containing only the E1A gene of nononcogenic adenovirus. All of these cell lines were effective in stimulating the generation of cytotoxic T lymphocytes (CTL) in vitro, provided that the cultures were supplemented with an exogenous source of lymphokine and that the responding lymphocytes were from syngeneic Fischer rats previously immunized with a cell line containing the intact E1A gene. HrA2, a Fischer BRK cell line immortalized by transfection with a plasmid containing only exon 1 of the E1A gene, did not generate, nor was it lysed by, E1A-specific CTL. The cytolytic activity of E1A-specific CTL was blocked by antiserum from Fischer rats immunized with purified E1A proteins synthesized in Escherichia coli, supporting the conclusion that an epitope on E1A proteins encoded by the intact E1A gene constitutes part of the CTL target structure on adenovirus-transformed cells. These data suggest that in addition to their functions within host cells, E1A gene products are important immunogenic determinants on the surfaces of adenovirus-transformed cells.     

Lytic and transforming functions of individual products of the adenovirus E1A gene.

To distinguish the individual roles of the 13S, 12S, and 9S adenovirus E1A gene products, we isolated the corresponding cDNA clones and recombined them into both plasmids and viruses. Only the expected E1A mRNA products were made from the corresponding 12S and 13S viruses. The 9S mRNA was detected when the 9S virus was coinfected with the 13S virus but not when either virus was infected alone. The 13S virus formed plaques equally well in 293 cells, HeLa cells, and A549 cells, a human lung oat cell carcinoma line. Plaque titers of the 12S virus were much reduced in HeLa and A549 cells compared with 293 cells, although the 12S virus is multiplicity-dependent leaky in both HeLa and A549 cells. A549 cells were significantly more permissive than HeLa cells for growth of the 12S virus. In A549 cells even at low multiplicities of infection the final yield of 12S virus eventually approached the maximum yield from 293 cells. Expression from the adenovirus early region 2 and early region 3 promoters in HeLa cells was activated in the presence of a 13S cDNA E1A region but not in the presence of a 12S E1A cDNA region. Although defective for lytic growth in HeLa cells, the 12S virus immortalized BRK cells at very high efficiency, whereas infection of these cells with 13S virus, as with wild-type E1A virus, resulted mainly in cell death. The 13S product does have an immortalization function, however, revealed in the absence of adenovirus lytic functions when a plasmid containing the E1A 13S cDNA region was transfected into BRK cells. The 9S virus failed to immortalize infected BRK cells or to interfere with focus formation when coinfected with the 12S virus.     

Expression of adenovirus E1a and E1b gene products and the Escherichia coli XGPRT gene in KB cells

The recombinant plasmid pSV2-gpt, which contains the Escherichia coli XGPRT gene under the control of a simian virus 40 early promoter, was modified to contain the type 2 adenovirus (Ad2) XhoI-C (0 to 15.5 map units) restriction endonuclease fragment. Plasmid (pLB206) DNA was introduced into human KB cells by Ca2+-mediated DNA transfection, and transformants were selected in medium containing xanthine, aminopterin, and mycophenolic acid, as a consequence of expression of the dominant, selectable XGPRT gene. A series of 13 gpt+ cell lines were isolated and tested for their ability to complement Ad5 deletion mutants in E1a (H5dl312) and E1b (H5dl315). Four classes of gpt+ KB cell lines were identified, including clones constitutively expressing both E1a and E1b, only E1a, or only E1b or not expressing either E1a or E1b. DNA and RNA filter transfer hybridization analysis substantiated the conclusions that those cell lines capable of complementing viral host range mutants contained the appropriate viral DNA sequences and cytoplasmic polyadenylated RNA species. DNA filter transfer hybridization studies also revealed that the transfected vector DNA was stably integrated into chromosomal DNA in the KB transformants and the number of integrated sites ranged from 1 to 3. The gpt+ KB cell line that only expressed E1b gene functions only contained viral E1b gene sequences; those cell lines that expressed neither E1a nor E1b gene function contained only small or no regions of Ad2 DNA. When weaned off the selective medium, transformed KB cell lines stably maintained their inserted DNA in the absence of selective pressure and could easily be adapted to growth in suspension culture.     

Control functions of adenovirus transformation region E1A gene products in rat and human cells.

Altered control of the rat cell cycle induced by adenovirus requires expression of transformation region E1A, but not of E1B, E2A, E2B, or late genes. We show here that neither E3 nor E4 is required, so the effect results directly from an E1A product. Mutants with defects in the 289-amino-acid (aa) E1A product had little or no effect on the rat cell cycle even at 1,000 IU per cell. A mutant (pm975) lacking the 243-aa E1A product altered cell cycle progression, but less efficiently than did wild-type virus. The 289-aa E1A protein is therefore essential for cell cycle effects; the 243-aa protein is also necessary for the full effect but cannot act alone. Mutants with altered 289-aa E1A proteins showed different extents of leak expression of viral early region E2A as the multiplicity was increased; each leaked more in human than in rat cells. dl312, with no E1A products, failed to produce E2A mRNA or protein at 1,000 IU per cell in rat cells but did so in some experiments in human cells. There appears to be a very strict dependence of viral early gene expression on E1A in rat cells, whereas dependence on E1A is more relaxed in HeLa cells, perhaps due to a cellular E1A-like function. Altered cell cycle control is more dependent on E1A function than is early viral gene expression.     

Ubiquitin-dependent degradation of adenovirus E1A protein is inhibited by BS69

Adenovirus E1A protein perturbs the cell cycle and promotes cell transformation. Although E1A is relatively unstable, regulation of E1A stability has not been fully elucidated. Here, we showed that E1A was ubiquitinated and degraded using a proteasome in vivo system. Interestingly, we found that BS69, one of the E1A-binding proteins, inhibited ubiquitination of E1A. BS69 mutants lacking the MYND domain could not bind to E1A and did not inhibit ubiquitination of E1A. Moreover, we demonstrated that overexpression of BS69 stabilized E1A in vivo. These results suggest that BS69 controls E1A stability via inhibition of ubiquitination.