John M. Routes

The expression of the Adenovirus serotype 5 (Ad5) E1A oncogene sensitizes tumor cells to natural killer (NK) cell–mediated killing and tumor rejection in vivo. These effects are dependent on the ability of E1A to bind the transcriptional coadaptor protein p300. To test the hypothesis that E1A up-regulates ligands recognized by the NKG2D-activating receptor, we stably transfected the highly tumorigenic mouse fibrosarcoma cell line MCA-205 with Ad5-E1A or a mutant form of E1A that does not interact with p300 (E1A-Δp300). Ad5-E1A, but not E1A-Δp300, up-regulated the expression of the NKG2D ligand retinoic acid early inducible (RAE)-1, but not murine ULBP-like transcript 1, another NKG2D ligand, in four independently derived MCA-205 transfectants. The up-regulation of RAE-1 by E1A targeted MCA-205 tumor cells to lysis by NK cells, resulting in NKG2D-dependent tumor rejection in vivo. Moreover, the up-regulation of NKG2D ligands by E1A was not limited to mouse tumor cells, as E1A also increased the expression of NKG2D ligands on primary baby mouse kidney cells, human MB435S breast cancer cells, and human H4 fibrosarcoma cells.

Patients who have common variable immunodeficiency (CVID) and granulomatous/lymphocytic interstitial lung disease (GLILD) are at high risk for early mortality and B cell lymphomas. Infection with human herpes virus type 8 (HHV8), a B cell lymphotrophic virus, is linked to lymphoproliferative disorders in people who have secondary immunodeficiencies. Therefore, we determined the prevalence of HHV8 infection in CVID patients with GLILD. Genomic DNA isolated from peripheral blood mononuclear cells was screened by nested- and real time-quantitative PCR (QRT-PCR) for the presence of HHV8 genome. It was positive in 6/9 CVID patients with GLILD (CVID-GLILD), 1/21 CVID patients without GLILD (CVID-control), and no patients receiving intravenous gamma globulin (n = 13) or normal blood donors (n = 20). Immunohistochemistry (IHC) demonstrated expression of the latency-associated nuclear antigen-1 (LANA-1) in the biopsies of the lung, liver, and bone marrow of four patients with CVID-GLILD. One CVID-GLILD patient developed a B cell lymphoma during the course of the study. QRT-PCR demonstrated high copy number of HHV8 genome and IHC showed diffuse staining for LANA-1 in the malignant lymph node. HHV8 infection may be an important factor in the pathogenesis of the interstitial lung disease and lymphoproliferative disorders in patients with CVID.

The expression of adenovirus serotype 2 or 5 (Ad2/5) E1A sensitizes cells to killing by NK cells and activated macrophages, a property that correlates with the ability of E1A to bind the transcriptional coadaptor proteins p300-CBP. The E6 oncoproteins derived from the high-risk human papillomaviruses (HPV) interact with p300 and can complement mutant forms of E1A that cannot interact with p300 to induce cellular immortalization. Therefore, we determined if HPV type 16 (HPV16) E6 could sensitize cells to killing by macrophages and NK cells. HPV16 E6 expression sensitized human (H4 and C33A) and murine (MCA-102) cell lines to lysis by macrophages but not by NK cells. The lysis of cells that expressed E6 by macrophages was p53 independent but dependent on the production of tumor necrosis factor alpha (TNF-α) or nitric oxide (NO) by macrophages. Unlike cytolysis assays with macrophages, E6 expression did not significantly sensitize cells to lysis by the direct addition of NO or TNF-α. Like E1A, E6 has been reported to sensitize cells to lysis by TNF-α by inhibiting the TNF-α-induced activation of NF-κB. We found that E1A, but not E6, blocked the TNF-α-induced activation of NF-κB, an activity that correlated with E1A-p300 binding. In summary, Ad5 E1A and HPV16 E6 sensitized cells to lysis by macrophages. Unlike E1A, E6 did not block the ability of TNF-α to activate NF-κB or sensitize cells to lysis by NK cells, TNF-α, or NO. Thus, there appears to be a spectrum of common and unique biological activities that result as a consequence of the interaction of E6 or E1A with p300-CBP.

Adenovirus (Ad) E1A and human papillomavirus (HPV) E7 express homologous conserved regions (CRs) that mediate their shared biological functions. Despite their similarities, the expression of E1A sensitizes tumor cells to killing by NK cells and macrophages but the expression of E7 does not, a factor that may contribute to the dissimilar oncogenicities of Ad and HPV. This study was undertaken to define molecular differences between E1A and E7 that are responsible for the ability of E1A and the inability of E7 to sensitize cells to killing by NK cells and macrophages. Genetic mapping studies using human fibrosarcoma cells (H4) that stably expressed mutant forms of E1A showed that only those forms of E1A that interacted with the transcriptional coadaptor protein p300 sensitized cells to killing by NK cells and macrophages. E7 lacks the N-terminal p300-binding region present in E1A. Therefore, a chimeric E1A/E7 gene was constructed that included the N terminus and the CR1 (p300-binding) domain of E1A fused to CR2 and the C-terminal sequences of E7. The E1A/E7 protein interacted with p300 and pRb and immortalized primary mouse embryo fibroblasts (MEF). The expression of E1A/E7 sensitized H4 and MEF cells to killing by activated macrophages but not to killing by NK cells. Therefore, N-terminal differences between E1A and E7 that map to the E1A-p300 binding region accounted for differences in their abilities to sensitize cells to killing by macrophages. However, regions in addition to the E1A-p300 binding region are required to sensitize cells to killing by NK cells.

Expression of the adenovirus serotype 5 (Ad5) E1A oncogene sensitizes cells to apoptosis by TNF-α and Fas-ligand. Because TNF-related apoptosis-inducing ligand (TRAIL) kills cells in a similar manner as TNF-α and Fas ligand, we asked whether E1A expression might sensitize cells to lysis by TRAIL. To test this hypothesis, we examined TRAIL-induced killing of human melanoma (A2058) or fibrosarcoma (H4) cells that expressed E1A following either infection with Ad5 or stable transfection with Ad5-E1A. E1A-transfected A2058 (A2058-E1A) or H4 (H4-E1A) cells were highly sensitive to TRAIL-induced killing, but Ad5-infected cells expressing equally high levels of E1A protein remained resistant to TRAIL. Infection of A2058-E1A cells with Ad5 reduced their sensitivity to TRAIL-dependent killing. Therefore, viral gene products expressed following infection with Ad5 inhibited the sensitivity to TRAIL-induced killing conferred by transfection with E1A. E1B and E3 gene products have been shown to inhibit TNF-α- and Fas-dependent killing. The effect of these gene products on TRAIL-dependent killing was examined by using Ad5-mutants that did not express either the E3 (H5dl327) or E1B-19K (H5dl250) coding regions. A2058 cells infected with H5dl327 were susceptible to TRAIL-dependent killing. Furthermore, TRAIL-dependent killing of A2058-E1A cells was not inhibited by infection with H5dl327. Infection with H5dl250 sensitized A2058 cells to TRAIL-induced killing, but considerably less than H5dl327-infection. In summary, expression of Ad5-E1A gene products sensitizes cells to TRAIL-dependent killing, whereas E3 gene products, and to a lesser extent E1B-19K, inhibit this effect.