Tumor necrosis factor (TNF) is a multifunctional immunoregulatory protein that is secreted by activated macrophages and is believed to have antiviral activities. We reported earlier that when mouse C3HA fibroblasts are infected with human adenoviruses, the 289R and 243R proteins encoded by region ElA render the cells susceptible to lysis by TNF, and a 14,700-molecular-weight protein (14.7K protein) encoded by region E3 protects the cells against lysis by TNF. We now report that the 19,000-molecular-weight (19K) (176R) protein encoded by the E1B transcription unit can protect human HEL-299 fibroblasts and human ME-180 cervical carcinoma cells against lysis by TNF. This was determined by infecting cells with adenovirus double mutants that lack region E3 and do or do not express the ElB-19K protein and by measuring cytolysis by using a short-term (18-h)-5Cr-release assay. Under these assay conditions, the-5Cr release was specific to TNF and was not a consequence of the cyt phenotype associated with ElB-19K protein-negative mutants. Also, by using virus double mutants that lack E3 in combination with other early regions, we found that ElA, the ElB-55K protein-encoding gene, E3, and E4 are not required to protect HEL-299 cells against TNF cytolysis. Three additional human cancer cell lines (HeLa, HCT8, and RC29) and a simian virus 40-transformed W138 cell line (VA-13) also required E1B for protection against TNF cytolysis, indicating that the ElB-19K protein is required to protect many if not all human cell types against lysis by TNF when infected by adenovirus. The ElB-19K protein was not able to protect six different adenovirus-infected mouse cell lines against TNF lysis, even though the protein was shown to be efficiently expressed in one of the cell lines. HEL-299 or ME-180 cells infected by a mutant that lacks the ElB-19K protein but retains region E3 were not lysed by TNF, indicating that one or more of the E3 proteins can protect these cells against TNF lysis in the absence of the ElB-19K protein. Thus, the E3-14.7K but not the EIB-19K protein can protect adenovirus-infected mouse cells against TNF cytolysis, whereas the ElB-19K protein as well as one or more of the E3 proteins can protect adenovirus-infected human cells against TNF cytolysis.

No abstract

No abstract

SV40 transformation of rodent fibroblasts generally produces cells that are highly sensitive to killing by activated macrophages. The cell line SV-COL-E8 (E8) is typical of SV40-transformed mouse fibroblasts in that it is readily lysed when exposed to activated macrophages. This killing is not due solely to TNF, because soluble TNF alone is incapable of lysing these cells. TNF is, however, necessary for lysis since antibodies to TNF will prevent macrophage-mediated lysis. Similarly, E8 is not sensitive to nitric oxide (NO); however, NO is also necessary for lysis since inhibition of NO generation (by coincubation with the arginine analogue NG-monomethyl-1-arginine) with Fe(II)) blocks lysis of E8 by activated macrophages. Cytolysis by macrophages is contact dependent, suggesting that the cell-associated TNF precursor may be involved in mediating cytolysis. However, transfected cell lines bearing cell-associated TNF precursor do not mediate killing of E8. Thus, killing of E8 either involves both TNF and NO in addition to a third, as yet unidentified, lytic mechanism, or killing requires the contact-dependent delivery of TNF and NO from the macrophage to its target.