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The adenovirus E3-14.7K protein is a general inhibitor of tumor necrosis factor-mediated cytolysis
L R Gooding1, I O Sofola, A E Tollefson
1Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322.
Abstract:
We have previously described a 14,700 m.w. protein (14.7K) encoded by the E3 region of adenovirus that prevents TNF-mediated cytolysis of adenovirus-infected C3HA mouse fibroblasts. In the studies described here we have extended our analysis of TNF cytolysis of C3HA cells and the circumstances under which 14.7K protects these cells from cytolysis. C3HA cells were killed by TNF in the presence of inhibitors of protein synthesis, in the presence of cytochalasin E (which disrupts the microfilaments), and when adenovirus E1A was expressed. As described for other cell types, pretreatment of C3HA cells with TNF prevented cytolysis by TNF plus cycloheximide or TNF plus cytochalasin E, indicating that TNF induces a response that protects against these treatments. Remarkably, when 14.7K was expressed in virus-infected cells, it also prevented TNF-induced lysis whether sensitivity to TNF was induced by inhibition of protein synthesis, disruption of the cytoskeleton by cytochalasin E, or expression of adenovirus E1A. The 14.7K protein also prevented TNF lysis of cells that are spontaneously sensitive to TNF lysis. Thus, 14.7K appears to be a general inhibitor of TNF cytolysis, and as such should be an important tool in unraveling the mechanism of TNF cytolysis. There was one exception; NCTC-929 cells were spontaneously sensitive to TNF lysis and that lysis was not affected by 14.7K even though the protein was made in large quantities and was metabolically stable in these cells. This suggests that there is heterogeneity among TNF-sensitive cell lines. The 14.7K protein was found in both the nuclear and cytosol fractions of TNF resistant as well as all spontaneously sensitive cells suggesting that 14.7K may have more than one site of action within the cell.
Insights
Adenovirus E3 region's 14.7K protein broadly inhibits tumor necrosis factor (TNF)-mediated cell death in various conditions. This finding offers a new tool for studying TNF cytolysis mechanisms.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Tumor necrosis factor (TNF) induces cell death (cytolysis) in various cell types.
- Adenovirus encodes a 14.7K protein that previously showed protection against TNF-mediated cytolysis in infected cells.
- The exact mechanisms and scope of 14.7K protein's protective effects against TNF cytolysis require further investigation.
Purpose of the Study:
- To investigate the circumstances under which the adenovirus 14.7K protein protects C3HA cells from TNF-mediated cytolysis.
- To determine if 14.7K protein acts as a general inhibitor of TNF cytolysis across different cellular conditions.
- To explore potential heterogeneity in TNF-sensitive cell lines and the cellular localization of the 14.7K protein.
Main Methods:
- Utilized C3HA mouse fibroblasts and NCTC-929 cells.
- Induced TNF-mediated cytolysis using TNF alone or in combination with protein synthesis inhibitors (cycloheximide) or cytoskeleton disruptors (cytochalasin E).
- Expressed adenovirus E1A and the 14.7K protein in cells to assess their impact on TNF sensitivity and lysis.
Main Results:
- Adenovirus 14.7K protein prevented TNF-induced lysis in C3HA cells, even when sensitivity was enhanced by protein synthesis inhibition, cytoskeleton disruption, or E1A expression.
- The 14.7K protein also inhibited TNF lysis in spontaneously sensitive cells.
- NCTC-929 cells, despite expressing 14.7K protein, remained sensitive to TNF lysis, indicating cell-line heterogeneity.
- 14.7K protein was detected in both nuclear and cytosol fractions of resistant and sensitive cells.
Conclusions:
- The adenovirus 14.7K protein functions as a general inhibitor of TNF-mediated cytolysis across various cellular stresses.
- Cellular heterogeneity exists among TNF-sensitive cell lines, as demonstrated by NCTC-929 cells.
- The dual localization of 14.7K protein suggests potential multiple sites of action within the cell, aiding in the elucidation of TNF cytolysis mechanisms.