The envelope protein of Ebola virus has been shown to mediate cell rounding and detachment in vitro and in vivo and has been implicated in the pathogenicity of Ebola virus infection (41
). Ebola virus GP cytotoxicity correlates with the downmodulation of several surface proteins important in adhesion and signaling, including certain integrins and EGFR (40
). It is also dependent on recycling and endocytosis mediated by the cytoskeletal modulator dynamin (41
). Although GP expression induces changes in cell surface protein levels, the mechanisms of surface protein downmodulation and cytotoxicity, as well as the cellular signaling events that mediate these effects, have not been defined. This study has analyzed the activation of several proteins that are pivotal to normal cell signaling. Ebola virus GP significantly decreased the activation of ERK2, and this effect was dependent on the mucin-like domain of the glycoprotein, which is also required for cellular cytotoxicity (40
). Inhibition of ERK2 activity, either through the overexpression of a catalytically inactive dominant negative form of ERK1/2 or through specific knockdown of endogenous ERK2, enhanced GP-induced αV integrin downregulation. Conversely, expression of a constitutively active ERK2 significantly decreased αV integrin downmodulation. Together, these data demonstrate that GP mediates its effect on cellular viability and integrin expression at least in part through ERK2 activity.
Cell adhesion is essential for proper cellular gene expression, growth, differentiation, and survival (19
). Integrins are pivotal to cell-cell and cell-matrix adhesion. Previously appreciated primarily for their role in the maintenance of adhesion, they are now well recognized as signaling receptors that regulate the cellular response to mitogens and intersect other signaling networks such as the ERK/MAPK cascade (22
). Several studies have shown that there is strong and sustained ERK activity in adherent cells (reviewed in references 4
). In contrast, endothelial cells treated with integrin αVβ3 agonists in vivo show diminished long-term ERK activation (15
). GP expression substantially reduced surface αV integrin levels as well as a loss of cell adherence. It may be that the observed GP-induced reduction in ERK activity is an upstream signaling event which subsequently leads to surface integrin downmodulation. However, given the importance of cell adhesion and integrins in the regulation of the ERK pathway, it is likely that the diminished ERK signaling is downstream of GP-induced cell rounding and detachment. This effect was most pronounced in ERK2 and was less obvious in other mitogen-activated kinases such as JNK and p38, which are not regulated by integrin-mediated adhesion (3
Our results suggest that GP affects the catalytic activity of the ERK2 protein. Given the 80% sequence homology between ERK1 and ERK2 (10
) and their similar modes of regulation, the preferential effect on ERK2 was not anticipated; however, several studies have shown more recently that despite their sequence homology, these two proteins have unique properties (16
). For instance, although the proteins are coexpressed in virtually all tissues, their relative abundances vary widely. In addition, ERK1-deficient mice have a different, significantly milder phenotype than ERK2-deficient mice, which die early in development (20
). These recent findings suggest that ERK1 and ERK2 are not redundant proteins and that they have individual functions in cell signaling. In addition, the results presented here show a similar effect, to a lesser degree, on ERK1, Thus, the finding that GP expression has a more pronounced effect on ERK2 activity is consistent with the current understanding of ERK1 and ERK2 function.
While this study demonstrates that GP expression reduces ERK2 phosphorylation and activation, the data also provide evidence that increasing or decreasing active ERK2 levels inversely affects GP-induced rounding and detachment. While the events that ensue to induce cell death are unknown, ERK2 is a pivotal protein within the ERK/MAPK signaling cascade, and it is likely that downstream substrates of ERK2 mediate GP cytotoxicity. Once activated, the ubiquitously expressed ERK1 and ERK2 MAPKs modify a diverse array of substrates that relay signals mediating critical cellular responses (reviewed in reference 48
). They phosphorylate approximately 160 proteins with substantial regulatory functions, including other protein kinases, transcription factors, cytoskeletal proteins, and other enzymes (10
). Reduced ERK2 activity during GP expression may lead to altered activity of one of these diverse substrates, thereby increasing cell rounding and detachment. The results from experiments utilizing constitutively active ERK2 indicate that at least one important substrate most likely localizes to the nucleus. The expression of a nuclear form of constitutively activated ERK2 decreased cell rounding and detachment, an effect not observed with a cytoplasmic version of active ERK2. A study by Robinson et al. demonstrated that certain functions of ERK2 are dependent on the nuclear localization of the active enzyme (32
). In addition, it has been shown that adhesion to the extracellular matrix is required for efficient accumulation of activated ERK in the nucleus (4
). This finding suggests that the loss of adherence in GP-expressing cells decreases active and nuclear ERK2 levels. Substrates of nuclear ERK2 include several transcription factors which regulate the expression of a large array of genes involved in diverse cellular processes and could affect cell viability. At the same time, we cannot exclude the possibility that a cytoplasmic protein, such as a cytoskeletal substrate, may be the target of phosphorylation. Indeed, one possible cytoplasmic target of ERK2 is dynamin, an important mediator of membrane internalization and integrin receptor endocytosis (12
). Previous results have shown that a dominant negative version of this protein reduces GP-induced integrin downmodulation (41
). However, we were unable to demonstrate an interaction between these two proteins (data not shown), suggesting that the observed effects of ERK activity on GP cytotoxicity are not mediated by dynamin.
Several viruses have been shown to modulate the ERK/MAPK signaling cascade, including influenza virus (31
), borna disease virus (30
), coxsackievirus (26
), visna virus (6
), human immunodeficiency virus (HIV) (28
), vaccinia virus (13
), Epstein-Barr virus (17
), cytomegalovirus (33
), and human herpesvirus 8 (1
). These viruses stimulate the activation of this pathway, which results in efficient cell cycle promotion as well as high cellular and viral gene production. Fewer studies have demonstrated a negative correlation between ERK activity and viral protein expression such as we have described. One such study demonstrated that the hepatitis C virus nonstructural NS5A protein inhibits EGF-stimulated activation of the ERK pathway by inhibiting EGFR complex formation (27
). A recent study by Yoshizuka et al. reported that the expression of the HIV type 1 (HIV-1) Vpr protein is associated with the downregulation of genes in the ERK/MAPK pathway and with decreased phosphorylation of ERK1/2 (49
). Vpr is associated with the induction of a G2
/M cell cycle arrest. That study suggests that an alternative mechanism of HIV-1 Vpr-induced cell cycle arrest is the Vpr-associated decrease in ERK1/2 activation, and the authors speculate that this function is important for HIV replication and pathogenesis. Currently, little is known about the effects of Ebola virus GP expression on cell cycle progression. The findings with HIV-1 Vpr, along with the known regulatory effects of adhesion and ERK activity on the activation of cyclin-dependent kinases, which regulate the cell cycle (5
), raise this possibility.
The results of this study demonstrate that Ebola virus GP reduces the phosphorylation and activation of the signaling molecule ERK2 and that reduction of ERK2 activity further enhances GP-induced αV integrin downmodulation, a correlate of cytopathicity. The observation that both GP-mediated cytotoxicity and ERK2 dephosphorylation are dependent on the mucin-like domain of GP further suggests that these processes may be intimately related. This is the first evidence of a GP-induced perturbation on a specific cellular signaling cascade. Future analysis of the activity of this signaling cascade during live viral infection may indicate an important role for this pathway not only in GP-mediated cytotoxicity but also in Ebola virus pathogenesis as well. A more complete understanding of the mechanisms involved in this phenomenon may facilitate the development of therapies for Ebola virus infection, which are currently unavailable.