Activation of the mitogen-activated protein kinase p38 by human cytomegalovirus infection through two distinct pathways: a novel mechanism for activation of p38

Recent evidence indicates activated mitogen-activated protein kinase (MAPK) p38 has a critical function in human cytomegalovirus (HCMV) viral DNA repl ication in infected human fibroblasts. To elucidate the mechanism of HCMV-m ediated p38 activation, we hale performed a detailed analysis of p38 activa tion and the kinases associated with this activation at different times pos tinfection. We demonstrate that p38 kinase activity is strongly increased f ollowing viral infection. Inhibition of this activity significantly inhibit ed HCMV-induced hyperphosphorylation of pRb and phosphorylation of heat sha ck protein 27, suggesting that p38 activation is invoked in virus-mediated changes in host cell metabolism throughout the course of infection, We then provide evidence that p38 activation is mediated by different mechanisms a t early times versus later times of infection. ht early times of infection (8 to 14 h postinfection [hpi]), when p38 activation is first observed, no significant activation of the three kinases which can directly phosphorylat e p38 (namely, MKK3, MKK6 and MKK4) is detected. Using vectors which expres s dominant negative proteins, we demonstrate that basal MKK6 kinase activit y is necessary for HCMV-mediated p38 activation at these early times of inf ection (12 hpi), Then,,ve use ATP depletion to show that at 12 hpi, HCMV: i nhibits dephosphorylation of activated p38. These two experiments suggest t hat HCMV activates p38 by inhibition of dephosphorylation of p38. In contra st to early times of infection, at later times of infection (48 to 72 hpi), increased MKK3/6, but not MKK4, activity is observed, These results indica te that at early times of HCMV infection, increased steady-state levels of activated p38 is mediated at least in part by inhibition of dephosphorylati on of p38, while at later times of infection p38 activation is due to incre ased activity of the upstream kinases MKK3 and MKK6. These findings indicat e that HCMV has dec-eloped multiple mechanisms to ensure activation of the MAPK p38, a kinase critical to viral infection.