Degradation of p21cip1 in cells productively infected with human cytomegalovirus

Human cytomegalovirus (HCMV) stimulates arrested cells to enter the cell cycle by activating cyclin-dependent kinases (Cdks), notably Cdk2, Several mechanisms are involved in the activation of Cdk2. HCMV causes a substantial increase in the abundance of cyclin E and stimulates translocation of Cdk2 from the cytoplasm to the nucleus. Further, the abundance of the Cdk inhibitors (CKIs) p21(cip1/waf1) (p21(cip1)) and p27(kip1) is substantially reduced, The activity of cyclin E/Cdk2 increases as levels of CKIs, particularly p21(cip1), fall. We have previously shown that these phenomena contribute to priming the cell for efficient replication of HCMV, In this study, the mechanisms responsible for the decrease in p21(cip1) levels after HCMV infection were investigated by measuring p21(cip1) RNA and protein levels in permissive human lung (LU) fibroblasts after HCMV infection. Northern blot analysis revealed that p21(cip1) RNA levels increased briefly at 3 h after HCMV infection and then decreased to their nadir at 24 h; thereafter, RNA levels increased to about 60% of the preinfection level. Western blot analysis demonstrated that the relative abundance of p21(cip1) protein roughly paralleled the observed changes in initial RNA levels; however, the final levels of protein were much lower than preinfection levels. After a transient increase at 3 h postinfection, p21(cip1) abundance declined sharply over the next 24 h and remained at a very low level through 96 h postinfection. The disparity between p21(cip1) RNA and protein levels suggested that the degradation of p21(cip1) might be affected in HCMV-infected tells. Treatment of HCMV-infected cells with MG132, an inhibitor of proteasome-mediated proteolysis, provided substantial protection of p21(cip1) in mock-infected cells, but MG132 was much less effective in protecting p21(cip1) in HCMV-infected cells. The addition of E64d or Z-Leu-Leu-H, each an inhibitor of calpain activity, to HCMV-infected cells substantially increased the abundance of p21(cip1) in a concentration-dependent manner. To verify that p21(cip1) was a substrate for calpain, purified recombinant p21(cip1) was incubated with either m-calpain or mu -calpain, which resulted in rapid proteolysis of p21(cip1). E64d inhibited the proteolysis of p21(cip1) catalyzed by either m-calpain or mu -calpain. Direct measurement of calpain activity in HCMV-infected LU cells indicated that HCMV infection induced a substantial and sustained increase in calpain activity, although there was no change in the abundance of either m- or mu -calpain or the endogenous calpain inhibitor calpastatin, The observed increase of calpain activity was consistent with the increases in intracellular free Ca2+ and phospholipid degradation in HCMV-infected LU cells reported previously from our laboratory. Considered together, these results suggest that the increase in calpain activity observed following HCMV infection contributes significantly to the reduction of p21(cip1) levels and the resultant cell cycle progression.