Degradation of p21 cip1 in Cells Productively Infected with Human Cytomegalovirus

Abstract

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 cells. 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 μ-calpain, which resulted in rapid proteolysis of p21^cip1. E64d inhibited the proteolysis of p21^cip1 catalyzed by either m-calpain or μ-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 μ-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.