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 cyclindependent 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 HCMVinfected 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 -calpain, which resulted in rapid proteolysis of p21 cip1 . E64d inhibited the proteolysis of p21 cip1 catalyzed by either m-calpain or -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 -calpain or the endogenous calpain inhibitor calpastatin. The observed increase of calpain activity was consistent with the increases in intracellular free Ca 2؉ 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.Human cytomegalovirus (HCMV) infection is wides...

Human Cytomegalovirus pUL29/28 and pUL38 Repression of p53-Regulated p21CIP1 and Caspase 1 Promoters during Infection

“…Initially, p21CIP1 protein levels are maintained within infected cells up to 9 hpi (42, 71) and then begin to decrease. Regulation occurs at the levels of both transcriptional control and protein degradation (71). Here, we demonstrate that steady-state p21CIP1 protein expression is maintained in the absence of pUL29/28 ( Fig.…”

“…Expression of p21CIP1 was previously demonstrated to be tightly regulated during infection (71). p21CIP1 contributes to HCMV IE gene expression during infection of cells in G 2 /M (24).…”

Inhibition of Cell Division by the Human Cytomegalovirus IE86 Protein: Role of the p53 Pathway or Cyclin-Dependent Kinase 1/Cyclin B1

“…Previous reports have shown that HCMV infection of permissive cells increases the level of p53 and phospho-Ser 15 p53 (17,24,35,48,55), but the levels of p21 decreased (10,40). The IE86 protein of HCMV also increases the level of p53 and interacts with p53 (3,8,55,58).…”

“…However, HCMV infection affects p53 because the cellular protein is sequestered in the nucleus in viral replication centers (17). Although the viral IE86 protein stabilizes p53 and activates p21, other proteins specified by the virus must inactivate p53 because HCMV infection causes a decrease in p21 levels (10,40).…”

Mitochondrial Cell Death Suppressors Carried by Human and Murine Cytomegalovirus Confer Resistance to Proteasome Inhibitor-Induced Apoptosis

“…Calpains, in particular, are activated during HCMV infection and underlie modulation of the cell cycle regulator p21 cip1 (15). Although constitutive proteasome constituents remain unaltered or increase during infection, immunoproteasome subunits LMP2, MECL1, and LMP7 are specifically downmodulated (33).…”