Stabilization of p53 in Human Cytomegalovirus-initiated Cells Is Associated with Sequestration of HDM2 and Decreased p53 Ubiquitination

Abstract

Human cytomegalovirus (HCMV) induces serum-or densityarrested human lung (LU) cells to traverse the cell cycle, providing it with a strategy to replicate in post-mitotic cells that are its cellular substrate in vivo. HCMV infection also induces high cellular levels of p53, seemingly in contradiction to the observed cell cycle progression. This study was undertaken to examine the mechanism(s) of the increased p53 abundance. HCMV infection caused a 4-fold increase in p53 that preceded a substantial increase in p53 transcripts by more than 24 h. p53 was stabilized in HCMV-infected cells (from a half-life of less than 30 min to about 8 h) and was less sensitive to proteasome-mediated degradation. Ubiquitination of p53 in mock-infected LU cells was sensitive to inhibition by trans-4-iodo, 4 -boranylchalcone, consistent with HDM2-catalyzing ubiquitination of p53. In HCMV-infected cells, ubiquitination of p53 was essentially undetectable. Although HDM2 had a nuclear distribution in mock-infected LU cells, in HCMV-infected cells HDM2 was translocated to the cytoplasm beginning at 12 h and demonstrated decreased cellular abundance thereafter. HDM2 was stabilized in the HCMV-infected cells by MG132, indicating a shift from p53 to HDM2 ubiquitination. p53 demonstrated a predominantly nuclear distribution in HCMV-infected cells through 48 h, resulting in p53 and HDM2 in distinct subcellular compartments. The principal mechanism responsible for increased p53 stabilization was nuclear export and degradation of HDM2. Thus, HCMV uses a shift from p53 to HDM2 ubiquitination and destabilization to obtain protracted high levels of p53, while promoting cell cycle traverse.Human cytomegalovirus (HCMV), 2 a large ␤-herpesvirus, infects the majority of humans, producing life long persistent infection (1, 2). Efficient replication of HCMV in stationary cells is dependent on the progression of infected cells through the cell cycle to a point at or near the G 1 /S boundary (3, 4). Consistent with this requirement, HCMV infection stimulates potent mitogenic signaling in infected cells, with some evidence suggesting that HCMV mitogenic signaling is more robust than that of serum growth factors, because HCMV, but not serum growth factors, induces the entry of density-arrested cells into the cell cycle. Among the immediate early and early signaling events induced by HCMV are hydrolysis of phosphatidylinositol 4,5-bisphosphate (5), activation of phospholipases (6), increased cellular levels of secondary messengers (3, 6, 7), production of robust and protracted signaling from some components of these pathways (8), stimulation of several protein kinases (6, 9), and activation of a number of cellular transcription factors (9, 10). Some of this signaling derives from HCMV binding to its cellular receptors (epidermal growth factor receptor (11) and integrin ␣v␤3 (12)), but HCMV infection also modifies the cell with induction of cellular (e.g. activation of m-and -calpains (13)) and viral (e.g. cellular transcriptional activation by IE2-86 (14)) ac...

Downregulation of Mdm2 and Mdm4 enhances viral gene expression during adenovirus infection

“…However, our data showed that both proteins are drastically stabilized in cells treated with MG132, suggesting that transcriptional repression might only IE2-86 protein binds Mdm2 and seems to facilitate its degradation in a proteasome-independent manner. 28 In another study, Chen et al showed that Mdm2 was downregulated in HCMVinfected cells, and its abundance was only slightly restored in the presence of proteasome inhibitor MG132, 29 a phenomenon similar to what we have observed with HAdV-infected cells (Fig. 4).…”

“…27 Although this epidemiological finding awaits experimental validation, it seems that increased Mdm2 expression might confer resistance to HPV infection, thereby reducing the risk of OSCC. Downregulation of Mdm2 has been observed in cells infected with human cytomegalovirus (HCMV) 28,29 and HAdVs. 9 Downregulation of Mdm2 could be due to inactivation and destruction of p53 in HAdV-infected cells, as Mdm2 gene expression is activated by p53.…”

HIV-1 viral infectivity factor interacts with TP53 to induce G2 cell cycle arrest and positively regulate viral replication

“…Furthermore, many reports have described a viral connection between TP53 and cell cycle arrest. Herpesviruses, such as herpes simplex virus (40), human herpesvirus 6 (41), and human cytomegalovirus (42)(43)(44), cause cell cycle arrest via the TP53 pathway; however, the viral proteins responsible for TP53 stabilization and the precise mechanisms and significance of cell cycle arrest remain unknown. The data in this study might provide a clue to the mechanism and the significance of cell cycle arrest induced by these viruses.…”

Stabilization of p53 in Influenza A Virus-infected Cells Is Associated with Compromised MDM2-mediated Ubiquitination of p53

“…Proteasome-mediated Degradation of p53 Was Inhibited in IAV-infected Cells-Previous studies demonstrated that the increase in p53 protein in IAV-infected cells was independent of increased transcription (4,11) and it is known that certain types of viral infection affect the stability of p53 protein (26,27). Therefore, in this study, we analyzed p53 stability in IAV-infected cells.…”

“…ibly disrupts Mdm2-p53 protein complexes (27,31), and measured its effect on p53 ubiquitination in mock-and IAV-infected cells. The ladders of polyubiquitinated p53 were detected in mock-infected cells in the presence of MG132 (Fig.…”