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Abstract
p53 is a well-known multi-functional transcription regulator and is critical in the induction of apoptosis in response to various stresses. Human cytomegalovirus (HCMV) infection induced the accumulation of p53, which was partly relocalized in cytoplasm, but no apparent cell death in human fibroblasts. p53 in HCMV-infected cells was mainly mono-ubiquitinated, which might be resulted from the decreased expression of MDM2 in the course of HCMV infection. Ubiquitinated p53 was also phosphorylated at serine 20. CRM1 increased in the cytoplasm of HCMV-infected cells. It was found that p53 and its mutant in nuclear export sequences were localized in the cytoplasm of cells when co-expressed with CRM1. Collectively, our data suggest that HCMV infection modifies p53 into a stable and exportable form and accumulates it in the cytoplasm, but does not result in apoptotic death of cells.
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Figure 1.
Characteristics of p53 induced by HCMV infection. (A) Western blot analysis with anti-p53 (DO-1) antibody for the detection of p53 accumulation in HEL 299 infected with HCMV during the course of infection. (B) Apoptosis in HCMV-infected human fibroblasts determined by annexin V staining during the course of infection. Experiments were performed twice independently, and results were expressed mean + standard deviation. Staurosporine (STS) was treated in fibroblast for a positive control of apoptosis. (C) mRNA expression pattern of TP53, MDM2, XPO1, BAX and PARC, in HEL 299 cells infected with HCMV. mRNA levels of each gene were detected by microarray assay as described in Materials and Methods. Values shown are the differences between those in HCMV- or mock-infected HEL 299 cells at the indicated time and in mock-infected HEL 299 cells at 0 h.p.i. (D) Subcellular localization of transfected GFP-p53, p53NLS(−) and p53NES(−) in U373MG after HCMV infection at 48 h.p.i. HCMV UL44 was stained with monoclonal anti-UL44 antibody and Alexa 568-conjugated anti-mouse IgG as a marker for HCMV infection.
Figure 2.
Nuclear export of p53. (A) MDM2 was stained with anti-MDM2 antibody and FITC-conjugated anti-mouse IgG in HEL 299 infected with HCMV at 48 h.p.i. (B) Ubiquitination of p53 was determined by immunoprecipitation with anti-p53 (FL-393) and western blot with anti-p53 (DO-1; lane DO), anti-p53 (FL-393; lane FL), and anti-ubiquitin (lane U) antibody, respectively, in the nuclear and cytoplasmic fraction of HCMV- and mock-infected HEL 299 at 48 h.p.i. (C) The presence of phospho-p53 serine 20 among the ubiquitinated proteins was determined by immunoprecipitation with agarose-conjugated anti-ubiquitin antibody and western blot with anti-phospho-p53 serine 20 antibody in the nuclear and cytoplasmic fraction of HCMV-infected HEL 299 during the course of infection. (D) The effect of proteasome inhibitor, MG132, and MDM2 antagonist, Nutlin-3, on the accumulation of p53 was determined by western blot with anti-p53 (FL-393) antibody in the nuclear fraction of HCMV-infected HEL 299 at 48 h.p.i. (E) CRM1 expression was determined by western blot with anti-CRM1 antibody in the nuclear and cytoplasmic fraction of HCMV-infected HEL 299 at 48 h.p.i. Doxorubicin (Dox) was treated as a control expression of CRM1. (F) The effect of a nuclear export inhibitor, leptomycin B, on the nuclear exportation of p53 was determined by western blot with anti-53 antibody in the cytoplasmic fraction of HCMV-infected HEL 299 at 48 h.p.i. (G) Subcellular localization of transfected GFP-p53, GFP-p53NLS(−) and GFP-p53NES(−) in H1299 transfected along with CRM1. CRM1 was stained with the monoclonal antibody and Alexa568-conjugated anti-mouse IgG.
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