Murine Cytomegalovirus M25 Proteins Sequester the Tumor Suppressor Protein p53 in Nuclear Accumulations

Abstract

To ensure productive infection herpesviruses utilize tegument proteins and nonstructural regulatory proteins to counteract cellular defense mechanisms and to reprogram cellular pathways. The M25 proteins of mouse cytomegalovirus (MCMV) belong to the beta-herpesvirus UL25 gene family that encodes viral proteins implicated with regulatory functions. Through affinity purification and mass spectrometric analysis, we discovered the tumor suppressor protein p53 as a host factor interacting with the M25 proteins. M25-p53 interaction in infected and transfected cells was confirmed by co-immunoprecipitation. Moreover, the proteins co-localized in nuclear dot-like structures upon both infection and inducible expression of the two M25 isoforms. p53 accumulated in wildtype MCMV-infected cells, while this did not occur upon infection with a mutant lacking the M25 gene. Both M25 proteins were able to mediate the effect, identifying them as the first CMV proteins responsible for p53 accumulation during infection. Interaction with M25 proteins led to substantial prolongation of the half-life of p53. Contrary to the higher abundance of the p53 protein in wildtype MCMV-infected cells, the transcript levels of the prominent p53 target genes Cdkn1a and Mdm2 were diminished compared to cells infected with the ΔM25 mutant, and this was associated with reduced binding of p53 to responsive elements within the respective promoters. Notably, the productivity of the M25 deletion mutant was partially rescued on p53-negative fibroblasts. We propose that the MCMV M25 proteins sequester p53 molecules in the nucleus of infected cells, reducing their availability for activating a subset of p53-regulated genes, thereby dampening the antiviral role of p53. IMPORTANCE Host cells use a number of factors to defend against viral infection. Viruses are, however, in an arms race with their host cells to overcome these defense mechanisms. The tumor suppressor protein p53 is an important sensor of cell stress induced by oncogenic insults or viral infections, which upon activation induces various pathways to ensure the integrity of cells. Viruses have to counteract many functions of p53, but complex DNA viruses such as cytomegaloviruses may also utilize some p53 functions for their own benefit. In this study, we discovered that the M25 proteins of mouse cytomegalovirus interact with p53 and mediate its accumulation during infection. Interaction with the M25 proteins sequesters p53 molecules in nuclear dot-like structures, limiting their availability for activation of a subset of p53-regulated target genes. Understanding the interaction between viral proteins and p53 may allow to develop new therapeutic strategies against cytomegalovirus and other viruses.

The regulation of shrimp metabolism by the white spot syndrome virus (WSSV)

“…Amino acids are the basic components of proteins, and they play crucial roles in viral pathogenesis 94,95 . As well as being involved in a wide array of metabolic processes including energy synthesis, they are also carbon and nitrogen sources for purine/pyrimidine biosynthesis and lipogenesis 96 .…”

Emerging Mechanisms of G 1 /S Cell Cycle Control by Human and Mouse Cytomegaloviruses

“…Both MCMV and HCMV have therefore devoted several gene products to this task. Although HCMV cell cycle regulation has been studied for many years, the cell cycle control mechanisms of MCMV have only recently begun to be elucidated, leading to the discovery of cell cycle effector functions of M25, M97, and M117 ( 20 , 69 , 91 , 116 ). Consistent with their similar requirements on the metabolic and replicative state of the host cell, both viruses share a number of common key targets in G 1 /S cell cycle regulation ( Fig.…”

“…CMV infection elicits profound stress responses within the infected cell ( 87 , 88 ), including the activation of the DNA damage checkpoint ( 89 ) and the accumulation of p53 ( 18 , 90 , 91 ). In the case of MCMV, gene products of the M25 locus were found to mediate this accumulation ( 91 ). M25 proteins interact with p53, leading to an increase of the p53 half-life.…”

“…M25 proteins interact with p53, leading to an increase of the p53 half-life. Concomitantly, p53 is sequestered in nuclear, dot-like accumulations by M25 and is prohibited from transcriptional activation of the p21 promoter ( 91 ). This suggests a scenario where M25 acts as a sponge for p53 molecules inhibiting their transcriptional activity.…”

“…The general concept of p53 upregulation, nuclear sequestration and downmodulation of its transcriptional targets, in particular p21, appears to be well conserved between HCMV and MCMV. However, based on experiments with p53 deficient cells, it was reported that p53 has a proviral role during HCMV infection, due to the dependence of some viral genes on p53 ( 106 , 107 ), but an antiviral function during MCMV infection ( 91 ). The negative effects of p53 deficiency on HCMV should be interpreted with caution because they were analyzed in a single immortalized p53 –/– cell strain and only partially rescued by reintroduction of the p53 wild-type gene ( 107 , 108 ).…”