Abstract

Literature has previously shown that Human cytomegalovirus (HCMV) interacts with the cellular transcription factor p53 in the context of a permissive infection. The interactions include rapid elevation of steady state levels of p53 and the sequestration of p53 into the viral replication centers upon their formation. The studies performed here further characterize this interaction between the virus and this cellular protein. These studies have revealed that the incoming viral genome does not induce a typical double stranded DNA break repair response from the host cell. However, it has been shown that the cell does respond to the incoming viral genome by rapid1y elevating the steady state levels of p53, followed by a change in cellular localization of p53 into a punctatated pattern at 3.5--5.5h post-infection (pi). These p53 punctations co-localized with two important viral proteins, IE2 and UL112/113, and additionally with 60% of the incoming viral genomes that were deposited in the host cell nucleus. This suggests that p53 may play a role in assisting the circularization of the linear viral genome.

Studies performed at later times pi revealed that the sequence-specific DNA binding domain of p53 was required for sequestration of p53 into the viral replication centers. A search for p53 response elements (RE) in the viral genome resulted in the definition of 21 potential p53 binding sites. Chromatin Immunoprecipitation analyses performed during infection precipitating DNA bound to p53 revealed that p53 was bound to 14 of the 21 sites during the course of infection. This data led us to believe that p53 potentially plays a role in viral gene expression.

In order to test this hypothesis we performed infections in the presence and absence of p53 (using WT and p53^-/- fibroblasts). Initial studies utilizing DNA microarray technology and Northern blot analyses to compare viral gene expression in these two different cell types strengthened this hypothesis that p53 was indeed playing a role in this process. The changes in gene expression between the two cell types theoretically would result in changes in viral protein production and Western blotting confirmed this to be true. The vast majority of viral proteins examined thus far had reduced levels, a delay in expression or both in the p53^-/- fibroblasts when compared to WT cells. These studies have therefore further elucidated the roles of p53 in a permissive HCMV infection.