Human cytomegalovirus UL97 kinase activity is required for the hyperphosphorylation of RB and inhibits the formation of nuclear aggresomes

Departments of Pediatrics, and Cell Biology, University of Alabama School of Medicine, Birmingham AL 35233; The Vaccine and Gene Therapy Institute, Oregon Health Sciences University, Portland, OR 97239; Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA

^* To whom correspondence should be addressed. Email: mprichard{at}peds.uab.edu.

	Abstract

Cells infected with human cytomegalovirus in the absence of UL97 kinase activity produce large nuclear aggregates that sequester considerable quantities of viral proteins. A transient expression assay suggested that pp71 and IE1 were also involved in this process and was significant since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma (RB) pocket proteins. PML bodies have been linked to the formation of nuclear aggresomes and colocalization studies suggested that viral proteins were recruited to these structures and that UL97 kinase activity inhibited their formation. Proteins associated with PML bodies were examined by western blot and pUL97 appeared to specifically affect the phosphorylation of RB in a kinase dependent manner. Three consensus RB binding motifs were identified in the UL97 kinase and recombinant viruses were constructed in which each was mutated to assess a potential role in the phosphorylation of RB and the inhibition of nuclear aggresome formation. Mutation of either the conserved LxCxE RB binding motif or the lysine required for kinase activity impaired the ability of the virus to stabilize and phosphorylate RB. We concluded from these studies that both UL97 kinase activity and the LxCxE RB binding motif are required for the phosphorylation and stabilization of RB in infected cells and that this affect can be antagonized by the antiviral drug, maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.