Human Cytomegalovirus UL97 Kinase Activity Is Required for the Hyperphosphorylation of Retinoblastoma Protein and Inhibits the Formation of Nuclear Aggresomes

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 this suggestion was significant, since both proteins have been reported to interact with components of promyelocytic leukemia (PML) bodies (ND10) and also interact functionally with retinoblastoma pocket proteins (RB). 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 analysis, and pUL97 appeared to specifically affect the phosphorylation of RB in a kinasedependent 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. The 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 effect can be antagonized by the antiviral drug maribavir. These data also suggest a potential link between RB function and the formation of aggresomes.All the human herpesviruses encode well-conserved serine/ threonine protein kinases that are important in viral infection (51) and are thought to phosphorylate substrates that are also targets of cdc2 (33). Herpes simplex virus (HSV) UL13 and Epstein-Barr virus BGLF4 phosphorylate eukaryotic elongation factor 1delta (34), and HSV UL13 and human cytomegalovirus (HCMV) pUL97 both phosphorylate the carboxylterminal domain of RNA polymerase II (4, 12). Many other interesting activities of cellular proteins have previously been described, such as the activation of cdc2 by HSV UL13 (1) as well as the inhibition of histone acetylation (57) and activation of protein kinase A (5, 43) by HSV US3. Viral proteins can also be substrates for these kinases; the DNA polymerase processivity factors are substrates, and it appears to be a common theme in the herpesviruses (21,27,39,47). Studies examining the function of these kinases suggest that they are not strictly required for viral infection; however, they perform important functions that are required for replication in vivo and suggest that the effects of these kinases on host and viral targets are important (53,54,62).The UL97 protein kinase in HCMV is particularly important because of its relevance to antiviral therapy. This enzyme phosphorylates and activates the antiviral drug ganciclovir, which is the treatment of choice for HCMV infections (44, 63). Although this drug...

Efficient Induction of Nuclear Aggresomes by Specific Single Missense Mutations in the DNA-binding Domain of a Viral AP-1 Homolog

“…Comparing Induction of Nuclear Aggresomes by Mutant ZEBRA and by Mutant Human Cytomegalovirus-Formation of nuclear aggresomes has been described during infection with a mutant version of a related herpesvirus, human cytomegalovirus (HCMV), lacking the viral UL97 kinase (46) Despite these similarities, however, significant differences between HCMV and EBV are likely to exist regarding their interactions with host aggresomal mechanisms. The ability of UL97 kinase to prevent aggresome formation during HCMV infection and to inhibit the aggregation of pp65 in a kinase-dependent manner indicates a role for UL97 as a virally encoded means of evading the nuclear aggresome response (53).…”

“…Aggresomes Induced by Z(R183E) Recruit SC35 but Not Nucleolin-Aggresomes selectively recruit properly folded wild-type proteins (46). We examined the ability of Z(R183E)-induced aggresomes to recruit and sequester two cellular proteins, SC35 and nucleolin, whose specific distribution within cell nuclei is well documented (31,(47)(48)(49).…”

Human cytomegalovirus UL69 protein facilitates translation by associating with the mRNA cap-binding complex and excluding 4EBP1

“…It may result from interaction with cellular cyclin-dependent kinases, such as CDK1, which phosphorylates pUL69 (6), or with the HCMVcoded pUL97 kinase (9). pUL97, which has a structural resemblance to cellular cyclin-dependent kinases, hyperphosphorylates and inactivates the cellular retinoblastoma protein, which favors cell cycle progression (10,11). Although the interaction of pUL97 and cellular cyclin-dependent kinases with pUL69 might simply reflect phosphorylation of pUL69 by the kinases, it is possible that the associations also modulate kinase behavior, influencing cell cycle progression.…”

Viral and cellular subnuclear structures in human cytomegalovirus-infected cells

“…A number of the observations discussed above rely on the study of transfection experiments using epitope-tagged versions of viral proteins in uninfected cells (Kuny et al, 2010;Prichard et al, 2008;Salsman et al, 2008). It is unclear what effect overexpression during transfection or epitope tagging might have on protein function.…”

“…UL97 phosphorylates and inactivates the PML-NB protein retinoblastoma (Rb) (Hume et al, 2008) and expression of epitope-tagged UL97 in uninfected simian cells (Prichard et al, 2008), but not uninfected human cells (Kuny et al, 2010), notably reduces the number of PMLNBs in the cell. Clarification of the roles, if any, of the UL97 and Rb proteins in PML-NB function in HCMVinfected cells is required.…”