Function of human cytomegalovirus UL97 kinase in viral infection and its inhibition by maribavir

Abstract

The serine/threonine kinase expressed by human cytomegalovirus from gene UL97 phosphorylates the antiviral drug ganciclovir, but its biological function is the phosphorylation of its natural viral and cellular protein substrates which affect viral replication at many levels. The UL97 kinase null phenotype is therefore complex, as is the mechanism of action of maribavir, a highly specific inhibitor of its enzymatic activity. Studies that utilise the drug corroborate results from genetic approaches and together have elucidated many functions of the UL97 kinase that are critical for viral replication. The kinase phosphorylates eukaryotic elongation factor 1delta, the carboxyl terminal domain of the large subunit of RNA polymerase II, the retinoblastoma tumour suppressor and lamins A and C. Each of these is also phosphorylated and regulated by cdc2/cyclin-dependent kinase 1, suggesting that the viral kinase may perform a similar function. These and other activities of the UL97 kinase appear to stimulate the cell cycle to support viral DNA synthesis, enhance the expression of viral genes, promote virion morphogenesis and facilitate the egress of mature capsids from the nucleus. In the absence of UL97 kinase activity, viral DNA synthesis is inefficient and structural proteins are sequestered in nuclear aggresomes, reducing the efficiency of virion morphogenesis. Mature capsids that do form fail to egress the nucleus as the nuclear lamina are not dispersed by the kinase. The critical functions performed by the UL97 kinase illustrate its importance in viral replication and confirm that the kinase is a target for the development of antiviral therapies.

Figure 1

Conserved domains in pUL97. The aa sequence for pUL97 (CAA35333.1) is shown with conserved kinase domains highlighted by gray blocks [70]. Amino terminal domains conserved among the primate cytomegalovirus UL97 homologs (HCMV (CAA35333.1), rhesus cytomegalovirus (AAC05259.1), and chimpanzee cytomegalovirus (NP_612729.1) are highlighted by yellow blocks. Sequences required for nuclear localisation signals are underlined in green (aa 48-110) [65], and the interaction domain with ppUL44 between aa 365-459 [38]. Phosphorylated serine and threonines are shown in red with asterisks below [88]. GCV resistance is associated with mutations in blue aa with deletions occurring in the area underlined in blue [73,74]. Mutations that confer resistance to MBV are shown in boxed red text [73]. Rb binding motifs are underlined bold text [67,68], and the invariant lysine 355 is boxed in black

Figure 2

Function of UL97 kinase in viral infection. Infection with HCMV results in the fusion of the virus particle with the cell membrane and the release of tegument proteins including pUL97 (shown as blue stars) into the host cell. This enzyme is also expressed early in infection and directs the phosphorylation of the viral proteins, ppUL44 and pp65, as well as cellular proteins. Phosphorylation of Rb results in the stimulation of the cell cycle and promotes the synthesis of cellular enzymes that help facilitate HCMV DNA replication. Phosphorylation of the RNA polymerase II large subunit as well as EF-1 is also thought to promote the expression of viral genes. The kinase also contributes to virion morphogenesis by inhibiting the formation of nuclear aggresomes, which sequester considerable quantities of viral structural proteins in the absence of its activity. The kinase then directs the phosphorylation and redistribution of the nuclear lamins to facilitate the egress of mature virions from the nucleus. The kinase is also thought to affect the formation of the assembly compartment in the cytoplasm. Inhibition of UL97 kinase activity by maribavir results in the accumulation of aggresomes and immature virions in the nucleus which are unable to pass through the nuclear cage; thus, very little infectious virus is produced or released from the cell. Although the kinase is delivered to the cell as a tegument protein, the kinase expressed during the infection appears to mediate most effects

Figure 3

Inactivation of retinoblastoma protein (Rb) by UL97 kinase and its impact on the cell. In quiescent cells, Rb binds and inhibits the activity of the transcription factor, E2F. During the cell cycle, Rb is phosphorylated by cyclin-dependent kinases (cdk) complexed with cyclins. This phosphorylation inactivates Rb, and leads to the release of active E2F, which promotes the transcription of factors involved in DNA synthesis, cell cycle regulation, mitosis and apoptosis. In infected cells, the UL97 kinase performs a similar function and also inactivates Rb, leading to the release of active E2F. Compounds, such as roscovitine, inhibit the enzymatic activity of cellular cdks and thus prevent the inactivation of Rb. Maribavir is a specific inhibitor of UL97 kinase and inhibits the inactivation of Rb only in infected cells