The Human Cytomegalovirus UL97 Protein Kinase, an Antiviral Drug Target, Is Required at the Stage of Nuclear Egress

Abstract

Human cytomegalovirus encodes an unusual protein kinase, UL97, that activates the established antiviral drug ganciclovir and is specifically inhibited by a new antiviral drug, maribavir. We used maribavir and a UL97 null mutant, which is severely deficient in viral replication, to determine what stage of virus infection critically requires UL97. Compared with wild-type virus, there was little or no decrease in immediate-early gene expression, viral DNA synthesis, late gene expression, or packaging of viral DNA into nuclease-resistant structures in mutant-infected or maribavir-treated cells under conditions where the virus yield was severely impaired. Electron microscopy studies revealed similar proportions of various capsid forms, including DNAcontaining capsids, in the nuclei of wild-type-and mutant-infected cells. However, capsids were rare in the cytoplasm of mutant-infected or maribavir-treated cells; the magnitudes of these decreases in cytoplasmic capsids were similar to those for virus yield. Thus, genetic and pharmacological evidence indicates that UL97 is required at the stage of infection when nucleocapsids exit from the nucleus (nuclear egress), and this poorly understood stage of virus infection can be targeted by antiviral drugs. Understanding UL97 function and maribavir action should help elucidate this interesting biological process and help identify new antiviral drug targets for an important pathogen in immunocompromised patients.Protein kinases are crucial for specific stages in the replication of many different viruses. Human cytomegalovirus (HCMV), a herpesvirus that causes severe disease in newborns and in immunocompromised adults (reviewed in reference 27), encodes an unusual protein kinase known as UL97 (so designated by the open reading frame in the HCMV genome that encodes it [5,12]). This enzyme, remarkably, phosphorylates and thereby activates the antiviral nucleoside analogs ganciclovir and acyclovir (17,37,38). On peptide substrates, it exhibits an unusual dependence on the identity of the residue five positions C terminal to the site of phosphorylation (1). Although not absolutely essential, UL97 is critical for viral replication in cell culture inasmuch as a UL97 deletion mutation causes a ϳ100-fold decrease in viral yield (28).The stages of HCMV infection resemble those of other herpesviruses. Following attachment to cells and entry, the internalized nucleocapsid travels to the nuclear membrane and deposits its DNA in the nucleus, wherein gene expression and DNA replication ensue (reviewed in reference 23). Viral genes are expressed in three temporally regulated classes: immediate early, early, and late. Viral DNA replication proceeds via highmolecular-weight, concatemeric intermediates, which are cleaved to genome-length units during packaging into capsids. Three capsid forms are observed in the nuclei of infected cells:nonproductive forms thought to result from failed packaging attempts (A capsids), productive intermediates that contain a scaffolding protein (B capsids), and ass...

Human papillomavirus 16 E7 inactivator of retinoblastoma family proteins complements human cytomegalovirus lacking UL97 protein kinase

“…HCMV mutants lacking UL97 produce approximately 10 to 1,000 fold fewer infectious particles than wild-type virus during replication in cultured cells (7,(15)(16)(17)(18). Specific defects of these mutants during viral replication include reduced viral DNA synthesis, impaired exit of capsids from the nucleus (nuclear egress), and altered intracellular localization of virion components; pharmacological inhibition of UL97 results in similar phenotypes (8,10,15,17,19,20).…”

“…Specific defects of these mutants during viral replication include reduced viral DNA synthesis, impaired exit of capsids from the nucleus (nuclear egress), and altered intracellular localization of virion components; pharmacological inhibition of UL97 results in similar phenotypes (8,10,15,17,19,20). Our discovery that UL97 directly phosphorylates pRb prompted us to examine the roles that both UL97 and pRb play during HCMV infection.…”

Kaposi's Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and Its Interaction with K-bZIP

“…Structural analysis of purified virions of several herpesviruses, including HSV, CMV, and EBV, indicate that these protein kinases are asso-ciated with virus particles (2,12,37,50,52); thus, the kinases are in a position to influence virion assembly and events that take place after entry of the virion into the cell. A number of studies using kinase-null viral mutants demonstrated the importance of the kinase for regulating viral gene expression, replication, tissue tropism, or infection in animal models (7,11,26,35,41,48). Various studies have implicated the viral protein kinase in influencing viral gene expression (6,58), viral DNA replication (27,52,53), or nucleocapsid egress from the nucleus during virus assembly (26,34,53).…”

“…Various studies have implicated the viral protein kinase in influencing viral gene expression (6,58), viral DNA replication (27,52,53), or nucleocapsid egress from the nucleus during virus assembly (26,34,53). The importance of the viral protein kinase (vPK) for HSV and CMV replication is supported by studies showing that changes in this gene can confer resistance to certain antiviral agents (e.g., ganciclovir) (7,26). Taken together, these studies demonstrate that the conserved herpesvirus protein kinases impact multiple steps in viral replication.…”

“…A number of studies using kinase-null viral mutants demonstrated the importance of the kinase for regulating viral gene expression, replication, tissue tropism, or infection in animal models (7,11,26,35,41,48). Various studies have implicated the viral protein kinase in influencing viral gene expression (6,58), viral DNA replication (27,52,53), or nucleocapsid egress from the nucleus during virus assembly (26,34,53). The importance of the viral protein kinase (vPK) for HSV and CMV replication is supported by studies showing that changes in this gene can confer resistance to certain antiviral agents (e.g., ganciclovir) (7,26).…”