Human cytomegalovirus UL97 kinase alters the accumulation of CDK1

Abstract

The UL97 protein kinase is a serine/threonine kinase expressed by human cytomegalovirus (CMV) that phosphorylates ganciclovir. An investigation of the subcellular localization of pUL97 in infected cells indicated that, early in infection, pUL97 localized to focal sites in the nucleus that transitioned to subnuclear compartments and eventually throughout the entire nucleus. When UL97 kinase activity was eliminated with a K355M mutation or pharmacologically inhibited with maribavir, the expansion and redistribution of pUL97 foci within the nucleus was delayed, nuclear reorganization did not occur and assembly complexes in the cytoplasm failed to form normally. As UL97 kinase and its homologues appear to be functionally related to CDK1, a known regulator of nuclear structural organization, the effects of the UL97 kinase on CDK1 were investigated. Expression of CDK1 in infected cells appeared to be induced by UL97 kinase activity at the level of transcription and was not tied to other virus life-cycle events, such as viral DNA replication or virion assembly. These results suggest that, in addition to phosphorylating CDK1 targets, the UL97 kinase modifies G₂/M cell-cycle checkpoint regulators, specifically CDK1, to promote virus replication.

Fig. 1.

Time course of pUL97 localization and expression during infection. HFF cells were infected with CMV strain AD169 at an m.o.i. of 1. Cells were fixed at 1, 8, 24, 48, 72 and 96 h p.i. Using confocal imaging, the cells were screened for the presence of pUL97 (left column, green in merge) or pp65 (middle column, red in merge), with the nuclei in the merged image (TO-PRO-3 staining; right column, blue in merge.) The arrows at 96 h highlight pUL97 (green arrow in pUL97 inset) and DNA (yellow arrow in merge inset) packaging within the assembly complex. Magnification ×600.

Fig. 2.

Time course of pUL97 localization and expression in MBV-treated WT infection. Cells were infected with CMV strain AD169 at an m.o.i. of 1 and treated with 20 µM MBV (WT+MBV). Time points are shown at 8, 24, 48, 72 and 96 h p.i. with pUL97 (left column, green in merge), pp65 (middle column, red in merge) and nucleic acid (TO-PRO-3 staining; right column, blue in merge). The yellow arrows on the 96 h p.i. merge image highlight the abnormal pp65-lined circular blebs outside the nucleus. Magnification ×600.

Fig. 3.

Time course of pUL97 localization and expression in a CMV strain AD169 RC314 infection. Cells were infected with kinase-inactivated virus RC314 (UL97^K355M) at an m.o.i. of 1. Infected cells were fixed and stained to detect pUL97 (left column, green in merge), pp65 (middle column, red in merge) and nucleic acid (TO-PRO-3 staining; right column, blue in merge). The yellow arrow on the 96 h p.i. merge image highlights an abnormal pp65-lined circular bleb outside the nucleus. Magnification ×600.

Fig. 4.

Time course of pUL97 localization and expression in CDV-treated CMV infection. Cells were infected with CMV strain AD169 at an m.o.i. of 1 and treated with 10 µM CDV (WT+CDV). Infected monolayers were fixed at the indicated time points and stained for pUL97 (left column, green in merge), pp65 (middle column, red in merge) and nucleic acid (TO-PRO-3 staining; right column, blue in merge). Magnification ×600.

Fig. 5.

pUL97 localizes to virus replication compartments. HFF cells were infected with CMV strain AD169 at an m.o.i. of 1. Infected cells were fixed at the indicated times and mAbs were used to detect pUL97 (left column, green in merge), replication compartments as defined by ppUL44 (middle column, red in merge) and nucleic acid (DAPI staining; right column, blue in merge). Magnification ×400.

Fig. 6.

CDK1 expression in CMV-infected HFFs is affected by UL97 kinase activity. (a) HFF cells were infected with CMV strain AD169 (WT) at an m.o.i. of 0.5 and treated with MBV or CDV at the concentrations indicated or were infected with the kinase-null virus RC319 (K355M). Cell extracts were harvested at 72 h, run on a gel and transferred to a nitrocellulose filter. Expression of CDK1 (p34 antibody, clone C-19), IE1/IE2 and actin was detected with mAbs as described in Methods. Both mock-infected (MI) cultures (−) and infected cultures (+) were treated with the drug as indicated. The quantities of CDK1 and IE1/IE2 detected were normalized to actin for each sample and expressed as a fraction compared with cells infected with the WT virus, as indicated below the blot. (b) At 72 h p.i. after infection at an m.o.i. of 1, the percentage of CDK1 and pUL97 double-positive cells was determined as the mean±sd of at least three separate experiments. MI cells were counted if positive staining for CDK1 (p34 antibody, clone B-6) was detected. Approximately 75 cells were counted per condition per experiment. All values were significant when compared with WT using Student’s t-test: WT/WT+MBV, P<0.0008; WT/K355M, P<0.0002; WT/MI, P<0.0001; WT+MBV/MI, P<0.0001; K355M/MI, P<0.0017; WT+MBV/K355M, P<0.04.

Fig. 7.

Transiently expressed pUL97 increases the expression of CDK1. (a) HEL299 cells were transfected with plasmids that expressed either WT UL97 (pMP92; WT) or UL97 with the K355M deletion (pMP307; K355M). One set of cells transfected with pMP92 was treated with 20 µM MBV (WT+MBV). Fixed cells were stained with mAbs to visualize pUL97 and CDK1 (p34 antibody, clone B-6), and the nuclei were stained with DAPI. Cells transfected with empty vector (pcDNA3.1) did not exhibit any significant change in CDK1 expression (data not shown). Magnification ×400. (b) The percentage of cells positive for both CDK1 and pUL97 was determined in three separate experiments (mean number of cells±sd counted per condition: pUL97 , n = 151; pUL97+MBV, n = 85, K355M, n = 183.) The results of Student’s t-test shown on the figure indicate the significance of differences between cells expressing pUL97 compared with those expressing pUL97 treated with MBV or kinase-null pUL97 (K355M). (c) Cos7 cells were transfected, imaged and counted as in (b), except that pcDNA3.1 cells were only counted for the presence of CDK1; the mean number of cells counted per condition was n = 176 for pUL97, n = 219 for pUL97+MBV, n = 223 for K355M and n = 252 for pcDNA3.1. The results of Student’s t-test shown in the figure compared pUL97-expressing cells with the vector control.