Cyclin-dependent kinase-like function is shared by the beta- and gamma- subset of the conserved herpesvirus protein kinases

Abstract

The UL97 protein of human cytomegalovirus (HCMV, or HHV-5 (human herpesvirus 5)), is a kinase that phosphorylates the cellular retinoblastoma (Rb) tumor suppressor and lamin A/C proteins that are also substrates of cellular cyclin-dependent kinases (Cdks). A functional complementation assay has further shown that UL97 has authentic Cdk-like activity. The other seven human herpesviruses each encode a kinase with sequence and positional homology to UL97. These UL97-homologous proteins have been termed the conserved herpesvirus protein kinases (CHPKs) to distinguish them from other human herpesvirus-encoded kinases. To determine if the Cdk-like activities of UL97 were shared by all of the CHPKs, we individually expressed epitope-tagged alleles of each protein in human Saos-2 cells to test for Rb phosphorylation, human U-2 OS cells to monitor nuclear lamina disruption and lamin A phosphorylation, or S. cerevisiae cdc28-13 mutant cells to directly assay for Cdk function. We found that the ability to phosphorylate Rb and lamin A, and to disrupt the nuclear lamina, was shared by all CHPKs from the beta- and gamma-herpesvirus families, but not by their alpha-herpesvirus homologs. Similarly, all but one of the beta and gamma CHPKs displayed bona fide Cdk activity in S. cerevisiae, while the alpha proteins did not. Thus, we have identified novel virally-encoded Cdk-like kinases, a nomenclature we abbreviate as v-Cdks. Interestingly, we found that other, non-Cdk-related activities reported for UL97 (dispersion of promyelocytic leukemia protein nuclear bodies (PML-NBs) and disruption of cytoplasmic or nuclear aggresomes) showed weak conservation among the CHPKs that, in general, did not segregate to specific viral families. Therefore, the genomic and evolutionary conservation of these kinases has not been fully maintained at the functional level. Our data indicate that these related kinases, some of which are targets of approved or developmental antiviral drugs, are likely to serve both overlapping and non-overlapping functions during viral infections.

Figure 1. Localization of the CHPKs in U-2 OS cells.

(A) U-2 OS cells plated on coverslips were transfected with expression plasmids encoding each of the eight CHPKs (denoted by their human herpesvirus number 1-8). Intracellular localization at 24 hours post transfection (hpt) was analyzed by indirect immunofluorescence microscopy after staining with anti-HA antibody (green) and counterstaining nuclei with Hoechst (blue). (B) At least 300 kinase positive cells from the experiment described above were scored for CHPK localization. The percentage of cells displaying the indicated localization pattern (primarily nuclear, primarily cytoplasmic, or both nuclear and cytoplasmic) for each CHPK (1–8) is shown.

Figure 2. The beta- and gamma-herpesvirus CHPKs phosphorylate Rb.

(A) Lysates from Saos-2 cells transfected with plasmids encoding Rb and either an empty vector (EV) or one encoding the indicated human herpesvirus (1–8) CHPKs were harvested 48 hpt and analyzed by Western blot with the indicated antibodies. Rb, total Rb; 807, Rb phosphorylated on serine residues 807 and 811; 780, Rb phosphorylated on serine 780; 821, Rb phosphorylated on threonine 821; HA, epitope tag; Tubulin, tubulin loading control. (B) Both wild type and kinase-deficient (KD) alleles of the beta- and gamma-herpesvirus CHPKs (4–8) were analyzed in the Saos-2 Rb phosphorylation assay as described above. (C) Lower levels of expression plasmids for the HHV-4 and HHV-7 CHPKs were analyzed in the Saos-2 Rb phosphorylation assay as described above. Please see the Materials and Methods section for details.

Figure 3. The beta- and gamma- herpesvirus CHPKs disrupt the nuclear lamina and phosphorylate lamin A.

(A) U-2 OS cells plated on coverslips were co-transfected with the indicated CHPK (1–8) or an empty vector (EV) and a plasmid expressing a GFP-lamin A fusion protein. Coverslips were harvested at 24 hpt, stained for indirect immunofluorescence with an HA antibody and Hoechst (not pictured), and visualized by fluorescence microscopy. Representative images showing GFP-lamin A (green) and the CHPK (red) are displayed. (B) At least 300 kinase and GFP-lamin A positive cells from the experiment described above were scored for nuclear lamina disruption. The percentage of cells displaying a disrupted GFP-lamin A signal for each CHPK (1–8) is shown. Error bars represent standard deviation. Double asterisks (**) indicate statistically significant differences from empty vector (EV) transfected cells (P≤0.01). (C) U-2 OS cells plated on coverslips were transfected with the indicated alpha-herpesvirus CHPK (1–3) or an empty vector (EV) and then cultured in low serum media. Coverslips were harvested at 36 hpt and stained for indirect immunofluorescence with an HA antibody to detect the CHPK (Kinase), an antibody that detects lamin A only when phosphorylated on Ser-22 (P-Ser 22), and Hoechst (Nuclei). (D) Beta-herpesvirus CHPKs (5–7) or their kinase deficient counterparts (KD) were analyzed as described above. (E) Gamma-herpesvirus CHPKs (4,8) or their kinase deficient counterparts (KD) were analyzed as described above. (F) Cells from the experiments described above (C–E) were scored for lamin A phosphorylation on serine-22. The percentage of cells displaying lamin phosphorylation for wild type (1–8) and KD (4–8) CHPKs is shown. Error bars represent standard deviation. Double asterisks (**) indicate statistically significant differences from empty vector (EV) transfected cells (P≤0.01).

Figure 4. A subset of CHPKs display CDK activity.

S. cerevisiae cdc28-13 strains growing in glucose at the permissive temperature and harboring galactose-inducible expression plasmids for the indicated proteins (Empty Vector, EV; human cyclin-dependent kinase 1, CDK1; CHPKs (1–8)) were transferred to galactose-containing media and shifted to the restrictive temperature for five hours, at which time budded cells (indicative of continued cell cycle progression) were counted by light microscopy. The percentage of budded cells is shown with standard deviation. Asterisks denote statistically significant differences from the empty vector control (* indicates P≤0.05, ** indicates P≤0.01).

Figure 5. The CHPKs show minor effects on the numbers of PML-NBs per cell.

(A) U-2 OS cells plated on coverslips were transfected with the indicated CHPK (1–8) or HCMV IE1 and 24 hours later were harvested and stained for indirect immunofluorescence with antibodies for PML, the HA epitope, and Hoechst (not pictured). Representative images showing PML (green) and the CHPK or IE1 (red) are displayed. Arrows denote CHPK/IE1 expressing cells. Images are grouped into the alpha-herpesvirus proteins (top), beta-herpesvirus proteins (middle), and gamma-herpesvirus proteins plus the IE1 control (bottom) (B) Using the same coverslips as in A, the number of PML-NB domains was counted in at least 100 kinase positive nuclei for three independent experiments. The frequency with which any specific number of PML-NBs was observed in an individual cell was plotted with Microsoft Excel as a best-fit curve. Control (dashed line) and IE1 expressing cells (black line) are shown in each graph for comparison. The average number of PML-NBs per cell was also determined from this data (see Table 3) and statistically significant differences from control cells are denoted with an asterisk (* indicates P≤0.05). Herpesvirus families are grouped as above.

Figure 6. Assorted CHPKs prevent nuclear and/or cytoplasmic aggresome formation.

(A) U-2 OS cells grown on coverslips were transfected with expression plasmids for Flag epitope tagged Ataxin-Q82 (a spontaneously aggregating protein) as well as the indicated CHPKs (1–8) or an empty vector (EV). Coverslips were harvested 24 hpt and stained for indirect immunofluorescence with antibodies against the Flag epitope, the HA epitope, and Hoechst. Representative images showing Ataxin-Q82 (green), the CHPK (red) and counterstained nuclei (blue) are displayed. (B) Using the same coverslips as in A, the number of cells that contained nuclear aggresomes was determined by counting between 200 and 300 Ataxin-Q82 positive cells in three independent experiments. The percentage of kinase and Ataxin-Q82 positive cells that formed nuclear aggresomes is shown with standard deviation. Asterisks denote statistically significant differences from the empty vector control (* indicates P≤0.05, ** indicates P≤0.01). (C) Cytoplasmic aggregates formed by the Ataxin-Q82-K772T were imaged as in (A) above. (D) Cytoplasmic aggresomes were quantitated as in B above except that four independent experiments were analyzed.

Figure 7. v-Cdks lack conserved cellular Cdk residues that allow for the regulation of kinase activity.

The amino acid sequences of the kinase domains of the indicated viral (top) and human (bottom) Cdks were aligned. Conserved kinase subdomains are indicated with Roman numerals . The residue corresponding to tyrosine 15 of Cdk2 is shown in green. The residue corresponding to threonine 160 of Cdk2 is shown in lavender. Amino acids that mediate cellular Cdk binding to p27 , a member of the Cip/Kip class of cyclin-dependent kinase inhibitors, are shown in red. Amino acids that mediate cellular Cdk binding to p16 , a member of the INK family of cyclin-dependent kinase inhibitors, are shown in blue. The cyclin-binding PSTAIRE sequence in the cellular Cdks is boxed , and other residues shown to be important for CDK2 binding to Cyclin A are shown in orange . The catalytic triad residues are marked with asterisks (*) .

Figure 8. Tree diagram of the human herpesvirus kinases.

Amino acid sequence comparison was used to generate a tree diagram showing the interrelatedness of sixteen human herpesvirus kinases. The segregation of the kinases into the Us3, TK, and CHPK families, and the separation of the CHPK family into the UL13 and v-Cdk clades is shown. Line distances represent relative evolutionary separation. The commonly-used names for the human herpesviruses are shown (see Table 2).