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. 2022 Oct 5;23(19):11814.
doi: 10.3390/ijms231911814.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

Martin Schütz  1 , Regina Müller  1 , Eileen Socher  1   2   3 , Christina Wangen  1 , Florian Full  4 , Emanuel Wyler  5 , Diana Wong  6   7 , Myriam Scherer  8 , Thomas Stamminger  8 , Sunwen Chou  9   10 , William D Rawlinson  6   7   11   12 , Stuart T Hamilton  6   11 , Heinrich Sticht  2 , Manfred Marschall  1
Affiliations

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

Martin Schütz et al. Int J Mol Sci. .

Abstract

The complex host interaction network of human cytomegalovirus (HCMV) involves the regulatory protein kinase pUL97, which represents a viral cyclin-dependent kinase (CDK) ortholog. pUL97 interacts with the three human cyclin types T1, H, and B1, whereby the binding region of cyclin T1 and the pUL97 oligomerization region were both assigned to amino acids 231-280. We further addressed the question of whether HCMVs harboring mutations in ORF-UL97, i.e., short deletions or resistance-conferring point mutations, are affected in the interaction with human cyclins and viral replication. To this end, clinically relevant UL97 drug-resistance-conferring mutants were analyzed by whole-genome sequencing and used for genetic marker transfer experiments. The recombinant HCMVs indicated conservation of pUL97-cyclin interaction, since all viral UL97 point mutants continued to interact with the analyzed cyclin types and exerted wild-type-like replication fitness. In comparison, recombinant HCMVs UL97 Δ231-280 and also the smaller deletion Δ236-275, but not Δ241-270, lost interaction with cyclins T1 and H, showed impaired replication efficiency, and also exhibited reduced kinase activity. Moreover, a cellular knock-out of cyclins B1 or T1 did not alter HCMV replication phenotypes or pUL97 kinase activity, possibly indicating alternative, compensatory pUL97-cyclin interactions. In contrast, however, cyclin H knock-out, similar to virus deletion mutants in the pUL97-cyclin H binding region, exhibited strong defective phenotypes of HCMV replication, as supported by reduced pUL97 kinase activity in a cyclin H-dependent coexpression setting. Thus, cyclin H proved to be a very relevant determinant of pUL97 kinase activity and viral replication efficiency. As a conclusion, the results provide evidence for the functional importance of pUL97-cyclin interaction. High selective pressure on the formation of pUL97-cyclin complexes was identified by the use of clinically relevant mutants.

Keywords: clinically relevant viral mutants; cyclin H functional significance; functional relevance; human cyclin complexes; human cytomegalovirus; kinase activity; mapping and knock-out analyses; pUL97/vCDK; pUL97–cyclin interaction; viral CDK-like kinase; viral replication efficiency.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Overview of identified and previously known drug resistance mutations in ORF-UL97. (A) HCMV ORF-UL97 drug resistance mutations detected in clinical specimens or cell culture. A schematic map shows resistance-conferring mutations in the globular domain (amino acids 337-707) that were analyzed in this study [16]. The resistance phenotype is color-marked. (B) Predicted structural allocation of pUL97 drug resistance mutations. The location of drug resistance mutations in the predicted structure of the pUL97 C-terminal region shows an accumulation in positions 342-480 around the ATP-binding site of the active center. Note that the sites of GCV-R and MBV-R mutations are not clearly separated in their structural locations (color code of resistance site identical with panel A). (C) The tabular view shows the resistance mutations sequenced in clinical isolates that were transferred into the TB40-IE2-YFP standard genome for further analysis in this study. The designation of clinical HCMV sequences refers to: Ulm, Germany, laboratory of T.S.; Portland, USA, laboratory of S.C.; Sydney, Australia, laboratory of W.D.R.; Erlangen, Germany, laboratory of M.M. Resistance mutation C480F was first sequenced by Houldcroft et al., 2016 [39].
Figure 2
Figure 2
Viral replication kinetics determined by HCMV-specific qPCR and quantitation of IE2-YFP positive cells. (A) 225,000 HFFs in 12-well plates were infected with parental HCMV strain TB40 WT or ORF-UL97 point mutants at a viral dose of 1 × 105 genome copies. Viral supernatants were harvested at indicated time points and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice. (B) Alternatively, 13,500 HFFs in 96-well plates were infected at an MOI of 0.01 and the number of HCMV-infected cells was measured by detection of YFP signal-positive cells at indicated time points, using ImageXpress Pico. Values are given as mean value ± SD of three independently infected wells. (C) HFFs were infected with parental HCMV strain Merlin WT or ORF-UL97 point mutations (A594V, conferring GCV resistance; P132L, not conferring drug resistance) at an MOI of 0.001. Viral genome copies in the supernatant were determined using qPCR. Each value is given as the mean ± SD of three independent biological replicates.
Figure 2
Figure 2
Viral replication kinetics determined by HCMV-specific qPCR and quantitation of IE2-YFP positive cells. (A) 225,000 HFFs in 12-well plates were infected with parental HCMV strain TB40 WT or ORF-UL97 point mutants at a viral dose of 1 × 105 genome copies. Viral supernatants were harvested at indicated time points and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice. (B) Alternatively, 13,500 HFFs in 96-well plates were infected at an MOI of 0.01 and the number of HCMV-infected cells was measured by detection of YFP signal-positive cells at indicated time points, using ImageXpress Pico. Values are given as mean value ± SD of three independently infected wells. (C) HFFs were infected with parental HCMV strain Merlin WT or ORF-UL97 point mutations (A594V, conferring GCV resistance; P132L, not conferring drug resistance) at an MOI of 0.001. Viral genome copies in the supernatant were determined using qPCR. Each value is given as the mean ± SD of three independent biological replicates.
Figure 3
Figure 3
Interaction analysis of TB40-IE2-YFP ORF-UL97 resistance mutants with human cyclins. HFFs in T75 flasks were infected with HCMV TB40-IE2-YFP WT or newly generated ORF-UL97 point mutants. Cells were harvested between 4 and 7 d.p.i., when approximately 80% of the cells showed a cytopathic effect. Cells were lysed, and human cyclins T1 (A), H (B), and B1 (C) were immunoprecipitated using specific antibodies. A chicken Fc fragment served as specificity control, and lysate taken prior to immunoprecipitation was used as input control. All samples were subjected to Wb analysis, and proteins were detected using specific monoclonal and polyclonal antibodies. (D) A summary is given of biological replicates (Rep1, Rep2, Rep3) monitoring the interaction of pUL97 mutants with human cyclins T1, H, and B1.
Figure 3
Figure 3
Interaction analysis of TB40-IE2-YFP ORF-UL97 resistance mutants with human cyclins. HFFs in T75 flasks were infected with HCMV TB40-IE2-YFP WT or newly generated ORF-UL97 point mutants. Cells were harvested between 4 and 7 d.p.i., when approximately 80% of the cells showed a cytopathic effect. Cells were lysed, and human cyclins T1 (A), H (B), and B1 (C) were immunoprecipitated using specific antibodies. A chicken Fc fragment served as specificity control, and lysate taken prior to immunoprecipitation was used as input control. All samples were subjected to Wb analysis, and proteins were detected using specific monoclonal and polyclonal antibodies. (D) A summary is given of biological replicates (Rep1, Rep2, Rep3) monitoring the interaction of pUL97 mutants with human cyclins T1, H, and B1.
Figure 4
Figure 4
Interaction analysis of AD169 pUL97 resistance mutants with human cyclins. HFFs in T75 flasks were infected with HCMV AD169 WT or UL97 point mutants (UL97 WT and mutants were derived from Sunwen Chou, Portland, OR, USA). Cells were harvested between 4 and 7 d.p.i., when approx. 80% of the cells showed a cytopathic effect. Cells were lysed, and human cyclins T1 (A), H (B), and B1 (C) were immunoprecipitated using specific antibodies. A chicken Fc fragment served as specificity control, and lysate taken prior to immunoprecipitation was used as input control. All samples were subjected to Wb analysis, and proteins were detected using the specific monoclonal and polyclonal antibodies indicated.
Figure 5
Figure 5
Viral replication kinetics of pUL97 WT and pUL97 Δ231-280 determined by HCMV-specific qPCR. In total, 225,000 HFFs in 12-well plates were infected with parental HCMV strain AD169-GFP WT or AD169-GFP pUL97 Δ231-280 at an MOI of 0.001. Viral supernatants were harvested at indicated time points and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; ****, p < 0.0001.
Figure 6
Figure 6
Interaction analysis with human cyclins, and viral replication kinetics of HCMV ORF-UL97 deletion mutants within the amino acid region 231-280. (A) A schematic depiction of newly generated HCMV ORF-UL97 deletion mutants in fragments of amino acids 231-280. (B) HFFs in T175 flasks were infected with HCMV AD169 WT, ORF-UL97 mutant versions, or remained mock-infected. Cells were harvested and lysed 4 d.p.i. Lysate control samples were taken before human cyclins H and T1 were immunoprecipitated using the specific antibodies indicated. A chicken Fc fragment served as negative control. All samples were subjected to Wb analysis, and proteins were detected using specific monoclonal and polyclonal antibodies as indicated below the panels. (C) A total of 225,000 HFFs in 12-well plates were infected with parental HCMV strain AD169-GFP WT or ORF-UL97 deletion mutants at a viral dose of 1 × 105 genome copies. Viral supernatants were harvested at indicated time points and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; ****, p < 0.0001.
Figure 7
Figure 7
HCMV AD169-GFP replication kinetics in HFF WT and cyclin B1/T1 KO populations. HFFs were infected with HCMV AD169. Viral replication kinetics were determined by collecting viral supernatants at the indicated time points, and viral genome equivalents were determined by qPCR. Each value represents the mean ± SD of two independent biological replicates, each measured twice: (A) replication kinetics obtained for cyclin B1 KO cell populations A and B compared to WT (MOI 0.025); (C) replication kinetics obtained for cyclin T1 KO cell populations A, B and C compared to WT (MOI 0.01); (B,D) effectivity of cyclin B1 KO (cell populations A and B) and cyclin T1 KO (cell populations A, B and C), respectively, was verified by standard SDS-PAGE and Wb analysis using specific antibodies as indicated.
Figure 8
Figure 8
HCMV AD169-GFP replication kinetics in HFF WT and in transiently transduced HFFs with a partial level of cyclin H KO. A total of 225,000 HFFs were seeded in 12-well plates. The next day, cells were incubated with lentiviral supernatant for 24 h, introducing the CRISPR/Cas9 system and a cocktail of cyclin H-specific gRNAs for transient transduction. Subsequently, cells were incubated with fresh medium for 24 h. Next, HFF WT and cyclin H KO populations were infected with HCMV AD169-GFP at an MOI of 0.5 and harvested at the indicated time points. (A) Cells were lysed and the GFP signal of lysates was measured in triplicate using a Victor multilabel reader. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; ****, p < 0.0001. (B) For comparison of intracellular cyclin H expression levels, the total cellular lysates used for GFP signal quantitation were subsequently denatured at the indicated time points and used for standard SDS-PAGE and Wb analysis. Cyclin H and β-actin were detected using specific antibodies as indicated.
Figure 9
Figure 9
Application of the qSox-IVKA for quantitative measurement of in vitro pUL97 kinase activity. The 293T cells were seeded in 10-cm2 dishes for transfection on the following day using plasmids coding for pUL97-Flag or empty vector control. At 1 day post-transfection (d.p.t.), cells were lysed and pUL97-Flag was immunoprecipitated using 25 µL Dynabeads® protein A coupled with Flag-tag specific antibodies. Dynabead-bound proteins were then eluted in 50 µL enzyme buffer and used for qSox-IVKA. For these reactions, 40 µL of kinase reaction mix was pipetted in Corning NBS 96-well half area microplates, before 10 µL Dynabeads protein solution was added. Kinase activity was measured using a Victor multilabel reader, at every 30 s of the kinetics for approximately 80 min. Values of pUL97 activity were normalized according to the expression levels on Wbs. (A) Absolute values of the measured RFUs for pUL97 and vector control, as treated with 5 µM of MBV or DMSO as the solvent control; measurements were performed in duplicate. (B) Empty vector controls from panel A were used for normalization of pUL97 signal intensities, to depict the specific activity. (C) The pUL97 kinase activity was determined by qSox-IVKA under conditions with or without cyclin H-HA coexpression (transient cotransfection setting); mean values of duplicates are given. (D) Relative pUL97 + cyclin H activity per min was calculated for the linear proportion of the curve obtained in panel C for 20–70 min, normalized to pUL97 + vector control. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; **, p < 0.01. (E) Mutant kinase activity of construct pUL97 Δ231-280 was compared with pUL97 WT (both Flag-tagged), by qSox-IVKA. Mean values are given of two independent experiments, with measurements taken in duplicate. (F) Relative pUL97 activity per minute was calculated for the linear proportion of the curve obtained in panel E for 20–60 min, normalized to pUL97-Flag. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; *, p < 0.05.
Figure 9
Figure 9
Application of the qSox-IVKA for quantitative measurement of in vitro pUL97 kinase activity. The 293T cells were seeded in 10-cm2 dishes for transfection on the following day using plasmids coding for pUL97-Flag or empty vector control. At 1 day post-transfection (d.p.t.), cells were lysed and pUL97-Flag was immunoprecipitated using 25 µL Dynabeads® protein A coupled with Flag-tag specific antibodies. Dynabead-bound proteins were then eluted in 50 µL enzyme buffer and used for qSox-IVKA. For these reactions, 40 µL of kinase reaction mix was pipetted in Corning NBS 96-well half area microplates, before 10 µL Dynabeads protein solution was added. Kinase activity was measured using a Victor multilabel reader, at every 30 s of the kinetics for approximately 80 min. Values of pUL97 activity were normalized according to the expression levels on Wbs. (A) Absolute values of the measured RFUs for pUL97 and vector control, as treated with 5 µM of MBV or DMSO as the solvent control; measurements were performed in duplicate. (B) Empty vector controls from panel A were used for normalization of pUL97 signal intensities, to depict the specific activity. (C) The pUL97 kinase activity was determined by qSox-IVKA under conditions with or without cyclin H-HA coexpression (transient cotransfection setting); mean values of duplicates are given. (D) Relative pUL97 + cyclin H activity per min was calculated for the linear proportion of the curve obtained in panel C for 20–70 min, normalized to pUL97 + vector control. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; **, p < 0.01. (E) Mutant kinase activity of construct pUL97 Δ231-280 was compared with pUL97 WT (both Flag-tagged), by qSox-IVKA. Mean values are given of two independent experiments, with measurements taken in duplicate. (F) Relative pUL97 activity per minute was calculated for the linear proportion of the curve obtained in panel E for 20–60 min, normalized to pUL97-Flag. Statistical analysis was performed using an ordinary two-way ANOVA and post-hoc Sidak correction; *, p < 0.05.
Figure 10
Figure 10
Schematic view of the generation of recombinant HCMVs. For a stepwise description of the procedure, see Results and Methods section. a–d, defined stretches of 50 nucleotides for primer design; adapted with permission from Tischer et al., 2010 [65]. © 2022, Springer Science+Business Media, LLC.
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