Figure 1.
In vitro phosphorylation of lamin A by GST-UL97.
(A) Recombinant His-tagged lamin A was incubated in kinase reaction buffer in the presence of γ-32P-ATP either alone (no kinase), with catalytically deficient GST-UL97 K355Q (K355Q), or with wild-type GST-UL97 (GST97 WT). GST-UL97 K335Q or wild-type GST-UL97 were also incubated in kinase buffer without lamin A. Following termination of kinase reactions, proteins were resolved by SDS-PAGE. Signal from incorporation of 32P was detected by exposure to a phosphorscreen (top panel), and total protein was detected by Coomassie brilliant blue staining (bottom panel). The positions of radiolabeled GST-UL97 (GST97) and lamin A, and Coomassie stained lamin A are indicated. (The amounts of GST-UL97 were too small to see on the stained gel.) (B) UL97 autophosphorylation and labeling of lamin A were quantified following in in vitro kinase reactions in the presence of varying concentrations of maribavir (MBV). Signal detected from 32P incorporation for autophosphorylation of GST-UL97 and phosphorylation of His-tagged lamin A are plotted as a percent of the signal detected in the absence of drug. The results taken together show that UL97 phosphorylates lamin A in vitro.
Figure 2.
Phosphorylation sites detected on lamin A/C from HCMV-infected cells and from UL97 treated lamin A in vitro.
(A) Mass spectrum from electrospray ionization (ESI)-MS-MS of SGAQASSTPLpSPTR tryptic peptide (amino acids 12–25 of native lamin A) after phosphorylation of His-lamin A in vitro with GST-UL97 indicating phosphorylation at Ser22. Diagnostic fragment ions used to verify detection of the sequence and the phosphorylation site are indicated in shaded circles and asterisks. A diagram of predicted fragment ions is shown below the spectrum. (B) Mass spectrum from ESI-MS-MS of LRLpSPSPTSQR tryptic peptide (amino acids 386–397 of native lamin A) after phosphorylation of His-lamin A in vitro with GST-UL97, indicating phosphorylation at Ser390. Diagnostic fragment ions are indicated as above. (C) Mass spectrum from ESI-MS-MS of LRLpSPSPTSQR tryptic peptide (amino acids 386–397 of native lamin A) after phosphorylation of His-lamin A in vitro with recombinant human CDK1/cyclin B complex, indicating phosphorylation at Ser390. Diagnostic fragment ions are indicated as above. (D) Representative mass spectrum from ESI-MS-MS of SGAQASSTPLpSPTR tryptic peptide (amino acids 12–25 of native lamin A) after immunoprecipitation of lamin A/C from HCMV-infected cells and iTRAQ labeling. Diagnostic fragment ions are presented as above. Note that the iTRAQ label alters the mass of the b-ions. The masses of the ions from this spectrum are presented in Figure S3. (E) Representative mass spectrum from ESI-MS-MS of LSPpSPTSQR tryptic peptide (amino acids 389–397 of native lamin A) after immunoprecipitation of lamin A/C from HCMV-infected cells. Diagnostic fragment ions are indicated as above. The results show that UL97, like CDK1 phosphorylates lamin A on Ser22 and Ser390 in vitro, but that phosphorylation occurs on Ser392 (and Ser22) in infected cells.
Table 1.
A-type lamin phosphopeptides detected by mass spectrometry.
Table 2.
Comparison of lamin A/C phosphorylation at positions Ser22 and Ser392 during replication of wild-type and UL97 mutant HCMVs.
Figure 3.
Orthophosphate labeling of lamin A/C during HCMV infection.
HCMV-infected cells (MOI = 3, 69 hours p.i.) were pulse labeled for 2 hours with [32P]-orthophosphate in the presence or absence of maribavir or roscovotine or both and then lysed. (A) Lamin A/C immunoprecipitates were prepared, resolved by SDS-PAGE, and transferred to a PVDF membrane. The membrane was exposed to a phosphorscreen (top panel, phosphorscreen) to detect 32P signal, and then probed with anti-lamin A/C antibodies to detect total lamin A/C in the immunoprecipitates (bottom panel, western blot). The signal from the phosphorscreen was quantified and the values for 32P incorporation into lamin A and lamin C are shown between the upper (phosphorscreen) and lower (western blot) panels. (B) HCMV UL44 immunoprecipitates were prepared from lysates of the same radiolabeled infected cells, resolved by SDS–PAGE and transferred to a PVDF membrane. The membrane was exposed to a phosphorscreen (top panel, phosphorscreen) to detect 32P signal, and then probed with anti-UL44 antibodies antibodies to detect total UL44 in the immunoprecipitates (bottom panel, western blot). The signal from the phosphorscreen was quantified and the values for 32P incorporation into UL44 are shown between the upper (phosphorscreen) and lower (western blot) panels. (C) The lysates used for immunoprecipitation in A were resolved by SDS-PAGE and transferred to a PVDF membrane that was probed with anti-actin antibodies. Abbreviations: IP, immunoprecipitate; MBV, maribavir; ROSC, roscovitine. The results show that transient inhibition of UL97 with maribavir reduces phosphorylation of lamin A/C by about 50%.
Figure 4.
Immunofluorescence of lamin A/C in cells infected with HCMV.
At 96 hours p.i. (MOI = 1), cells were fixed and double-stained with a mouse monoclonal antibody against UL44, a replication compartment marker (red, left panels) and a goat antibody against lamin A/C (green, center panels). Signals from both antibodies were merged (right panels). (A) Wild-type HCMV strain AD169 (AD169) infected cells show characteristic deformation of the nuclei. The nuclei of cells infected with AD169 in the presence of 5 µM maribavir (AD169+MBV) (B) or in cells infected with UL97 deletion mutant virus RCΔ97 (Δ97) (C) retain the oval shape characteristic of mock-infected cells (mock) (D).
Figure 5.
Confocal microscopy of UL97-dependent nuclear lamina alterations in HCMV-infected cells.
At 96 hours p.i. (MOI = 1), cells were fixed and double stained for UL44 (red) and lamin A/C (green) and imaged by confocal microscopy. Discontinuities in lamin A/C staining were detected in wt AD169-infected cells (A) and (B) and are labeled with white arrows. Lamin A/C staining was more uniform and intense around the nuclear rim and gaps in staining were rarely observed in cells infected with AD169 in the presence of maribavir (MBV) (C), when a UL97 deletion mutant virus was used (Δ97) (D), or in mock-infected cells (E).
Figure 6.
Quantification of UL97-dependent nuclear lamina perturbations during HCMV infection.
(A) Occurrence of nuclear deformity in fibroblasts infected with HCMV strain AD169 (AD169), AD169 in the presence of maribavir (AD169+MBV), and independent isolates of UL97 deletion mutant RCΔ97 (RCΔ97.08) and (RCΔ97.19). Asterisk denotes Fisher's exact test p-value of <0.001 compared to AD169. Numbers above bars indicate numbers of cells scored. (B) Occurrence of gaps in the nuclear lamina in the same panel of cells scored above for nuclear deformity. Asterisk denotes Fisher exact test p-value of <0.001. These results show that UL97 is required for nuclear deformation and disruptions of lamina during HCMV infection.