The endogenous nuclear lamina is partially disrupted during baculovirus infection

To investigate the effects of baculovirus infection on the dynamics of the Sf9 cell endogenous nuclear lamina, the subcellular localization of Sf9 lamin B upon infection of the model baculovirus, Autographa californica multiple nucleopolyhedrovirus (AcMNPV), was analyzed by immunofluorescence staining using the anti-lamin B antibody ADL101. As shown in Fig 1A, in uninfected cells, lamin B was predominantly localized to the nuclear rim, with a slight distribution in the nucleoplasm. In cells infected with vAcWT-mCh, the distribution of lamin B in the nucleoplasm gradually increased as infection progressed: while most lamin B was still localized at the nuclear rim, an increased nucleoplasmic signal was observed compared with that in uninfected cells at 12 h postinfection (p.i.); at 24 h p.i., lamin B was obviously distributed in the nucleoplasm in the majority of cells, and only a few cells exhibited a moderate nucleoplasmic distribution; by 48 h p.i., the nucleoplasmic signal of lamin B further intensified, and the proportion of cells with an obvious lamin B distribution in the nucleoplasm further increased (Fig 1A). To quantitatively estimate the alterations in the subcellular localization of Sf9 cell lamin B upon viral infection, the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B was calculated. As shown in Fig 1B, the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B increased significantly (all P < 0.0001 vs. uninfected) upon viral infection, from 45.1% ± 5.8% in uninfected cells to 53.5% ± 6.0%, 65.3% ± 6.6%, and 70.2% ± 5.8% at 12, 24, and 48 h p.i., respectively. These results indicate that baculovirus infection leads to an alteration in the subcellular localization of endogenous lamin B and increases its distribution in the nucleoplasm of Sf9 cells.

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Fig 1. The endogenous nuclear lamina is partially disrupted during baculovirus infection.

(A and B) Subcellular localization of Sf9 lamin B. Cells were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell or remained uninfected. (A) At the indicated time points p.i., the cells were fixed, permeabilized, and immunolabeled with the ADL101 antibody against lamin B. vAcWT-mCh-infected cells were monitored by mCherry fluorescence. DNA was stained with Hoechst 33258. Scale bar, 5 μM. (B) The nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B was quantified using ImageJ software. The data were pooled from two independent experiments with similar results and are presented as means ± standard deviation (SD) (n = 60). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (****, P < 0.0001). (C and D) Time course analysis of Sf9 lamin B expression. Sf9 cells were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell or remained uninfected. (C) At the indicated time points p.i., the cells were harvested and subjected to Western blotting. Lamin B was detected with the ADL101 antibody, and tubulin was probed with an anti-tubulin antibody as a loading control. (D) The signal intensity of lamin B was quantified using Image Studio Ver 5.2 software and normalized against that of tubulin. The quantified intensities are presented in arbitrary units relative to those for uninfected cells, which were set as 1. The data were obtained from three independent experiments and are presented as means ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (ns, not significant).

https://doi.org/10.1371/journal.ppat.1013991.g001

To assess whether the alteration in the subcellular localization of endogenous lamin B was associated with changes in the amount of lamin B in the context of viral infection, lamin B protein levels in Sf9 cells infected with vAcWT-mCh or remained uninfected were analyzed by Western blotting using the ADL101 antibody. As shown in Fig 1C, a protein band corresponding to lamin B was detected in both uninfected cells and infected cells at different time points p.i. at comparable levels. To further assess the change in the total amount of lamin B upon infection, three independent Western blot assays were performed, and the intensities of the immunoreactive bands were quantified by a densitometric analysis. As shown in Fig 1D, the values at each time point p.i. were not significantly different (P > 0.05) from those of uninfected cells, indicating that no quantitative changes in the amounts of lamin B occurred following baculovirus infection, which suggests that the increased nucleoplasmic lamin B distribution observed by immunofluorescence staining during viral infection was derived from the nuclear rim.

Taken together, these findings indicate that the endogenous nuclear lamina of Sf9 cells is partially disrupted during baculovirus infection and that lamin B disassembly gradually increases as infection progresses.

S47 of lamin B is a key phosphorylation site for baculovirus-induced lamina disruption

The disassembly of the nuclear lamina either during cellular mitosis or induced by herpesvirus and circovirus infections is correlated with lamin phosphorylation [26,37,58]. Having established that baculovirus infection disrupts the endogenous nuclear lamina, we sought to determine whether endogenous lamin B phosphorylation was increased during infection. Cells that were either uninfected or infected with vAcWT-mCh were harvested at designated time points p.i. and subjected to immunoprecipitation with protein A/G agarose beads conjugated with the ADL101 antibody. The immunocomplexes were assessed by Western blotting with an anti-pan phospho-serine/threonine antibody to detect phosphorylated lamin B and with the ADL101 antibody to detect the total lamin B (Fig 2A). The relative signal intensities of phosphorylated lamin B were determined from three independent immunoblotting experiments by a densitometric analysis. As shown in Fig 2B, the level of phosphorylated lamin B was increased by a factor of 1.429 ± 0.056 (P < 0.001) at 12 h p.i., 1.609 ± 0.079 (P < 0.0001) at 24 h p.i., and 2.115 ± 0.120 (P < 0.0001) at 48 h p.i. compared with that in uninfected cells. This result indicates that baculovirus infection induced increased phosphorylation of endogenous lamin B in Sf9 cells, consistent with the changes in GFP-Drosophila lamin B phosphorylation observed in baculovirus-infected Sf9-L cells [53].

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Fig 2. Baculovirus infection induces increased phosphorylation of lamin B at S47.

(A and B) Time course analysis of the pan-serine/threonine phosphorylation of Sf9 lamin B. Cells were infected with vAcWT-mCh at an MOI of 20 TCID50/cell or remained uninfected. At the indicated time points p.i., the cells were harvested and subjected to immunoprecipitation with Protein A/G agarose beads conjugated with the ADL101 antibody. (A) The immunocomplexes were subjected to Western blotting with an anti-pan phospho-serine/threonine antibody and the ADL101 antibody to detect pan-serine/threonine phosphorylated lamin B (pS/T-lamin B) and total lamin B. (B) The signal intensity of pS/T-lamin B was quantified using Image Studio Ver 5.2 software and normalized against that of total lamin B. The quantified intensities are presented in arbitrary units relative to those for uninfected cells, which were set as 1. The data were obtained from three independent experiments and are presented as means ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (***, P < 0.001; ****, P < 0.0001). (C) The specificity of an anti-human phospho-lamin A/C (S22) antibody in Sf9 cells. The cells were mock-transfected or transfected with the pIB-GFP:lamin B or pIB-GFP:lamin B (S47A) plasmid. At 36 h posttransfection (p.t.), the cells were harvested and subjected to Western blotting with an anti-phospho-lamin A/C (S22) antibody, anti-GFP antibody, and the ADL101 antibody. Tubulin was probed with an anti-tubulin antibody as a loading control. (D and E) Time course analysis of lamin B phosphorylation at S47. The cells were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell or remained uninfected. (D) At the indicated time points p.i., the cells were harvested and subjected to Western blotting with an anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody to detect phospho-S47 lamin B (pS47-lamin B) and total lamin B. (E) The signal intensity of pS47-lamin B was quantified using Image Studio Ver 5.2 software and normalized against that of total lamin B. The quantified intensities are presented in arbitrary units relative to those for uninfected cells, which were set as 1. The data were obtained from three independent experiments and are presented as means ± SD. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (*, P < 0.05; ***, P < 0.001; ****, P < 0.0001). (F) Subcellular localization of pS47-lamin B. At 24 h p.i., the cells were fixed, permeabilized, and immunolabeled with an anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody to detect pS47-lamin B and total lamin B. vAcWT-mCh-infected cells were monitored by mCherry fluorescence. DNA was stained with Hoechst 33258. Scale bar, 5 μM.

https://doi.org/10.1371/journal.ppat.1013991.g002

The N-terminal “mitotic site” is crucial for lamina disassembly during mitosis [18,22,23,26,27], and this site is repurposed by herpesviruses and circoviruses to facilitate the local disruption of the nuclear lamina and nuclear egress of progeny viral capsids [3,29,37]. The N-terminal “mitotic site” is located at position S22 in human lamin A/C [18,20], and the corresponding position is S47 in Spodoptera frugiperda lamin B based on amino acid sequence alignment (S1A Fig). To determine whether the S47 phosphorylation site of Sf9 lamin B is involved in baculovirus-induced lamina disruption, the phosphorylation of this site during viral replication was analyzed. Considering that no commercial site-specific phospho-antibody is available for phospho-S47 Sf9 lamin B and that this residue is highly conserved, an anti-phospho-lamin A/C (S22) antibody raised against human lamin A/C was selected to detect phospho-S47 Sf9 lamin B. To evaluate the specificity of this commercially available antibody in Sf9 cells, we generated a transient expression plasmid, pIB-GFP:lamin B (S47A), which expresses GFP-tagged lamin B with a phospho-deficient mutation at S47 (substituting serine with alanine). pIB-GFP:lamin B, a transient expression vector expressing GFP-tagged wild-type lamin B, was generated as a control. Sf9 cells were transfected with the pIB-GFP:lamin B or pIB-GFP:lamin B (S47A) plasmid. At 36 h p.t., the cells were harvested and subjected to Western blotting with an anti-phospho-lamin A/C (S22) antibody, an anti-GFP antibody, and the ADL101 antibody. As shown in Fig 2C, wild-type GFP:lamin B and the GFP:lamin B mutant were expressed as fusion proteins with comparable amounts in the cells transfected with the above plasmids. While the anti-phospho-lamin A/C (S22) antibody reacted with endogenous lamin B and wild-type GFP:lamin B in Sf9 cells, it failed to recognize the GFP:lamin B (S47A) mutant (Fig 2C). The results indicated that the anti-phospho-lamin A/C (S22) antibody raised against human lamin A/C could specifically cross-react with phospho-S47 Sf9 lamin B. Based on these results, Sf9 cells were either uninfected or infected with vAcWT-mCh, harvested at the indicated time points p.i., and analyzed by Western blotting with the anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody to detect phospho-S47 lamin B and total lamin B (Fig 2D). Relative signal intensities for S47 phosphorylation were determined from three independent immunoblotting experiments by a densitometric analysis. As shown in Fig 2E, a time course-dependent increase in S47 phosphorylation was detected in vAcWT-mCh-infected cells compared with uninfected cells, and the phospho-S47 lamin B level was increased by a factor of 2.128 ± 0.009 (P < 0.05) at 12 h p.i., 3.983 ± 0.464 (P < 0.001) at 24 h p.i., and 6.721 ± 0.944 (P < 0.0001) at 48 h p.i. This result indicated that the phosphorylation of lamin B at S47 significantly increased in Sf9 cells during baculovirus infection.

In addition to the Western blot analysis, cells were subjected to immunofluorescence staining with the anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody to investigate alterations in the subcellular distribution of phospho-S47 lamin B upon viral infection. As shown in Fig 2F, the intensity and distribution of phospho-S47 lamin B in infected cells clearly differed from those in uninfected cells. In uninfected cells, the signal of phospho-S47 lamin B was quite weak in the nucleoplasm. In contrast, in virus-infected cells, phospho-S47 lamin B presented a highly abundant nucleoplasmic distribution. Correspondingly, the majority of the total lamin B was eliminated from the nuclear rim and dispersed into the nucleoplasm in infected cells. This result indicates that baculovirus infection promotes the nucleoplasmic accumulation of phospho-S47 lamin B, coinciding with the increased dispersal of total lamin B into the nucleoplasm. These findings suggest that S47 phosphorylation is highly relevant to the baculovirus-induced redistribution of lamin B from the nuclear rim to the nucleoplasm.

To further detect the contribution of lamin B S47 phosphorylation to the lamina disruption induced by baculovirus infection, we investigated the effects of the lamin B S47 phospho-deficient mutant on lamina disruption. Sf9 cells were transfected with the pIB-GFP:lamin B or pIB-GFP:lamin B (S47A) plasmid. At 24 h p.t, the transfected cells were either left uninfected or infected with vAcWT-mCh. The cells were fixed at 36 h p.i., and the distributions of wild-type GFP:lamin B and its S47A mutant were analyzed by confocal microscopy. In uninfected cells, both wild-type GFP:lamin B and the S47A mutant were mainly localized to the nuclear rim, with a slight distribution in the nucleoplasm (Fig 3A). Upon infection, wild-type GFP:lamin B underwent a dramatic redistribution from the nuclear rim to the nucleoplasm (Fig 3A), recapitulating the distribution pattern of endogenous lamin B (Fig 1A and B). Strikingly, this virus-induced redistribution was markedly suppressed in cells expressing the S47A mutant, which largely retained its nuclear rim localization despite some nucleoplasmic distribution (Fig 3A). To quantitatively estimate the effect of phospho-deficient mutation of lamin B at S47 on GFP:lamin B disassembly, the nucleoplasm/whole nucleus fluorescence intensity ratio of GFP:lamin B was calculated to reflect the degree of lamina disruption. As shown in Fig 3B, in uninfected cells, the nucleoplasm/whole nucleus fluorescence intensity ratio of the S47A mutant exhibited a slight yet statistically nonsignificant reduction compared to the wild-type GFP:lamin B (30.4% ± 6.5% vs. 33.4% ± 7.6%). In infected cells, the nucleoplasm/whole nucleus fluorescence intensity ratio of wild-type GFP:lamin B increased markedly to 64.0% ± 6.4%, whereas the nucleoplasm/whole nucleus fluorescence intensity ratio of the S47A mutant was only 41.8% ± 6.7%, a much lower (P < 0.0001) ratio than that of wild-type GFP:lamin B in infected cells but much closer to its own ratio in uninfected cells (30.4% ± 6.5%). Importantly, the nucleoplasm/whole nucleus fluorescence intensity ratio of GFP:lamin B in infected cells was decreased in 95% of the cells expressing the S47A mutant compared with those expressing wild-type GFP:lamin B. These data indicate that the phospho-deficient mutation of lamin B at S47 significantly inhibited the virus-induced increase in lamin B disassembly.

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Fig 3. Phosphorylation of lamin B at S47 is critical for baculovirus-induced lamina disruption.

The cells were transfected with the pIB-GFP:lamin B, pIB-GFP:lamin B (S47A), or pIB-GFP:lamin B (S47D) plasmid and then either left uninfected or infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell at 24 h p.t. (A and C) At 36 h p.i., the cells were fixed, permeabilized, and stained with Hoechst 33258 to visualize the nuclear DNA. vAcWT-mCh-infected cells were monitored by mCherry fluorescence. The subcellular localization of wild-type and mutant GFP:lamin B was detected by GFP fluorescence. For simplicity, in this figure, GFP:lamin B, GFP:lamin B (S47A), and GFP:lamin B (S47D) are denoted as WT, S47A, and S47D, respectively. Scale bar, 5 μM. (B and D) The nucleoplasm/whole nucleus fluorescence intensity ratio of GFP:lamin B was quantified using ImageJ software. The data were pooled from two independent experiments with similar results and are presented as means ± SD (n = 60). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (ns, not significant; ****, P < 0.0001).

https://doi.org/10.1371/journal.ppat.1013991.g003

To further validate the role of lamin B S47 phosphorylation in lamina disruption, we investigated the effects of the lamin B S47 phospho-mimetic mutant (S47D) mutant on this process. Sf9 cells were transfected with plasmids expressing wild-type GFP:lamin B or the GFP:lamin B (S47D) mutant. At 24 h p.t, the transfected cells were either left uninfected or infected with vAcWT-mCh. Confocal microscopy observations at 36 h p.i. showed that the S47D mutant exhibited a more pronounced nucleoplasmic distribution than wild-type GFP:lamin B in both uninfected and infected cells (Fig 3C). To quantify these observations, we measured the nucleoplasm/whole nucleus fluorescence intensity ratio of wild-type GFP:lamin B and its S47D mutant. In uninfected cells, the ratio for wild-type GFP:lamin B was 38.3% ± 7.4%, whereas the S47D mutant displayed a significantly higher ratio of 59.3% ± 10.5% (P < 0.0001; Fig 3D). Following infection, the ratio of wild-type GFP:lamin B increased to 60.5% ± 6.4%. Notably, the ratio for the S47D mutant reached 74.8% ± 6.4%, which was significantly higher than that of the infected wild-type control (P < 0.0001; Fig 3D). These data demonstrated that the phospho-mimetic mutation of lamin B at S47 is sufficient to promote lamin B disassembly in the absence of viral infection and further enhances baculovirus-induced disassembly of lamin B.

Taken together, the results described above demonstrate that S47 of lamin B is a key phosphorylation site that is required for baculovirus-induced lamina disruption.

CDK1 directly mediates the phosphorylation of lamin B at S47 during baculovirus infection

Site-specific phosphorylation of S22 on human lamin A/C and its equivalent position in other lamins, which promotes disassembly of the nuclear lamina, is mainly mediated by CDK1 during mitosis [18–20,22,24,28]. Baculovirus infection arrests cells in G2/M phase of the cell cycle [59] and therefore probably utilizes the host cell CDK1 pathway for the phosphorylation of lamin B at S47 and the disruption of the nuclear lamina for nuclear egress. To test this hypothesis, we first assessed whether CDK1 could directly phosphorylate Sf9 lamin B at S47 using an in vitro kinase assay. Due to the poor solubility of full-length lamin B, a truncated form of lamin B encompassing the N-terminal 1–150 amino acids was constructed. Both the N-terminally His-tagged truncated wild-type lamin B and its S47A mutant were expressed and purified from Escherichia coli (Fig 4A). As CDK1 requires binding to cyclin B for activation [60], we expressed the Sf9 CDK1–cyclin B complex using the eukaryotic baculovirus expression vector system (BEVS) to ensure proper kinase activity. Specifically, two recombinant baculoviruses were constructed: one driving the expression of CDK1 (C-terminally His-tagged) and the other driving the expression of cyclin B (C-terminally Twin-Strep II-tagged). Co-infection of Sf9 cells with these viruses enabled assembly of the CDK1–cyclin B complex, which was purified by nickel affinity chromatography (Fig 4B). Incubation with the purified CDK1–cyclin B complex induced robust phosphorylation at S47 in the truncated wild-type lamin B, whereas no phosphorylation was detected in reactions lacking the kinase complex. Critically, this phosphorylation was completely abolished in the S47A mutant regardless of the presence of CDK1–cyclin B (Fig 4C, left). Furthermore, addition of RO-3306, a specific CDK1 inhibitor, to the reactions containing CDK1–cyclin B complex and truncated wild-type lamin B led to a marked reduction in S47 phosphorylation in a dose-dependent manner, with complete abrogation observed at a concentration of 5 μM (Fig 4C, Right). Taken together, these results demonstrated that CDK1 directly phosphorylates Sf9 lamin B at S47 in vitro.

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Fig 4. CDK1 directly phosphorylates lamin B at S47 in vitro.

(A) Quality assessment of recombinant truncated lamin B (N-terminal 1–150 aa) and its S47A mutant. Left: SDS–PAGE with Coomassie brilliant blue staining. Right: immunoblot probed with an anti-His antibody. (B) Quality assessment of the recombinant CDK1–cyclin B complex expressed in Sf9 cells via the eukaryotic baculovirus expression vector system. Left: SDS–PAGE with Coomassie brilliant blue staining. Right: immunoblot probed with an anti-His antibody (detecting C-terminally His-tagged CDK1) or an anti-Strep antibody (detecting C-terminally Twin-Strep II-tagged cyclin B). (C) In vitro kinase assay for CDK1–cyclin B-mediated phosphorylation of lamin B at S47. Left: recombinant truncated lamin B or its S47A mutant was incubated in the presence (+) or absence (–) of purified Sf9 CDK1–cyclin B complex. Right: recombinant truncated wild-type lamin B was incubated with CDK1–cyclin B complex in the presence of the indicated concentrations of RO-3306 (0–5 μM). The samples were analyzed by Western blotting with an anti-phospho-lamin A/C (S22) antibody (detecting phospho-S47 lamin B) or an anti-His antibody (detecting His-tagged lamin B).

https://doi.org/10.1371/journal.ppat.1013991.g004

To investigate whether CDK1 mediates lamin B S47 phosphorylation during baculovirus infection, a CDK1-specific inhibitor, RO-3306, was used to repress CDK1 activity in Sf9 cells, and the phosphorylation of lamin B at S47 was analyzed. The data derived from a trypan blue exclusion assay indicated that RO-3306-induced cytotoxicity occurred only at drug concentrations of 15 μM or higher in Sf9 cells (S2 Fig). Thus, Sf9 cells were treated with 10 μM RO-3306 to repress CDK1 activity. We designed four experimental conditions to assess the contribution of CDK1 to both basal and baculovirus-induced lamin B phosphorylation: uninfected and baculovirus-infected cells, each treated with either RO-3306 or DMSO control. Given that baculovirus late protein expression is strictly dependent on viral DNA replication—a process that ceases at ~20 h p.i. [39,61], RO-3306 was added at 24 h p.i. to minimize potential effects on viral late protein expression, which could otherwise contribute to lamin B phosphorylation. This treatment time point was validated by our finding that while RO-3306 treatment at 11 h p.i. significantly reduced viral late protein expression, treatment at 24 h p.i. did not (S3 Fig). To capture the most pronounced lamin B phosphorylation, which becomes evident at later stages of infection (Fig 2D and E), and to ensure sufficient time for RO-3306 to act, cells were harvested at 48 h p.i. and subjected to Western blotting with an anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody (Fig 5A), and the relative signal intensities for S47 phosphorylation were determined from three independent immunoblotting experiments by a densitometric analysis (Fig 5B). Compared with DMSO-treated controls, RO-3306 treatment decreased the basal level of lamin B S47 phosphorylation in uninfected cells by 63.3% ± 6.2% (P < 0.0001), indicating that CDK1 activity is required for basal phosphorylation of lamin B at this site. As expected, baculovirus infection dramatically increased S47 phosphorylation by a factor of 6.769 ± 0.131 (P < 0.0001) relative to uninfected DMSO-treated cells. Strikingly, RO-3306 treatment reduced this virus-induced phosphorylation by 94.5% ± 1.1% (P < 0.0001) compared with virus-infected DMSO-treated cells, bringing it down to a level comparable to the suppressed basal state in uninfected RO-3306-treated cells (Fig 5A and B). These results demonstrated that CDK1 mediates the phosphorylation of lamin B at S47 during baculovirus infection.

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Fig 5. CDK1 mediates the phosphorylation of lamin B at S47 during baculovirus infection.

(A and B) CDK1 inhibition with RO-3306 inhibits lamin B S47 phosphorylation. Cells were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell or remained uninfected. Both groups were treated with DMSO or 10 μM RO-3306 (treatment timed to 24 h p.i. for infected cells) and harvested at 48 h p.i. (C and D) Dose-dependent suppression of lamin B S47 phosphorylation by RO-3306 in baculovirus-infected cells. vAcWT-mCh-infected cells (MOI = 20 TCID₅₀/cell) were treated with the indicated concentrations of RO-3306 (0–10 μM) at 24 h p.i. and harvested at 48 h p.i. (E and F) Time-dependent suppression of lamin B S47 phosphorylation by RO-3306 in baculovirus-infected cells. vAcWT-mCh-infected cells (MOI = 20 TCID₅₀/cell) were treated with 10 μM RO-3306 at 47, 45, 42, 36, 30, and 24 h p.i., corresponding to treatment durations of 1, 3, 6, 12, 18, and 24 h, respectively. The vehicle control (DMSO) was added at 24 h p.i. and used as the reference for quantification. All samples were harvested at 48 h p.i. (G and H) Dominant-negative CDK1 inhibits lamin B S47 phosphorylation in baculovirus-infected cells. Cells were infected with vAcWT-mCh2, vFLAG:CDK1, or vFLAG:CDK1 (DN) at an MOI of 20 TCID₅₀/cell and harvested at 48 h p.i. (A, C, E, and G) Representative immunoblots of cell lysates probed with an anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody to detect phospho-S47 lamin B (pS47-lamin B) and total lamin B. (B, D, F, and H) Quantification of pS47-lamin B signals using Image Studio Ver 5.2 software and normalized against that of total lamin B. Data are presented in arbitrary units relative to those for DMSO-treated uninfected control (B), 0 μM RO-3306 (vehicle control, DMSO) (D), DMSO vehicle control (added at 24 h p.i. for a 24-h treatment) (F), or vAcWT-mCh2-infected control (H), set to 1. Values are means ± SD from three independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test (ns, not significant; ****, P < 0.0001).

https://doi.org/10.1371/journal.ppat.1013991.g005

To establish the dose- and time-dependence of RO-3306 inhibition on CDK1-mediated lamin B S47 phosphorylation during baculovirus infection, we performed detailed kinetic analyses using the inhibitor RO-3306. For the dose-response analysis, virus-infected cells were treated with increasing concentrations of RO-3306 (1–10 μM) at 24 h p.i. and harvested at 48 h p.i. Cells treated with vehicle (DMSO) at 24 h p.i. were used as a negative control. As shown in Fig 5C and D, treatment with RO-3306 resulted in a significant reduction in lamin B S47 phosphorylation in a strict concentration-dependent manner. A modest yet significant reduction (58.6% ± 5.2%, P < 0.0001) in S47 phosphorylation was observed even at 1 μM, with inhibition continuing to strengthen as concentrations increased and reaching a 93.2% ± 2.3% reduction at 10 μM (P < 0.0001). For the time-course analysis, infected cells were treated with 10 μM RO-3306 at various times p.i., resulting in treatment durations of 1–24 h before a common harvest at 48 h p.i.; the DMSO vehicle control was added at 24 h p.i. (24-h treatment). As shown in Fig 5E and F, the inhibition of S47 phosphorylation exhibited a clear time-dependent manner. A 1-hour treatment led to a modest yet significant reduction (34.1% ± 7.0%, P < 0.0001), while longer durations resulted in progressively stronger inhibition, with the 24-hour treatment achieving a near-complete (92.5% ± 1.2%, P < 0.0001) reduction. Taken together, these results demonstrated that the inhibitory effect of the CDK1-specific inhibitor RO-3306 on baculovirus-induced lamin B S47 phosphorylation is tightly dependent on both treatment dose and duration. This strict dose- and time-dependence further confirms that CDK1 critically mediates lamin B S47 phosphorylation during baculovirus infection.

Mutation of the key amino acid aspartate in the active center of CDK1 has been reported to generate a dominant-negative mutant [62–64], and such a mutation in human CDK1 was found to arrest cells at the G2 to M phase transition [65]. This residue is highly conserved in CDK1 among species [65], and amino acid sequence alignment revealed that the corresponding position is the aspartic acid at residue 146 (D146) in S. frugiperda CDK1 (S4 Fig). To further confirm CDK1’s mediatory role in baculovirus-induced lamin B S47 phosphorylation, we validated this role genetically by expressing a dominant-negative mutant of CDK1 in Sf9 cells and assessed its impact on this phosphorylation event, thereby ruling out potential off-target effects of the pharmacological inhibitor RO-3306. To this end, a recombinant virus that expressed a FLAG-tagged dominant-negative mutant of CDK1 under the control of the hr5 enhancer and, i.e.,1  promoter was generated and designated vFLAG:CDK1 (DN). As a control, vFLAG:CDK1, a recombinant virus expressing FLAG-tagged wild-type CDK1, was generated. In these constructs, the fluorescent protein mCherry under the control of the gp64 promoter was used to monitor viral infection. A pseudo-wild-type virus expressing mCherry under the control of the gp64 promoter (vAcWT-mCh2) was also generated as a control. Sf9 cells were infected with vAcWT-mCh2, vFLAG:CDK1, or vFLAG:CDK1 (DN) and subjected to Western blotting with an anti-FLAG antibody. The results showed that FLAG-tagged wild-type CDK1 and its dominant-negative mutant were expressed as fusion proteins with comparable amounts (S5 Fig). Then, Sf9 cells infected with the above viruses were collected at 48 h p.i. and subjected to Western blotting with an anti-phospho-lamin A/C (S22) antibody and the ADL101 antibody (Fig 5G), and the relative signal intensities for S47 phosphorylation were determined from three independent immunoblotting experiments by a densitometric analysis (Fig 5H). No significant difference (P > 0.05) in the level of S47 phosphorylation was observed between vFLAG:CDK1- and vAcWT-mCh2-infected cells, whereas the level of S47 phosphorylation was reduced by 45.6% ± 3.2% (P < 0.0001) in vFLAG:CDK1 (DN)-infected cells compared with vAcWT-mCh2-infected cells (Fig 5G and H). These results demonstrated that expression of dominant-negative CDK1 mutant significantly reduces baculovirus-induced lamin B phosphorylation at S47.

Collectively, our in vitro kinase assay, pharmacological inhibition, and dominant-negative genetic approach provide strong and concordant evidence that CDK1 directly mediates baculovirus-induced lamin B phosphorylation at S47.

CDK1 mediates the baculovirus-induced disruption of the nuclear lamina

Given that S47 of lamin B is a key phosphorylation site for baculovirus-induced lamina disruption and that CDK1 mediates the phosphorylation of lamin B at S47, we reasoned that CDK1 would be required for the baculovirus-induced disruption of the nuclear lamina. To test this issue, Sf9 cells were infected with vAcWT-mCh and incubated with 10 μM RO-3306 at 24 h p.i. At 48 h p.i., the cells were fixed and processed for immunofluorescence staining with the ADL101 antibody to reveal the distribution of lamin B. Cells infected with vAcWT-mCh and treated with the DMSO-containing vehicle were used as a control. As expected, in vAcWT-mCh-infected cells treated with the DMSO-containing vehicle, lamin B was obviously distributed in the nucleoplasm in the majority of cells, with only a few cells showing a moderate nucleoplasmic distribution of lamin B. However, in vAcWT-mCh-infected cells treated with RO-3306, lamin B predominantly localized to the nuclear rim with a slight nucleoplasmic distribution in the majority of cells, and only a few cells exhibited a moderate distribution of lamin B in the nucleoplasm (Fig 6A). Quantification of the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B showed that in DMSO-treated cells, the ratio was 73.9% ± 7.2%; however, in RO-3306-treated cells, the ratio was 60.4% ± 8.2%, which was 18.3% ± 13.7% lower (P < 0.0001) than that in DMSO-treated cells (Fig 6B). These results indicated that the CDK1 inhibitor RO-3306 significantly inhibited the virus-induced increase in lamin B disassembly, suggesting that CDK1 mediates the baculovirus-induced disruption of the nuclear lamina.

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Fig 6. CDK1 mediates the baculovirus-induced disruption of the nuclear lamina.

(A and C) Subcellular localization of lamin B in baculovirus-infected cells following CDK1 inhibition. (A) Cells were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell and treated at 24 h p.i. with DMSO or 10 μM RO-3306. (C) Cells were infected with vAcWT-mCh2, vFLAG:CDK1, or vFLAG:CDK1 (DN) at an MOI of 20 TCID₅₀/cell. At 48 h p.i., the cells from (A) and (C) were fixed, permeabilized, and immunolabeled with the ADL101 antibody against lamin B. Virus-infected cells were monitored by mCherry fluorescence. DNA was stained with Hoechst 33258. Scale bar, 5 μM. (B and D) Quantification of the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B in (A) and (C) using ImageJ software. The data were pooled from two independent experiments with similar results and are presented as means ± SD (n = 60). Statistical significance was determined by unpaired two-tailed t-test (B) or one-way ANOVA followed by Tukey’s multiple comparison test (D) (ns, not significant; ****, P < 0.0001).

https://doi.org/10.1371/journal.ppat.1013991.g006

To further confirm the contribution of CDK1 to the baculovirus-induced disruption of the nuclear lamina, Sf9 cells were infected with vFLAG:CDK1, vFLAG:CDK1 (DN), or vAcWT-mCh2, and the distribution of lamin B was analyzed by immunofluorescence staining at 48 h p.i. As shown in Fig 6C and D, no significant difference (P > 0.05) in the nucleoplasmic distribution of lamin B was observed between vFLAG:CDK1- and vAcWT-mCh2-infected cells, and the nucleoplasm/whole nucleus fluorescence intensity ratios of lamin B were 73.6% ± 10.0% and 74.0% ± 9.0%, respectively; whereas the nucleoplasmic distribution of lamin B decreased in vFLAG:CDK1 (DN)-infected cells, and the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B was 63.9% ± 9.0%, which was 13.1% ± 17.0% lower (P < 0.0001) than that in vAcWT-mCh2-infected cells. These results indicated that the dominant-negative CDK1 mutant significantly inhibited the virus-induced increase in lamin B disassembly, which confirmed that CDK1 mediates the baculovirus-induced disruption of the nuclear lamina.

The nuclear lamina is a barrier to baculovirus replication

Baculovirus nucleocapsids are 30–60 nm in diameter and 250–300 nm in length; thus, they are too large to traffic through the nuclear lamina network (with an average crossover spacing of 5 nm) to access the budding site at the nuclear membrane [38,66]. Therefore, the nuclear lamina appears to be a barrier to baculovirus nuclear egress and subsequent BV production. To address this possibility, the disruption of the nuclear lamina was inhibited by suppressing CDK1 activity with RO-3306 or the dominant-negative CDK1 mutant, and the effect on infectious BV production was investigated. Sf9 cells were infected with vAcWT-mCh and incubated with 10 μM RO-3306 or DMSO at 24 h p.i. The supernatants were collected at 24, 48, 72, 96, and 120 h p.i., and virus titers were determined by 50% tissue culture infective dose (TCID₅₀) endpoint dilution assays. As shown in Fig 7A, in DMSO-treated cells, the virus titer surged between 24 and 48 h p.i., from 2.731 (± 0.402) × 10⁶ to 2.053 (± 0.407) × 10⁸, indicating a period of intensive nuclear egress of nucleocapsids. This robust replication was severely compromised by RO-3306 treatment, and this inhibition exhibited distinct kinetics. Specifically, while titers at 24 h p.i. were comparable to that in DMSO-treated cells (P > 0.05), a 91.8% ± 2.3% reduction was observed at the critical 48 h p.i. time point (P < 0.0001), with significant suppression persisting at 72 (88.9% ± 3.3%), 96 (72.9% ± 4.1%), and 120 h p.i. (68.4% ± 3.5%); all P < 0.0001. This phenotype was recapitulated genetically. vFLAG:CDK1-infected cells exhibited viral replication comparable to vAcWT-mCh2-infected controls (P > 0.05), confirming that FLAG-tagged wild-type CDK1 did not affect viral replication. In contrast, vFLAG:CDK1 (DN)-infected cells exhibited a major defect in BV production (Fig 7B), a kinetic profile that paralleled the one observed with pharmacological CDK1 inhibition. While titers at 24 h p.i. were comparable to vAcWT-mCh2-infected controls (P > 0.05), the most severe reduction (87.9% ± 5.3%, P < 0.0001) occurred at 48 h p.i.—mirroring the pharmacological inhibition. Significant suppression was maintained at 72 (84.3% ± 5.3%), 96 (80.5% ± 10.2%), and 120 h p.i. (65.0 ± 10.7%); all P < 0.0001. Collectively, these results indicated that the inhibition of CDK1 activity decreased infectious BV production, suggesting that the disruption of the nuclear lamina is critical for efficient baculovirus replication. The striking temporal correlation—where the peak defect in virus production at 48 h p.i. coincides with the period of most active nuclear egress (24–48 h p.i.) in controls—strongly suggested that the replication defect upon CDK1 inhibition may stem from a failure in nucleocapsid nuclear egress.

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Fig 7. The nuclear lamina is a barrier to infectious BV production.

The cells were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell and treated at 24 h p.i. with DMSO or 10 μM RO-3306 (A); the cells were infected with the indicated viruses at an MOI of 20 TCID₅₀/cell (B and C). The supernatants were harvested at the designated time points p.i., and BV titers were determined by TCID₅₀ endpoint dilution assays. The data were obtained from three independent experiments and are presented as means ± SD. Statistical significance was determined by two-way ANOVA followed by Tukey’s multiple comparison test (ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001).

https://doi.org/10.1371/journal.ppat.1013991.g007

To further confirm the contribution of lamina disruption to baculovirus replication, we examined whether phospho-deficient mutation of lamin B at S47, which also impairs baculovirus-induced disruption of the nuclear lamina, affects virus replication. To this end, a recombinant virus expressing GFP-tagged lamin B (S47A) under the control of the hr5 enhancer and, i.e.,1  promoter was constructed and designated vGFP:lamin B (S47A). As a control, a recombinant virus expressing GFP-tagged wild-type lamin B was generated and designated vGFP:lamin B. A pseudo-wild-type virus, vAcWT-GFP, was generated and used as an additional control. Sf9 cells were infected with the above viruses, and virus titers were determined at 24, 48, 72, 96, and 120 h p.i. As shown in Fig 7C, virus titers in vGFP:lamin B-infected cells were comparable to the vAcWT-GFP control (P > 0.05), indicating that exogenous wild-type lamin B does not interfere with viral replication. In contrast, viral replication was significantly impaired in cells expressing the S47A mutant, mirroring the kinetic inhibition profile of the aforementioned two CDK1-targeted interventions. Titers were reduced by 53.6% ± 15.3% (P < 0.05) at 24 h p.i., 63.9% ± 3.8% (P < 0.0001) at 48 h p.i., and 57.8% ± 22.0% (P < 0.01) at 72 h p.i. Although the reductions at 96 and 120 h p.i. did not reach statistical significance (P > 0.05), the persistent downward trend indicated a sustained inhibitory effect. Notably, the rapidly expressed exogenous nuclear lamina protein exerted an early inhibitory effect, restricting nucleocapsid nuclear egress as early as 24 h p.i. and thereby reducing BV production. This inhibition peaked at 48 h p.i., coinciding with the period of most active nuclear egress (24–48 h p.i.)—a temporal profile that reinforces the lamina barrier’s role in restricting nucleocapsid export. These results provide direct genetic evidence that the phosphorylation-dependent disassembly of the nuclear lamina is a requisite step for efficient baculovirus replication. The lamin B S47A mutant, by resisting lamina disruption, severely restricted virus production, suggesting that the nuclear lamina sustains a structural barrier that impedes nucleocapsid nuclear egress and thereby limits progeny virus production.

To further confirm whether the impaired production of infectious progeny viruses caused by inhibiting nuclear lamina disassembly was due to a defect in nucleocapsid nuclear egress, TEM analysis was performed to compare the nuclear egress of nucleocapsids between cells with inhibited lamina disassembly and control cells. Given that the CDK1 inhibitor RO-3306 exhibited the most pronounced inhibitory effect in BV production among the aforementioned experimental settings, we compared the nuclear egress of nucleocapsids in vAcWT-mCh-infected cells treated with either RO-3306 or the DMSO vehicle control. As shown in Fig 8A, numerous electron-dense rod-shaped nucleocapsids were observed in the nucleus of each sample, and the nucleocapsids in vAcWT-mCh-infected cells either treated with RO-3306 or treated with vehicle were morphologically indistinguishable. However, the number of egressing nucleocapsids, including those penetrating the nuclear membrane, transiting the cytoplasm, or budding at the plasma membrane, differed in the presence or absence of RO-3306. Egressing nucleocapsids were frequently observed in DMSO-treated cells but were only occasionally observed in cells treated with RO-3306. To quantify this observation, twenty infected cells in thin sections of each sample were randomly chosen, and the number of nucleocapsids in the nucleoplasm, penetrating the nuclear membrane, transiting the cytoplasm, and budding at the plasma membrane was counted. As shown in Fig 8B, the number of nucleocapsids observed in the nucleoplasm of each sample was comparable, and no significant difference (P > 0.05) was found in the number of nucleocapsids in the presence or absence of RO-3306, indicating that nucleocapsid assembly and production were not affected by the CDK1 inhibitor RO-3306. However, the number of egressing nucleocapsids in RO-3306-treated cells was much lower than that in DMSO-treated cells. Compared with that in DMSO-treated cells, the mean number of egressing nucleocapsids decreased by 66.3% ± 33.6% (P < 0.0001) in RO-3306-treated cells. This deficit was reflected across all egress stages by a significant reduction in nucleocapsids penetrating the nuclear membrane, transiting through the cytoplasm, and budding at the plasma membrane (all P < 0.01; Fig 8C). These results indicated that the CDK1 inhibitor RO-3306 significantly decreased efficient baculovirus nuclear egress, suggesting that the disruption of the nuclear lamina is required for this process.

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Fig 8. The nuclear lamina is a barrier to baculovirus nuclear egress.

(A) Localization of nucleocapsids in vAcWT-mCh-infected cells treated with DMSO or the CDK1 inhibitor RO-3306. vAcWT-mCh-infected cells (MOI of 20 TCID₅₀/cell) in the absence (DMSO) or presence of 10 μM RO-3306 (added at 24 h p.i.) were subjected to TEM at 48 h p.i. Nucleocapsids in the process of egress, including those penetrating the nuclear membrane (NM), transiting the cytoplasm, or budding at the plasma membrane (PM), are indicated with white arrowheads. Nu, nucleoplasm; Cy, cytoplasm. Scale bars, 500 nm. (B and C) Quantification of the nucleocapsids in (A). (B) Numbers of nucleocapsids residing in the nucleoplasm and in the process of egressing. (C) Numbers of nucleocapsids penetrating the NM, transiting the cytoplasm, or budding at the PM. The data were pooled from two independent experiments with similar results and are presented as means ± SD (n = 20). Statistical significance was determined by unpaired two-tailed t-test (ns, not significant; **, P < 0.01; ****, P < 0.0001).

https://doi.org/10.1371/journal.ppat.1013991.g008

Taken together, these data indicate that the disruption of the nuclear lamina is required for the efficient egress of nucleocapsids from the nucleus and the subsequent production of infectious BVs. The nuclear lamina represents a barrier to baculovirus replication.

Cells, viruses, strains, and antibodies

S. frugiperda IPLB-Sf21-AE clonal isolate 9 (Sf9) cells [113] were cultured at 27°C in Grace’s insect medium (Thermo Fisher Scientific, Waltham, USA) supplemented with 10% fetal bovine serum, 100 μg/mL penicillin, and 30 μg/mL streptomycin. The pseudo-wild-type virus vAcWT-mCh was constructed by inserting the polyhedrin (polh) and mCherry genes into the polh locus of the commercial bacmid bMON14272 (containing an AcMNPV genome) in the E. coli strain DH10B [53]. To prevent the random insertion of insertion sequence (IS) elements in DH10B [114], which may cause potential disruption of the baculovirus genome, both bMON14272 and vAcWT-mCh were extracted from DH10B cells, electroporated into the IS-free E. coli strain MDS42 ΔrecA Blue (Scarab Genomics, Wisconsin, USA) [115,116], and propagated exclusively in this strain for all downstream experiments. The E. coli strain MDS42 ΔrecA Blue, DH5α (RuiBiotech, Beijing, China), and BL21(AI) (Weidi Bio, Shanghai, China) were used for bacmid construction, plasmid construction, and protein expression, respectively.

The mouse monoclonal anti-lamin B antibodies ADL101 and ADL67 were purchased from the Developmental Studies Hybridoma Bank (Iowa, USA). The mouse monoclonal anti-FLAG antibody, rabbit polyclonal anti-pan phospho-serine/threonine antibody, mouse monoclonal anti-His antibody, mouse monoclonal anti-Strep-tag II antibody, and 50% slurry of Protein A/G agarose beads were purchased from Abmart (Shanghai, China). The mouse monoclonal anti-tubulin antibody, mouse monoclonal anti-GP64 AcC6 antibody, rabbit polyclonal anti-GFP antibody, and rabbit monoclonal anti-phospho-lamin A/C (S22) antibody were purchased from Beyotime (Shanghai, China), eBioscience (San Diego, USA), Proteintech (Chicago, USA), and Cell Signaling Technology (Massachusetts, USA), respectively. The rabbit polyclonal anti-AcMNPV VP39 antibody was generated by Li et al. [117]. The Alexa Fluor 488-conjugated donkey anti-rabbit antibody and the Alexa Fluor 647-conjugated donkey anti-mouse antibody were purchased from Thermo Fisher Scientific. The IRDye 680RD-conjugated goat anti-mouse or anti-rabbit IgG and IRDye 800CW-conjugated goat anti-mouse or anti-rabbit IgG were purchased from LI-COR (Nebraska, USA).

Construction of plasmids and viruses

To evaluate the specificity of the anti-human-phospho-lamin A/C (S22) antibody in Sf9 cells and to detect the contribution of the lamin B S47 phospho-deficient mutant to baculovirus-induced lamina disruption, a plasmid expressing N-terminally GFP-tagged wild-type Sf9 lamin B or its phospho-deficient mutant (Ser to Ala at position 47) was generated. The lamin B open reading frame (ORF) was PCR amplified from Sf9 cDNA with the primers lamin B-F and lamin B-R (the sequences of the primers used in this study are listed in S1 Table) and cloned into pIB-GFP [118] to generate the transient expression plasmid pIB-GFP:lamin B. The plasmid expressing the GFP-tagged lamin B phospho-deficient mutant, pIB-GFP:lamin B (S47A), was constructed by inserting the mutant fragment generated using overlap extension PCR as previously described [119] into the pIB-GFP vector. Briefly, the fragment GFP:lamin B (S47A) was amplified from pIB-GFP:lamin B by overlap PCR with the primers lamin B-F, S47A-R, S47A-F, and lamin B-R. The internal primers S47A-R and S47A-F were designed to generate intermediate fragments bearing overlapping, complementary 3′ ends and to introduce a targeted nucleotide substitution (AGC to GCA). The resulting full length mutant fragment was subsequently cloned into pIB-GFP to generate pIB-GFP:lamin B (S47A). Similarly, to assess the contribution of the lamin B S47 phospho-mimetic mutant to baculovirus-induced lamina disruption, the plasmid expressing the GFP-tagged lamin B phospho-mimetic mutant, pIB-GFP:lamin B (S47D), was constructed following the same overlap PCR approach. The mutant fragment GFP:lamin B (S47D) was amplified from pIB-GFP:lamin B with the primers lamin B-F, S47D-R, S47D-F, and lamin B-R, and then cloned into pIB-GFP.

To perform the in vitro kinase assay, plasmids expressing N-terminally His-tagged truncated wild-type Sf9 lamin B (N-terminal 1–150 aa) or its S47A mutant were constructed to provide the substrate component for the assay. The coding sequences of truncated wild-type Sf9 lamin B and its S47A mutant were PCR amplified from pIB-GFP:lamin B or pIB-GFP:lamin B (S47A) with the primers Hlamin B-F and Hlamin B-R. The resulting fragments were cloned into the pET-28a(+) vector (Novagen, Madison, USA), which contains an N-terminal 6 × His tag, to generate plasmids pET28a-His:lamin B (1–150) and pET28a-His:lamin B (1–150, S47A). For the kinase component, recombinant viruses expressing C-terminally His-tagged mutant CDK1 (vCDK1:His) and C-terminally Twin-Strep II-tagged cyclin B (vcyclin B:Strep) were generated by site-specific transposition, as previously described [120,121]. For vCDK1:His, a donor plasmid (pFB1^ts-CDK1:His) was constructed as follows: (i) a modified parental vector designated pFB1^ts-M was constructed from pFB1^ts-PH-GFP [122] by site-directed mutagenesis [123] using the primers 2KO-Ft, 2KO-Rs, 2KO-Fs, and 2KO-Rt to remove the polh and gfp genes, eliminating potential interference from Polh protein and the GFP fluorescent reporter; this vector retains the SV40 poly(A) signal and introduces several additional restriction sites to facilitate subsequent cloning. (ii) A fusion fragment containing the p6.9 promoter (from bMON14272) and the Sf9 cdk1 ORF tagged with an 8 × His sequence at the 3′ end (amplified from Sf9 cDNA) was generated by overlap PCR with the primers p6.9-F, p6.9-R, CDK1His-F, and CDK1His-R. (iii) To abolish inhibitory phosphorylation sites, which suppress CDK1 kinase activity, threonine 14 (T14) and tyrosine 15 (Y15) in the fusion fragment from step (ii) were mutated to alanine (T14A) and phenylalanine (Y15F), as previously reported [124], by overlap PCR using primers p6.9-F, 1415-R, 1415-F, and CDK1His-R. This final fragment was cloned into the vector pFB1^ts-M to yield the donor plasmid pFB1^ts-CDK1:His. For vcyclin B:Strep, a donor plasmid, pFB1^ts-cyclin B:Strep, was constructed. A fragment containing the p6.9 promoter (amplified from bMON14272) and the Sf9 cyclin B ORF tagged with a Twin-Strep II sequence at the 3′ end (amplified from Sf9 cDNA) was generated by overlap PCR with the primers p6.9-F2, p6.9-R2, cyclin B-F, and cyclin B-R. The resulting fragment was cloned into pFB1^ts-M to yield the donor plasmid pFB1^ts-cyclin B:Strep. These donor plasmids, pFB1^ts-CDK1:His and pFB1^ts-cyclin B:Strep, were transformed into electrocompetent MDS42 ΔrecA Blue cells harboring bMON14272 and the helper plasmid pMON7124^ts [122] to generate the recombinant viruses vCDK1:His and vcyclin B:Strep.

To inhibit the activity of CDK1 in baculovirus-infected cells, a recombinant virus expressing N-terminally FLAG-tagged wild-type CDK1 or its dominant-negative mutant, in which Asp at position 146 of CDK1 was replaced with Asn, was generated by site-specific transposition, as previously described [120]. To this end, a donor plasmid for the generation of recombinant virus expressing wild-type CDK1, designated pFB1^ts-PH-FLAG:CDK1-mCh2, was constructed as follow: (i) The fragment containing the cdk1 ORF tagged with the FLAG coding sequence at the 5′ end was PCR amplified from Sf9 cDNA with the primers FCDK1-F and CDK1-R and subsequently cloned into pIB/V5-His (Thermo Fisher Scientific) to generate the plasmid pIB-FLAG:CDK1. (ii) The fragment containing the hr5 enhancer-i.e.,1 promoter, FLAG:CDK1, and a simian virus 40 (SV40) poly(A) signal was amplified from bMON14272, pIB-FLAG:CDK1, and vAcWT-mCh by overlap PCR with the primers hr5-F, hr5-R, Pie1-F, Pie1-R, FCDK1-F2, CDK1-R2, SV40-F, and SV40-R. The resulting fragment was subsequently cloned into pFB1^ts-PH, which contains the polh gene and was modified from the plasmid pFB1^ts-PH-GFP [122] using site-directed mutagenesis, as previously described [123], with the primers GFPKO-Ft, GFPKO-Rs, GFPKO-Fs, and GFPKO-Rt, to generate the plasmid pFB1^ts-PH-FLAG:CDK1. (iii) The fragment containing the gp64 promoter, mCherry ORF, and SV40 poly(A) signal was amplified from vAcWT-mCh by overlap PCR with the primers Pgp64-F, Pgp64-R, mCh-F, and SV40-R2 and then cloned into the pFB1^ts-PH-FLAG:CDK1 to yield the final donor plasmid pFB1^ts-PH-FLAG:CDK1-mCh2. In parallel, a donor plasmid for the virus expressing a dominant-negative CDK1 mutant was subsequently constructed by overlap PCR, as previously described [119]. The mutant CDK1 sequence tagged with the FLAG coding sequence at the 5′ end was amplified from the plasmid pFB1^ts-PH-FLAG:CDK1 by overlap PCR with the primers hr5-F, D146N-R, D146N-F, and SV40-R3. The internal primers D146N-R and D146N-F were designed to produce intermediate segments with overlapping complementary 3′ ends and to introduce a targeted nucleotide substitution (AGC to GCA). The resulting fragment was subsequently cloned into pFB1^ts-PH-mCh2 to generate the donor plasmid pFB1^ts-PH-FLAG:CDK1 (DN)-mCh2. As a control, pFB1^ts-PH-mCh2 was generated by inserting a fragment containing the gp64 promoter, mCherry ORF, and SV40 poly(A) signal into pFB1^ts-PH. The above donor plasmids were transformed into electrocompetent MDS42 ΔrecA Blue cells harboring bMON14272 and the helper plasmid pMON7124^ts [122] to generate the recombinant viruses vFLAG:CDK1, vFLAG:CDK1 (DN), and vAcWT-mCh2.

To investigate the role of nuclear lamina disruption in baculoviral replication, we generated recombinant viruses expressing GFP:lamin B and GFP:lamin B (S47A). The DNA fragment containing the hr5 enhencer-i.e.,1 promoter, GFP-tagged wild-type or mutant lamin B, and SV40 poly(A) signal was amplified from vAcWT-mCh2, pIB-GFP:lamin B or pIB-GFP:lamin B (S47A), and vAcWT-mCh by overlap PCR with the primers hr5-F, Pie1-R2, GFP-F, lamin B-R2, SV40-F, and SV40-R2. The resulting fragment was subsequently cloned into pFB1^ts-PH to generate the donor plasmid pFB1^ts-PH-GFP:lamin B or pFB1^ts-PH-GFP:lamin B (S47A). As a control, a fragment containing the hr5 enhencer-i.e.,1 promoter, gfp ORF, and SV40 poly(A) signal was amplified from vAcWT-mCh2, pIB-GFP:lamin B, and vAcWT-mCh by overlap PCR with the primers hr5-F, Pie1-R2, GFP-F, GFP-R, SV40-F, and SV40-R2, and the resulting fragment was inserted into pFB1^ts-PH to generate the donor plasmid pFB1^ts-PH-GFP2. The above donor plasmids were transformed into electrocompetent MDS42 ΔrecA Blue cells harboring bMON14272 and the helper plasmid pMON7124^ts to generate the recombinant viruses vGFP:lamin B, vGFP:lamin B (S47A), and vAcWT-GFP via site-specific transposition.

All of the constructs were confirmed by PCR and sequencing. Plasmid DNA was extracted using an Endo-Free Plasmid Mini Kit (Omega, Georgia, USA) and quantified using a NanoDrop spectrophotometer. Bacmid DNA was isolated and purified using a Large-Construct Kit (Qiagen, California, USA) and quantified using a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific).

Immunofluorescence microscopy

Sf9 cells (5 × 10⁵) seeded on a 35-mm-diameter glass-bottom dish were transfected with 4 μg of purified plasmid DNA or 2 μg of purified bacmid DNA or infected with the recombinant virus at an MOI of 20 TCID₅₀/cell. At the indicated time points, the cells were processed for immunofluorescence staining and microscopy at room temperature, as previously described [118], with some modifications. Briefly, the cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 12 min and permeabilized with 0.2% Triton X-100 in PBS for 10 min. After being blocked in 3% bovine serum albumin in PBS for 30 min, the cells were incubated with the primary antibody for 1 h, washed twice with PBS for 5 min each, and then incubated with an Alexa Fluor 488-conjugated donkey anti-rabbit antibody (1:100) or an Alexa Fluor 647-conjugated donkey anti-mouse antibody (1:100) as the secondary antibody for 1 h, followed by two washes with PBS for 5 min each. Prior to analysis, the labeled cells were stained with Hoechst 33258 (Thermo Fisher Scientific) to identify the nucleus and DNA-rich regions. Images were captured using a Zeiss LSM 880 (Carl Zeiss, Baden-Württemberg, Germany) or Leica TCS SP8 (Leica Microsystems, Wetzlar, Germany) confocal laser scanning microscope with the same parameter settings used in each experiment. Three primary antibodies were used in this study: the mouse monoclonal anti-lamin B antibody ADL101 (1:50); the mouse monoclonal anti-lamin B antibody ADL67 (1:50); and a rabbit monoclonal anti-phospho-lamin A/C (S22) antibody (1:30).

Calculation of the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B

ImageJ software was used for region selection and lamin B fluorescence intensity quantification on the lamin B fluorescence channel. For the purpose of this study, we defined the nuclear rim as the peripheral region of the nucleus exhibiting high lamin B fluorescence intensity, and the nucleoplasm as the internal region excluding the nuclear rim (i.e., devoid of the high-intensity rim signal). Accordingly, the whole nucleus was defined as the area encompassing both the nuclear rim and the nucleoplasm. For whole nucleus region selection, the Wand (tracing) tool was applied to all cells. Specifically, we clicked the area immediately adjacent to the outer edge of the nuclear rim—this operation leveraged the high lamin B fluorescence signal at the nuclear rim to ensure precise delineation of the whole nucleus. For nucleoplasmic region selection, a tool-dependent two-step selection strategy was adopted to ensure objectivity and consistency across all cells. Initially, the Wand tool (with the same tolerance value as used for whole nucleus selection) was employed: we clicked the central area of the nucleoplasm that was sufficiently distant from the nuclear rim to avoid interference from its high-intensity signals. The automatic selection result was retained only if it met the following criteria: strictly corresponding to the nucleoplasmic area, excluding all nuclear rim signals, covering the entire nucleoplasm without omission, and exhibiting a clear boundary with the nuclear rim. If the automatic selection by the Wand tool failed to meet the above criteria (typically due to an indistinct intensity gradient between the nucleoplasm and nuclear rim), the result was discarded. The nucleoplasmic region was then manually traced using the Polygon selection tool, strictly adhering to the same definition as above by carefully outlining the area immediately adjacent to the inner edge of the nuclear rim, ensuring complete coverage of the nucleoplasm while carefully avoiding overlap with the high-intensity signals of the nuclear rim. The nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B was calculated by dividing the fluorescence intensity of the nucleoplasmic region by that of the whole nucleus. For quantitative reliability, 30 cells per replicate were randomly selected from two independent biological replicates for each experimental group. Representative examples of the region selection strategy (i.e., automatic selection with the Wand tool and manual tracing with the Polygon tool), which clearly depict the delineation of the whole nucleus, nucleoplasm, and nuclear rim, are provided in S7 Fig.

Western blot analysis

Sf9 cells (1 × 10⁶ cells/35-mm-diameter dish) were transfected with 8 μg of purified plasmid DNA or 4 μg of purified bacmid DNA or infected with recombinant virus at an MOI of 20 TCID₅₀/cell. At the indicated time points, the cells were collected via centrifugation at 2,000 × g for 5 min at 4°C. After being washed once with prechilled PBS, the pelleted cells were resuspended in PBS, mixed with 5 × protein sample buffer [250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue, 50% glycerol, and 5% 2-mercaptoethanol], and boiled at 100°C for 10 min. Equal volumes of each sample were resolved by SDS‒PAGE and electrophoretically transferred to 0.22-μm polyvinylidene difluoride membranes (Millipore, Darmstadt, Germany). Immunostaining was performed using monoclonal or polyclonal primary antibodies combined with IRDye-conjugated secondary antibodies. Proteins were visualized using an LI-COR Odyssey CLx scanner, and band intensities were quantified using Image Studio Ver 5.2 software. Ten primary antibodies were used in this study, including the mouse monoclonal anti-lamin B antibody ADL101 (1:1,000), a mouse monoclonal anti-tubulin antibody (1:1,000), a rabbit polyclonal anti-pan phospho-serine/threonine antibody (1:250), a rabbit monoclonal anti-phospho-lamin A/C (S22) antibody (1:500), a rabbit polyclonal anti-GFP antibody (1:1,000), a mouse monoclonal anti-FLAG antibody (1:1,000), a mouse monoclonal anti-His antibody (1:1,000), a mouse monoclonal anti-Strep-tag II antibody (1:1,000), a mouse monoclonal anti-GP64 antibody (1:1,000), and a rabbit polyclonal anti-VP39 antibody (1:5,000). Four secondary antibodies were used in this study, including an IRDye 680RD-conjugated goat anti-mouse IgG (1:10,000), an IRDye 680RD-conjugated goat anti-rabbit IgG (1:10,000), an IRDye 800CW-conjugated goat anti-mouse IgG (1:10,000), and an IRDye 800CW-conjugated goat anti-rabbit IgG (1:10,000).

Immunoprecipitation

To investigate whether endogenous lamin B phosphorylation was increased during baculovirus infection, Sf9 lamin B in virus-infected cells was enriched by immunoprecipitation, and its phosphorylation was detected by Western blotting with an anti-pan phospho-serine/threonine antibody. Briefly, Sf9 cells (1 × 10⁷ cells/100-mm-diameter dish) were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell. At the indicated time points p.i., the cells were harvested, centrifuged at 2,000 × g for 5 min at 4°C, washed once with prechilled PBS, and lysed in RIPA buffer (Thermo Fisher Scientific) supplemented with 2 μg/mL complete EDTA-free protease inhibitor cocktail and 10 μg/mL phosphatase inhibitor cocktail (Roche, Basel, Switzerland) on a vertical rotating mixer for 30 min at 4°C. The cell lysates were sonicated for 2 min on ice and then centrifuged at 17,000 × g for 10 min at 4°C. The supernatant was transferred to a fresh Eppendorf tube and mixed with 70 μL of a 50% slurry of Protein A/G agarose beads and 50 μL of the ADL101 antibody. After an incubation at 4°C for 8 h while rotating, the beads were collected by centrifugation at 1,000 × g for 5 min at 4°C, washed twice with 1 mL of prechilled PBS, and boiled in 40 μL of 1 × protein sample buffer for 10 min. The samples were subjected to Western blot analysis using an anti-pan phospho-serine/threonine antibody and the ADL101 antibody, as described above.

Protein expression and purification

N-terminally His-tagged truncated wild-type Sf9 lamin B (N-terminal 1–150 aa) and its S47A mutant were expressed in E. coli strain BL21(AI). Cells were cultured in LB medium supplemented with 100 μg/mL kanamycin at 37°C and with shaking at 200 rpm until the optical density at 600 nm reached ~0.5. Protein expression was induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside (IPTG) and 0.02% L-(+)-arabinose at 25°C for 8 h. Cells were collected by centrifugation (3,000 × g, 10 min, 4°C) and lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 20 mM imidazole, 10% TieChui E. coli lysis buffer (ACE Biotechnology, Hunan, China), and 2 μg/mL complete EDTA-free protease inhibitor cocktail (Roche). The suspension was rotated at 4°C for 30 min and then sonicated using a Sonics Vibra-Cell ultrasonic processor (Thermo Fisher Scientific) at 40% amplitude (40 cycles of 3 s on and 10 s off). Cell debris was removed by centrifugation (6,000 × g, 5 min, 4°C), and the supernatant was filtered through a 0.45 μm membrane. The clarified lysate was incubated with 4 mL Ni-NTA agarose resin (TransGen, Beijing, China) for 30 min at 4°C with gentle rotation. The resin was washed sequentially with wash buffers containing 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and increasing concentrations of imidazole (20, 40, 60, 80, 100, and 150 mM). Bound proteins were eluted with elution buffer containing 250 mM imidazole. Fractions containing His-tagged proteins were identified by SDS–PAGE, pooled, and concentrated using an Amicon Ultra centrifugal filter (3 kDa MWCO, Millipore). The buffer was exchanged to storage buffer [50 mM Tris-HCl (pH 8.0), 300 mM NaCl, and 10% glycerol].

The CDK1–cyclin B complex was expressed in Sf9 insect cells using the BEVS, consistent with the standard method for commercially available human CDK1–cyclin B complexes. 100 mL of Sf9 cells (2 × 10⁶ cells/mL) were co-infected with 10 mL each of recombinant P2 baculoviruses vCDK1:His and vcyclin B:Strep and incubated at 27°C with shaking at 120 rpm for 48 h. Cells were harvested by centrifugation (2,000 × g, 5 min, 4°C) and lysed in buffer containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl₂, 1 mM DTT, 10% glycerol, 0.2% Triton X-100, 2 μg/mL EDTA-free protease inhibitor cocktail, and 10 μg/mL phosphatase inhibitor cocktail (Roche). The suspension was rotated at 4°C for 30 min and then sonicated at 30% amplitude (8 cycles of 5 s on and 15 s off). Cell debris was removed by centrifugation (5,000 × g, 5 min, 4°C), and the supernatant was filtered through a 0.45 μm membrane. The clarified lysate was incubated with 4 mL Ni–NTA agarose resin (TransGen) for 30 min at 4°C with gentle rotation. The resin was washed sequentially with buffers containing 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl₂, 1 mM DTT, 10% glycerol, and increasing concentrations of imidazole (20, 40, 60, and 80 mM). Bound proteins were eluted with elution buffer containing 100 mM imidazole. Fractions containing the CDK1–cyclin B complex were identified by SDS–PAGE, pooled, and concentrated using an Amicon Ultra centrifugal filter (10 kDa MWCO, Millipore), and the buffer was exchanged to 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl₂, 1 mM DTT, and 10% glycerol.

Protein concentrations were determined by a Bradford protein assay (Bio-Rad, Hercules, USA). The purified proteins were resolved by SDS–PAGE, stained with Coomassie Brilliant Blue, imaged using a ChemiDoc MP imager (Bio-Rad), and verified by Western blotting with the indicated antibodies. The proteins were flash-frozen in liquid nitrogen and stored at -80°C.

In vitro kinase assay

The in vitro kinase assay was performed as previously described [28] with some modifications. Briefly, 0.2 μg of recombinant CDK1–cyclin B complex was incubated with 10 μg of His-tagged truncated wild-type lamin B (N-terminal 1–150 aa) or its S47A mutant in 100 μL of kinase reaction mixture containing kinase buffer [50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM EGTA, and 1 mM DTT] and 100 μM ATP. The reaction was carried out at 25°C for 10 min, terminated by adding 5 × protein sample buffer, followed by boiling at 100°C for 10 min. Proteins were resolved by SDS–PAGE and analyzed by immunoblotting with the indicated antibodies.

Analysis of viral propagation

Sf9 cells (1 × 10⁶ cells/35-mm-diameter dish) were infected with vAcWT-mCh (treated with DMSO or 10 μM RO-3306 at 24 h p.i.), vAcWT-mCh2, vFLAG:CDK1, vFLAG:CDK1 (DN), vAcWT-GFP, vGFP:lamin B, or vGFP:lamin B (S47A) at an MOI of 20 TCID₅₀/cell. At the designated time points p.i., the BV-containing supernatants were harvested and centrifuged at 3,000 × g for 10 min to remove the cell debris. The BV titers were determined by a TCID₅₀ endpoint dilution assay in Sf9 cells [125].

Transmission electron microscopy

Sf9 cells (1 × 10⁶ cells/35-mm-diameter dish) were infected with vAcWT-mCh at an MOI of 20 TCID₅₀/cell. At 24 h p.i., the cells were treated with DMSO or 10 μM RO-3306. At 48 h p.i., the cells were dislodged with a rubber policeman and pelleted by centrifugation at 1,000 × g for 5 min at 4°C. Then, the cell pellets were fixed, dehydrated, embedded, sectioned, and stained, as previously described [126]. The sections were collected at 20 μm intervals to avoid analyzing the same cell in multiple sections. The samples were examined with a JEM-100CX/II transmission electron microscope (JEOL, Tokyo, Japan) at an accelerating voltage of 120 kV.

Statistical analysis

The data are presented as means ± standard deviation (SD) from at least two independent experiments. Statistical significance was determined by unpaired two-tailed t-test for comparisons between two groups and by one-way or two-way ANOVA followed by Tukey’s multiple comparison test for comparisons between multiple groups. All the statistical analyses were performed using GraphPad Prism 9.0.0, with P < 0.05 considered statistically significant.