Baculoviruses exploit the mitotic kinase CDK1 to disrupt the nuclear lamina
Mei Mo, Silan Yu, Yushan Yang, Jiongjiong Liu, Kai Yang, Meijin Yuan
TL;DR
Baculoviruses use the host's CDK1 kinase to disrupt the nuclear lamina, enabling efficient viral replication and nucleocapsid egress.
Contribution
Baculoviruses are shown to exploit CDK1 to phosphorylate lamin B at S47, a novel mechanism for nuclear lamina disruption.
Findings
Baculovirus infection triggers endogenous nuclear lamina disassembly via phosphorylation of lamin B at S47.
CDK1 is directly used by baculoviruses to phosphorylate lamin B, enabling efficient viral nucleocapsid egress.
Lamina disruption is essential for the production of infectious baculovirus particles.
Abstract
The nuclear lamina is disassembled during mitosis, and certain DNA viruses exploit this process to facilitate replication. While we previously showed that baculoviruses disrupt the exogenously integrated lamina, their impact on the endogenous structure, the underlying mechanism, and the functional consequences for viral replication remained unknown. Here, we demonstrate that baculovirus infection triggers endogenous nuclear lamina disassembly, and that phosphorylation of lamin B at the N-terminal “mitotic site” serine 47 (S47) is the key event driving this process. Using in vitro phosphorylation assays, phospho-specific reagents, and site-directed mutagenesis, we further show that baculoviruses exploit the mitotic kinase cyclin-dependent kinase 1 (CDK1) to directly phosphorylate S47, thereby disrupting the lamina. Critically, this baculovirus-induced lamina disruption is not an…
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Fig 8- —http://dx.doi.org/10.13039/501100001809National Natural Science Foundation of China
- —http://dx.doi.org/10.13039/501100001809National Natural Science Foundation of China
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Taxonomy
TopicsNuclear Structure and Function · Microtubule and mitosis dynamics · Biotin and Related Studies
Introduction
The nuclear lamina is a rigid protein meshwork underlying the nucleoplasmic face of the inner nuclear membrane (INM) in all metazoans [1–3]. The principal components of the nuclear lamina are type V intermediate filament proteins known as lamins [4–7]. Lamins are categorized into two types: A-type lamins, including lamin A and lamin C, and B-type lamins, including lamin B1, lamin B2, and lamin B3 [8,9]. While most vertebrates have one A-type lamin gene and two B-type lamin genes, invertebrates generally possess only a single lamin gene of the B-type, with some exceptions, such as Drosophila, which expresses one B-type lamin (lamin Dm0) and one A-type lamin (lamin C) encoded by two distinct genes [10–12]. Like all intermediate filaments, lamins are composed of a short N-terminal head domain, a long coiled-coil central rod domain, and a globular C-terminal tail domain [4,13]. The nuclear lamina plays an important role in maintaining the structural integrity of the nucleus [11,14]. Accordingly, such a rigid meshwork must be disassembled during mitosis and then reassembled after mitosis along with the nuclear envelope [15–17]. These dynamic processes are regulated by the site-specific phosphorylation of lamins. The phosphorylation of lamins by cyclin-dependent kinase 1 (CDK1) at the N-terminal “mitotic site” is crucial for lamina disassembly during mitosis [18–24]. This phosphorylation site is highly conserved and shared by all lamins except for a single Caenorhabditis elegans lamin, and the corresponding site in human lamin A/C, the serine at residue 22 (S22), has been extensively studied for its role in lamina disassembly [18,20,25–28]. Mechanistic investigations have revealed that S22 phosphorylation of lamin A/C mediates lamina disassembly through two distinct pathways: by recruiting the peptidyl-prolyl cis/trans isomerase (PPIase) Pin1 to induce conformational changes [29]; and by disrupting intramolecular ionic interactions between lamin dimers [30].
Disassembly of the nuclear lamina has been observed not only during mitosis but also during infection with various DNA viruses, which assemble their nucleocapsids in the host cell nucleus, facing the rigid lamina meshwork underlying the INM as a critical barrier to nuclear egress of intranuclear nucleocapsids [27,31]. For example, herpesviruses encode conserved herpesvirus protein kinases (CHPKs)—e.g., pUL97 in human cytomegalovirus (HCMV) and BGLF4 in Epstein-Barr virus (EBV)—that mimic CDK1 activity to phosphorylate lamin A/C at the N-terminal “mitotic site”, disrupting the nuclear lamina to facilitate nuclear egress [32–34]; cellular kinases such as protein kinase C (PKC) have been reported to be recruited for lamin phosphorylation during herpesvirus infection, though direct evidence for PKC targeting the “mitotic site” is lacking [35,36]. In contrast, circoviruses, with limited coding capacity, rely on host PKC to phosphorylate lamin A/C at the N-terminal “mitotic site”, promoting lamina rearrangement for viral nuclear egress [37]. Baculoviruses are a group of large DNA viruses with circular double-stranded genomes packaged within enveloped, rod-shaped nucleocapsids, and their genome replication and nucleocapsid assembly also take place within the host cell nucleus [38–40]. Some progeny nucleocapsids are subsequently transported from the nucleus to the cytoplasm and obtain their envelopes at the cell surface to produce budded virions (BVs) [41–45]. A critical step in BV production is the nuclear egress of nucleocapsids. The results of a recent study indicated that baculoviruses do not egress from the nucleus via the nuclear pore complex but instead locally disrupt the nuclear envelope and release nucleocapsids into the cytoplasm [46]. Meanwhile, large transmission electron microscopy (TEM) studies of NPVs have shown that the nuclear egress of baculovirus nucleocapsids involves a budding process at the nuclear membrane [47–52]. Nucleocapsids have been observed budding at the evaginations of the nuclear membrane toward the cytoplasm, producing transport vesicles containing nucleocapsid(s) surrounded by two membranes in the cytoplasm [47,48]. Nucleocapsids budding through the INM into an enlarged perinuclear space and “enveloped” nucleocapsids residing in the perinuclear space have also been observed [49–52]. Regardless of whether baculovirus nucleocapsids egress from the nucleus by disrupting the nuclear envelope or by budding from the nuclear membrane, the rigid nuclear lamina meshwork underlying the nucleoplasmic face of the INM represents a barrier to nuclear egress. In addition, baculovirus nucleocapsids are 30–60 nm in diameter and 250–300 nm in length [38] and thus are too large to traverse the crossover spacing of the nuclear lamina. Therefore, similar to herpesviruses and circoviruses, baculoviruses should modify the nuclear lamina to gain access to the egress site at the nuclear membrane.
Our previous study showed that the exogenously integrated nuclear lamina was partially disrupted and that some green fluorescent protein (GFP)-lamin B was redistributed from the nuclear rim into the nucleoplasm of baculovirus-infected Sf9-L cells stably expressing GFP-tagged Drosophila lamin B, as monitored by GFP fluorescence [53]. In addition, a recent study revealed the localized dissolution of mCherry-lamin B during baculovirus infection, which validates our findings [46]. However, Wei et al. found that the nuclear lamina of Sf9 cells did not undergo remarkable structural alterations, and no lamin B was observed within the nucleoplasm during baculovirus infection using ADL67, a mouse monoclonal antibody that recognizes the tail domain of Drosophila lamin B, to detect the distribution of the endogenous Sf9 lamin B [54]. Given that herpes simplex virus (HSV) infection masks the epitope within the tail domain of lamin A/C and reduces its antibody staining [55,56], the inability to detect lamina structural alterations upon baculovirus infection using the ADL67 antibody may likewise result from epitope masking in the lamin B tail domain. Therefore, whether baculovirus infection induces endogenous nuclear lamina disruption and how the nuclear lamina undergoes disruption in Sf9 cells remain unknown. In this study, we address these unresolved questions by identifying a distinct mechanism: baculoviruses uniquely co-opt the host mitotic kinase CDK1 to disrupt the nuclear lamina in Sf9 cells.
We report the following key findings that define this mechanism. We first confirmed that the endogenous nuclear lamina is partially disrupted during baculovirus infection, using ADL101—a mouse monoclonal antibody that recognizes the rod domain of Drosophila lamin B and cross-reacts with Sf9 lamin B [57]. Building on this observation, we uncovered the molecular mechanism underlying this disruption: unlike other previously studied viruses, baculoviruses exploit the host cell mitotic pathway to disrupt the nuclear lamina. Specifically, they utilize CDK1, the kinase that phosphorylates lamins during mitosis, to phosphorylate the conserved N-terminal “mitotic site” serine at residue 47 (S47) in lamin B to disrupt the nuclear lamina. We further investigated the role of lamina disruption in baculovirus replication and found that it is required for the efficient nuclear egress of nucleocapsids and the subsequent production of infectious BVs. Thus, we provide the first evidence that the nuclear lamina represents a barrier to baculovirus replication. Baculovirus is, to our knowledge, the first virus identified to employ CDK1 to disrupt the nuclear lamina. These findings offer valuable insights into virus‒host interactions, expanding our understanding of viral exploitation of the host cellular machinery.
Results
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.
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).
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].
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.
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.
*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).
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.
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).
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.
*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).
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.
*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).
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 (TCID_50_) 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.
*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).
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.
*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).
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.
Discussion
The nuclear lamina represents a physical barrier against certain DNA viruses, restricting nucleocapsid access to the nuclear membrane and thereby impairing nuclear egress and viral replication. Herpesviruses and circoviruses overcome this barrier through the phosphorylation of lamins and subsequent disruption of the nuclear lamina [29,31,32,37]. In the case of baculoviruses, our previous study showed that baculovirus infection induces the disruption of the exogenously integrated nuclear lamina [53]. However, whether the endogenous nuclear lamina undergoes disruption during baculovirus infection and the underlying mechanism remain to be elucidated. In the present study, we demonstrated that baculovirus infection induces the disruption of the endogenous nuclear lamina through a mechanism involving CDK1-mediated phosphorylation of lamin B at the conserved N-terminal “mitotic site” S47. Furthermore, we present evidence that the nuclear lamina serves as a critical barrier to baculovirus nuclear egress and that lamina disruption is required for efficient nuclear exit of nucleocapsids.
Immunofluorescence staining using the ADL101 antibody, an antibody that recognizes the rod domain of lamin B, revealed that baculovirus infection induces a partial disruption of the nuclear lamina, with a redistribution of lamin B from the nuclear rim to the nucleoplasm (Fig 1A and B). However, no fluorescence signal was detected within the nucleoplasm when the ADL67 antibody that recognizes the tail domain of lamin B was used (S6 Fig), which was consistent with the results of Wei et al*.*, who employed the same antibody [54]. These results suggest that the ADL101 antibody is capable of recognizing disassembled lamin B, whereas the ADL67 antibody is not. Similarly, inconsistencies in experimental outcomes due to antibody targeting of distinct antigen epitopes have also been reported in studies of herpesvirus-induced lamina disruption. An immunofluorescence analysis of lamin A/C during HSV-1 infection showed that the intensity of lamin staining was greatly reduced in virus-infected cells when a monoclonal antibody that recognizes an epitope in the lamin A/C tail domain was used, whereas virtually no difference in lamin staining was detected between infected and uninfected cells when a polyclonal antibody raised against full-length lamin A/C was used [55]. Moreover, similar results were observed in HSV-2-infected cells with a monoclonal antibody recognizing an epitope in the lamin A/C tail domain and a polyclonal antibody recognizing an epitope in the rod domain of lamin A/C [56]. The authors proposed that HSV infection induces conformational changes in lamin A/C that mask its epitope for the monoclonal antibody in the tail domain [55,56]. Because the ADL101 antibody targets an epitope in the rod domain of lamin B, while the ADL67 antibody recognizes the tail domain of lamin B, it is probable that baculovirus infection also induces conformational changes in the tail domain of lamin B. In addition to conformational changes, the hyperphosphorylation of lamins is thought to contribute to the loss of immunoreactivity of lamin by some monoclonal antibodies in virus-infected cells [67]. Consistent with this finding, peptide competition experiments provided direct evidence that phosphorylation of serine at residue 25 impedes lamin binding by the anti-lamin B antibody ADL84 [68], indicating that phosphorylation at the recognition site interferes with antibody binding. Based on these findings, it cannot be ruled out that one or more sites in the tail domain of lamin B are phosphorylated in baculovirus-infected cells, thereby preventing recognition by the ADL67 antibody. Further work will be necessary to confirm whether the loss of lamin B recognition by the ADL67 antibody in virus-infected cells was due to either a conformational change or hyperphosphorylation at the tail domain and to determine the details and mechanism underlying these events.
Time course analysis of the phosphorylation of lamin B at S47 (Fig 2D and E) and the subcellular localization of lamin B (Fig 1A and B) revealed a progressive increase in both the levels of S47 phosphorylation and the disassembly of lamin B from 24 to 48 h p.i. On this basis, when virus-infected cells were incubated with a CDK1 inhibitor at 24 h p.i., both the level of S47 phosphorylation and the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B at 48 h p.i. were expected to be inhibited at levels between 24 and 48 h p.i. However, after treatment with the CDK1 inhibitor RO-3306, the level of S47 phosphorylation and the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B were reduced by 94.5% and 18.3% (Fig 5B and Fig 6B), with much greater reductions than the changes observed between 24 and 48 h p.i. from time course analyses, as the level of S47 phosphorylation and the nucleoplasm/whole nucleus fluorescence intensity ratio of lamin B at 24 h p.i. were 40.7% and 7.01% lower than those at 48 h p.i. (Fig 2E and Fig 1B). One possible explanation for these pronounced decreases in the presence of a CDK1 inhibitor is that the baculovirus-induced lamina disruption is a dynamic event in which lamin B undergoes continuous phosphorylation and dephosphorylation. While CDK1 inhibitor treatment suppressed the phosphorylation of lamin B at S47, the dephosphorylation processes remained active. This combination led to a significant decrease in the overall phosphorylation of lamin B at S47, dropping it below the level observed prior to the inhibitor treatment. Subsequently, the dephosphorylated lamin B gradually reassembled into the nuclear lamina, leading to a corresponding decrease in the amount of lamin B retained within the nucleoplasm. This explanation is consistent with the results of our previous study, which showed a dynamic alteration in lamin B distribution in which a portion of GFP-Drosophila lamin B at the nuclear rim diffused and then quickly recovered during baculovirus infection in Sf9-L cells [53]. As a typical baculovirus infection cycle results in the production of two forms of virions, BVs and occlusion-derived virions (ODVs), and the morphogenesis of ODVs occurs in the nucleoplasm [40,48,69], the manipulation of nuclear lamina dynamics by baculovirus not only partially disrupts the nuclear lamina to promote the nuclear egress of nucleocapsids but also maintains the nuclear integrity for ODV morphogenesis within the nucleus, thereby facilitating the baculovirus biphasic replication cycle.
Extensive studies have provided evidence that the N-terminal “mitotic site”, a canonical phosphoacceptor residue that is highly conserved among lamins, is highly relevant to lamina disassembly. During mitosis, the phosphorylation of this site by CDK1 triggers lamina disassembly. For example, S22 in human lamin A/C [18,20,25,28], S16 in chicken lamin B2 [19,21,22], and S45 in Drosophila lamin B [24] have been identified as CDK1-mediated phosphorylation sites; the phosphorylation of these sites induces lamina disassembly in the corresponding species, and the serine-to-alanine substitutions within these sites block mitotic disassembly of lamin filaments in vivo. The phosphorylation of the N-terminal “mitotic site” is not only essential for mitotic lamina disassembly but also plays a crucial role in virus-induced lamina disruption. For example, the vertebrate viruses circoviruses [37] and herpesviruses [29,32,33] target S22 of lamin A/C to mediate lamina disruption; inhibition of porcine lamin A/C phosphorylation at S22 blocks the circovirus-induced redistribution of lamin A/C from the nuclear rim to the nucleoplasm [37], whereas the phospho-deficient mutation of human lamin A/C at S22 is resistant to the herpesvirus-induced redistribution of lamin A/C [29]. In the present study, we demonstrated that S47, the N-terminal “mitotic site” in the S. frugiperda (insect) lamin B protein, is a key phosphorylation site for baculovirus-induced lamina disruption; the phospho-deficient mutation of lamin B at S47 significantly inhibited the virus-induced increase in lamin B disassembly. Our data, together with those of previous studies, suggested that lamina disassembly is primarily mediated by the conserved N-terminal “mitotic site”, regardless of whether it occurs during mitosis or viral infection and whether it is in vertebrates or invertebrates, implying the high evolutionary conservation of N-terminal “mitotic site”-triggered lamina disassembly.
Analysis of the effect of lamin B S47 phospho-mimetic mutant expression on Sf9 lamina disassembly showed that mimicking phosphorylation at S47 promotes lamin B nucleoplasmic distribution, but does not lead to its complete nuclear localization (Fig 3C and D); that is, it fails to induce full lamina disassembly. This finding suggests that S47 alone is insufficient to drive complete Sf9 lamina disassembly, and additional phosphorylation sites must act synergistically with S47 to mediate this process. This multi-site synergistic regulatory mechanism for lamina disassembly is evolutionarily conserved, as supported by studies in other species [18,24,25,28,30,70,71]. In humans, lamin A/C—the most well-characterized lamin family member—relies on synergistic phosphorylation of multiple sites for lamina disassembly: mutant lamin proteins display progressively enhanced nucleoplasmic distribution as the number of phospho-mimetic mutations increases; single-site phospho-mimetic mutation of S22 fails to trigger full lamina disassembly, and even simultaneous mutation of four key phosphorylation sites (T19, S22, S390, and S392 in lamin A) is insufficient for complete lamina disassembly [28]. Sequence alignment shows that human lamin A residues T19 and S390 correspond to S44 and T415 in Sf9 lamin B, whereas S392 is not conserved (S1A and S1B Fig). In insects, Drosophila lamin B also employs a multi-site phosphorylation mechanism for lamina disassembly—besides the conserved N-terminal “mitotic site” S45, phosphorylation at S42 and S50 has been validated to promote lamina disassembly [24], and these two sites are homologous to S44 and T52 in Sf9 lamin B (S1B Fig). Combined with our initial finding on S47, these data collectively imply that homologous sites (S44, T52, and T415) in Sf9 lamin B may synergize with S47 to regulate lamina disassembly. Our studies on the contribution of S47 phosphorylation to lamina disassembly during baculovirus infection further reinforce this multi-site regulatory hypothesis: GFP-tagged S47A mutant lamin B exhibited reduced rather than completely abrogated lamina disassembly upon viral infection compared to uninfected cells (Fig 3A and B), demonstrating that in addition to S47, other phosphorylation sites are indeed involved in lamina disassembly during viral infection. Taken together, these findings suggest that baculovirus infection may induce the phosphorylation of S47 and the aforementioned sites (S44, T52, and T415) in Sf9 lamin B. Future studies will validate whether the synergistic phosphorylation among S47 and these candidate sites is required for complete lamina disassembly during baculovirus infection.
Although N-terminal “mitotic site”-mediated lamina disruption is highly conserved during virus infections, the kinases utilized vary among different viruses. Circoviruses utilize the host kinase PKC to phosphorylate lamin A/C, thereby inducing lamina disruption [37]. For herpesviruses, in addition to utilizing PKC [35,36], they encode CHPKs (e.g., pUL97 in HCMV and BGLF4 in EBV) to phosphorylate lamin A/C and disrupt the nuclear lamina [29,32,33]. In the present study, we found that baculovirus-induced lamina disruption is mediated by CDK1, the enzyme that is responsible for the phosphorylation of lamins and the breakdown of the nuclear lamina during mitosis. These findings prompt the question of why viruses selectively exploit distinct kinases to disrupt the nuclear lamina. This property could be associated with the cell cycle arrest in distinct phases induced by different viruses. Circovirus and herpesvirus infections arrest the cell cycle in interphase [72,73]. Therefore, they ingeniously exploit PKC [35–37], the key kinase for lamin phosphorylation during interphase [74–76], to phosphorylate lamin A/C and thereby disrupt the nuclear lamina. In addition, as herpesviruses are unable to utilize the CDK1 pathway for lamina disruption due to G1/S arrest, they have evolved to encode CHPKs to mimic CDK1 activity to overcome this limitation [32]. Unlike the viruses mentioned above, baculoviruses arrest cells in G2/M phase of the cell cycle [59]. Therefore, they ingeniously exploit the host CDK1-mediated mitotic lamina disassembly pathway to phosphorylate lamin B and disrupt the nuclear lamina. This CDK1-dependent strategy stands in stark contrast to the herpesvirus reliance on encoded CHPKs, and reveals a distinct paradigm: unlike herpesviruses that must devote genetic resources to encoding mimics to access mitotic machinery, baculovirus have evolved to co-opt the host’s endogenously CDK1 pathway. This direct co-option of an endogenously host kinase during G2/M arrest represents a fundamentally different and more economical viral strategy for viral nuclear egress. The fact that viruses utilize kinases specific to the host cell cycle phase they arrest to facilitate their replication illustrates their precise evolutionary adaptation to exploit the host cellular machinery.
While our findings confirmed that baculoviruses exploit the G2/M phase-specific host kinase CDK1 as a key mediator of lamin B phosphorylation and subsequent nuclear lamina disassembly, consistent with their evolutionary adaptation to utilize cell cycle-matched host machinery, the precise molecular mechanism by which baculoviruses utilize CDK1 to achieve this biological effect remains unclear. CDK activity is tightly regulated by a complex upstream signaling network, including cyclin-dependent activation (e.g., cyclin B), phosphatase-mediated dephosphorylation (e.g., Cdc25), and kinase-mediated phosphorylation (e.g., CDK-activating kinase, CAK) [77–80]. Viruses frequently subvert this regulatory signaling network to manipulate host CDK activity for their own replication [81–84]. For instance, Kaposi’s sarcoma-associated herpesvirus (KSHV) expresses a viral cyclin (v-cyclin) that activates CDK6, driving cells into S phase to facilitate viral DNA replication [85–88]. Similarly, HCMV directly encodes a functional CDK homolog, the UL97 kinase, which not only modulates host cell cycle regulatory pathways but also phosphorylates lamin A/C to disrupt the nuclear lamina [32,34,73,89–91]. Based on these well-characterized viral strategies, it is plausible to propose that baculoviruses adopt a similar strategy by encoding viral proteins that mimic CDK1 regulators—such as cyclin B homolog, phosphatase, or CDK-activating kinase—to enhance CDK1 activity and mediate lamin B phosphorylation. Beyond direct modulation of CDK1 activity, viruses also frequently utilize scaffold complexes to spatially recruit kinases to the nuclear lamina—a mechanism best characterized in herpesviruses. In herpesvirus-infected cells, the nuclear egress complex (NEC) functions as a central scaffold. It consists of two conserved viral proteins (e.g., HSV-1 pUL34–pUL31, HCMV pUL50–pUL53, and EBV BFRF1–BFLF2), assembles at the INM, and recruits kinases such as CHPKs and PKC to the lamina to promote localized lamin phosphorylation and lamina disassembly [35,58,92–97]. This spatial coordination is critical for avoiding global disruption of host cell architecture while enabling viral egress. It remains an open question whether baculoviruses assemble a functionally analogous NEC-like complex that recruits CDK1 to the nuclear lamina for lamin B phosphorylation, thereby promoting lamina disassembly. To address these gaps, a promising future research direction would involve identifying protein complexes associated with lamin B and CDK1 during baculovirus infection, using approaches such as co-immunoprecipitation coupled with mass spectrometry. This strategy could reveal potential cellular and viral interactors including candidate CDK1 regulators and NEC-like scaffold proteins, providing crucial insights into how baculoviruses utilize CDK1 for nuclear lamina disassembly.
Although our findings establish CDK1 as a core kinase mediating baculovirus-induced lamin B S47 phosphorylation and lamina disassembly, compelling evidence suggests the existence of a more complex kinase network orchestrating this process. Specifically, Western blot and immunofluorescence analyses showed that lamin B phosphorylation at S47 and lamina disassembly were not completely inhibited when cells were treated with the CDK1 inhibitor RO-3306 at 24 h p.i. (Fig 5A and B; Fig 6A and B). While one plausible explanation for this partial inhibition is the relatively late timing of inhibitor addition, as S47 phosphorylation and lamina disassembly had already initiated by 24 h p.i. (Fig 2D and E; Fig 1A and B), we cannot rule out the possibility that other kinases (host-derived or virus-encoded) also contribute to this process. Among host kinases, several candidates have been reported to phosphorylate homologous sites of Sf9 lamin B S47 in other species, though their relevance to baculovirus infection requires careful consideration. For instance, it has been shown that CDK5 is capable of phosphorylating human lamin A/C at S22, but its activity is primarily restricted to terminally differentiated cells (e.g., neurons), making it less likely to act in the context of baculovirus-infected cells. Similarly, mitogen-activated protein kinases (MAPKs) have been reported to phosphorylate chicken lamin B2 at S16 [98]; however, subsequent studies have revealed that MAPKs act as upstream regulators of CDK1 [99–102], and thus its effect on the S16 site is most likely indirect. Additionally, PKC has been implicated in phosphorylating the S22 homolog of lamin A/C during circovirus infection [37]; however, as discussed above, PKC functions predominantly in interphase [74–76], whereas baculoviruses arrest cells in G2/M phase of the cell cycle, creating a temporal mismatch that limits PKC’s potential role in baculovirus-induced lamin B S47 phosphorylation and lamina disassembly. Taken together, these considerations suggest that while host kinases beyond CDK1 may contribute, their relevance in the specific context of baculovirus-induced lamina disruption remains uncertain. Nevertheless, we cannot exclude the possibility that other, as-yet-unidentified host kinases may also participate in this process. Beyond host kinases, virus-encoded kinases also merit consideration. Many viruses encode kinases to directly manipulate host structural proteins, with lamin phosphorylation being a well-documented target—for instance, during herpesvirus infection, the viral kinases CHPKs directly phosphorylate lamin A/C at S22 to promote lamina disassembly [29,32,33,58,103]. Intriguingly, baculoviruses encode two putative protein kinases, PK1 and PK2, whose roles in viral replication are only partially characterized [104–106]. Given their predicted kinase activity and precedents from other viruses, it is reasonable to hypothesize that PK1 and/or PK2 may directly or indirectly participate in lamin B S47 phosphorylation, complementing CDK1 to drive efficient lamina disassembly. These observations highlight the need to identify additional kinases involved in lamin B S47 phosphorylation and lamina disassembly during baculovirus infection. Notably, although in vitro biochemical and cellular evidence strongly supports a direct role for CDK1 in phosphorylating lamin B at S47, we acknowledge that CDK1’s contribution could also be partially indirect, given the complex regulation of nuclear lamina dynamics and the fact that CDK1 activity is integrated into broader signaling networks [17,25,107–110]. Lamin B dynamics, as we have discussed earlier in this section, are likely governed by a complex kinase network; CDK1, as a master mitotic regulator [108,111,112], may function as a key node within this network, orchestrating or priming the activity of other host or virus-encoded kinases (e.g., the putative viral kinases PK1/PK2) that subsequently converge on S47. Such indirect effects might cooperate with direct CDK1-mediated phosphorylation to ensure efficient nuclear lamina disruption. To determine the indirect involvement of CDK1 and fully delineate the signaling cascades governing lamin B S47 phosphorylation during baculovirus infection, future studies utilizing kinome-wide screening combined with targeted validation assays will be required to dissect the intricate regulatory interplay between CDK1 and other kinase pathways involved in this process.
In conclusion, the data presented in this work demonstrate that baculoviruses exploit the CDK1-mediated mitotic lamina disassembly pathway to phosphorylate lamin B and disrupt the nuclear lamina, thereby adapting to the cell cycle arrest at G2/M phase they induce. Although many DNA viruses utilize the conserved N-terminal “mitotic site” for lamina disruption, to the best of our knowledge, baculovirus is the first virus discovered to exploit the canonical CDK1 pathway for the phosphorylation of this site. The viral repurposing of this critical cellular process not only provides new insights into the complex interaction between viral pathogens and the host cellular machinery but also suggests an evolutionary adaptation that enables baculoviruses to optimize their life cycle within the host cellular environment.
Materials and methods
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 × 10^5^) 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 TCID_50_/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 × 10^6^ 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 TCID_50_/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 × 10^7^ cells/100-mm-diameter dish) were infected with vAcWT-mCh at an MOI of 20 TCID_50_/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 × 10^6^ 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 TCID_50_/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 TCID_50_ endpoint dilution assay in Sf9 cells [125].
Transmission electron microscopy
Sf9 cells (1 × 10^6^ cells/35-mm-diameter dish) were infected with vAcWT-mCh at an MOI of 20 TCID_50_/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.
Supporting information
S1 FigMultiple sequence alignment of the N-terminal head (A) and partial C-terminal tail (B) domains of selected lamins.The alignment was performed with Geneious Prime software using ClustalW alignment. The residue numbers are shown on both sides. The conserved “mitotic site” is denoted with a black triangle. S44, T52, and T415 in Spodoptera frugiperda lamin B are indicated by a black arrow, a white arrow, and a white diamond, respectively. The black, dark gray, and light gray shading indicate 100%, 80–100%, and 60–80% conservation, respectively. Protein sequences were obtained from the NCBI database with the following accession numbers: LMNA_Homo sapiens, NP_733821.1; LMNB1_Homo sapiens, NP_005564.1; LMNB2_Gallus gallus, NP_990616.1; LMNB_Drosophila melanogaster, NP_476616.1; and LMNB_Spodoptera frugiperda, XP_050555002.1.(TIF)
S2 FigEffects of different concentrations of RO-3306 on Sf9 cell viability.Sf9 cells were treated with 0, 5, 10, 15, or 20 μM RO-3306 for 24 h. Cell viability was measured by a trypan blue exclusion assay. 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; ***, P < 0.001; ****, P < 0.0001).(TIF)
S3 FigEffects of CDK1 inhibitor RO-3306 treatment at different time points on viral protein expression.(A and C) Western blot analyses of viral proteins GP64 and VP39. Sf9 cells were infected with vAcWT-mCh at an MOI of 20 TCID_50_/cell and treated with DMSO or 10 μM RO-3306 at 11 h p.i. (A) or 24 h p.i. (C). Cells were harvested 24 h post-treatment (i.e., at 35 h p.i. for A and 48 h p.i. for C) and subjected to Western blotting using anti-GP64 and anti-VP39 antibodies. Tubulin was probed with an anti-tubulin antibody as a loading control. (B and D) Quantification of GP64 and VP39 expression levels in (A) and (C), respectively. The signal intensities of GP64 and VP39 were normalized to tubulin. The normalized values are presented in arbitrary units relative to those of DMSO-treated 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 unpaired two-tailed t-test (ns, not significant; ****, P < 0.0001).(TIF)
S4 FigAlignment of CDK1 residues 140–165 in the indicated species.The alignment was performed with Geneious Prime software using ClustalW alignment. The residue numbers are shown on the left and right, with residue D146 denoted by a black triangle. The black, dark gray, and light gray shading indicate 100%, 80–100%, and 60–80% conservation, respectively. Protein sequences were obtained from the NCBI database with the following accession numbers: CDK1_Homo sapiens, NP_001777.1; CDK1_Mus musculus, NP_031685.2; CDK1_Gallus gallus, NP_990645.2; CDK1_Xenopus laevis, NP_001080093.1; CDK1_Drosophila melanogaster, NP_476797.1; and CDK1_Spodoptera frugiperda, XP_035448974.1.(TIF)
S5 FigExpression of FLAG:CDK1 and FLAG:CDK1 (DN) in Sf9 cells.The cells were infected with vAcWT-mCh2, vFLAG:CDK1, or vFLAG:CDK1 (DN) at an MOI of 20 TCID_50_/cell. At 48 h p.i., the cells were harvested and subjected to Western blotting with an anti-FLAG antibody. Tubulin was probed with an anti-tubulin antibody as a loading control.(TIF)
S6 FigSubcellular localization of endogenous lamin B detected by immunofluorescence staining with the ADL67 antibody.The cells were infected with vAcWT-mCh at an MOI of 20 TCID_50_/cell or remained uninfected. At the indicated time points p.i., the cells were fixed, permeabilized, and immunolabeled with the ADL67 antibody against lamin B. vAcWT-mCh-infected cells were monitored by mCherry fluorescence. DNA was stained with Hoechst 33258. Scale bar, 5 μM.(TIF)
S7 FigThe selection strategy for lamin B fluorescence intensity ratio calculation.Two representative cells (Cell 1 and Cell 2) illustrate the application of the strategy described in Materials and methods. Cell 1 exhibits a distinct nucleoplasm (NP)-nuclear rim (NR) boundary. This allowed both the whole nucleus and the nucleoplasm to be accurately and automatically selected using the Wand (tracing) tool (wavy solid lines, a characteristic of the intensity-based automatic tracing). Cell 2 exhibits an indistinct NP-NR boundary. In this case, while the whole nucleus was successfully selected with the Wand tool (wavy solid lines), the boundary ambiguity precluded reliable automatic nucleoplasm selection. Therefore, following the strategy, the nucleoplasm was manually traced using the Polygon selection tool (smooth solid lines). For each cell, three views are shown: whole nucleus selection, nucleoplasm selection, and a merged view (showing spatial relationships). Scale bar, 5 μm.(TIF)
S1 TablePrimers used in this study.(DOCX)
S1 DataThe raw data used in the figures.(XLSX)
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