Redundant and Specific Roles of A-Type Lamins and Lamin B Receptor in Herpes Simplex Virus 1 Infection

ABSTRACT

We investigated whether A-type lamins (lamin A/C) and lamin B receptor (LBR) are redundant during herpes simplex virus 1 (HSV-1) infection in HeLa cells expressing lamin A/C and LBR. Lamin A/C and LBR double knockout (KO) in HSV-1-infected HeLa cells significantly impaired expressions of HSV-1 early and late genes, maturation of replication compartments, marginalization of host chromatin to the nuclear periphery, enlargement of host cell nuclei, and viral DNA replication. Phenotypes of HSV-1-infected HeLa cells were restored by the ectopic expression of lamin A/C or LBR in lamin A/C and LBR double KO cells. Of note, lamin A/C single KO, but not LBR single KO, promoted the aberrant accumulation of virus particles outside the inner nuclear membrane (INM) and viral replication, as well as decreasing the frequency of virus particles inside the INM without affecting viral gene expression and DNA replication, time-spatial organization of replication compartments and host chromatin, and nuclear enlargement. These results indicated that lamin A/C and LBR had redundant and specific roles during HSV-1 infection. Thus, lamin A/C and LBR redundantly regulated the dynamics of the nuclear architecture, including the time-spatial organization of replication compartments and host chromatin, as well as promoting nuclear enlargement for efficient HSV-1 gene expression and DNA replication. In contrast, lamin A/C inhibited HSV-1 nuclear export through the INM during viral nuclear egress, which is a unique property of lamin A/C.

IMPORTANCE This study demonstrated that lamin A/C and LBR had redundant functions associated with HSV-1 gene expression and DNA replication by regulating the dynamics of the nuclear architecture during HSV-1 infection. This is the first report to demonstrate the redundant roles of lamin A/C and LBR as well as the involvement of LBR in the regulation of these viral and cellular features in HSV-1-infected cells. These findings provide evidence for the specific property of lamin A/C to inhibit HSV-1 nuclear egress, which has long been considered but without direct proof.

INTRODUCTION

Herpesviruses undergo productive infection through transcription, replication of their viral genomes, and encapsidation within the nucleus. After they enter a host cell, the released viral genome from the nucleocapsid is transported through a nuclear pore into the nucleus, where viral gene expression is initiated (1). Herpes simplex virus 1 (HSV-1) encodes approximately 100 different proteins (1,to 3) that can be characterized into three major classes: immediate early (IE), early (E), and late (L), which are expressed as a regulated cascade (1). Specific nuclear compartments are formed in the nuclei of HSV-1-infected cells allowing HSV-1 DNA replication and L gene transcription and packaging of replicated viral genomes into nascent capsids to occur (1, 4). Similar to other herpesviruses, HSV-1 becomes an infectious virion after acquiring a final envelope in the cytoplasm, and therefore, nascent nucleocapsids in the nucleus need to be exported to the cytoplasm by nuclear egress, a nuclear pore-independent and sequential envelopment-deenvelopment process. Thus, nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space located between the INM and the outer nuclear membrane (ONM) to acquire the primary envelope, a process termed primary envelopment (1, 5,to 7). Then, the envelopes of the perinuclear virions, termed perinuclear enveloped virions (PEVs), fuse with the ONM, which allows the release of nucleocapsids into the cytoplasm, a process termed deenvelopment (1, 5 to 7). Achieving nuclear egress requires an HSV-1 heterodimeric complex termed the nuclear egress complex (NEC), consisting of UL34 and UL31 (1, 5 to 7).

The nuclear lamina, a dense filamentous network mostly composed of lamins, is present under the INM and functions by maintaining the integrity of the nucleus (8 to 10). When mitosis occurs in uninfected cells, the disassembly of the nuclear lamina is promoted by cellular protein kinases that phosphorylate the lamins (11). Two types of lamins have been characterized, A- (lamins A and C) and B-types (lamins B1 and B2). Lamins A and C (lamin A/C) are alternative splice variants encoded by the LMNA gene, and lamins B1 and B2 are encoded by two separate genes (LMNB1 and LMNB2, respectively). During the primary envelopment of herpesviruses, nucleocapsids dock to and secure an area of deformable INM (1, 5 to 7); however, a nuclear lamina underneath the INM highly likely inhibits nucleocapsid interactions with the INM. Therefore, the disruption of the nuclear lamina is considered necessary for successful viral primary envelopment. In agreement with this idea, HSV-1 infection induced conformational changes, and the phosphorylation and redistribution of lamins (12 to 21). During mitosis in uninfected cells, lamins are completely disrupted; however, during HSV-1 infection, alterations of lamins are limited to discrete regions (12, 13, 15, 17). Furthermore, during HSV-1 infection, virally encoded Us3 and cellular protein kinases, including protein kinase C isoforms (PKCs) that were reported to phosphorylate lamins in vitro and/or phosphorylate lamins in HSV-1-infected cells, are recruited to the NM (15 to 17, 19 to 21). Moreover, UL13, a viral protein kinase encoded by HSV-2, directly phosphorylated lamins in vitro and promoted the partial redistribution of lamins when it was ectopically expressed in the absence of other viral proteins (22). The NEC and HSV-1 ICP34.5 are required for the local alterations of lamins and recruitment of PKCs to the NM during HSV-1 infection (12, 13, 16, 20, 21). Thus, evidence for the alterations of lamins by HSV-1 infection has increased. However, there is little evidence directly demonstrating that the nuclear lamina is an obstacle to HSV-1 nuclear egress and that local alterations of lamins induced by HSV-1 infection promote viral nuclear egress. A simple approach to address the former question is to investigate the effects of depleting lamins on HSV-1 nuclear egress. However, lamins have pleiotropic functions associated with gene expression, chromatin regulation, and signaling in addition to providing structural integrity to the nucleus in uninfected cells (8 to 10). A previous study reported that lamin A/C knockout (KO) HSV-1-infected murine embryonic fibroblasts (MEFs) had smaller replication compartments, which prevented their expansion to the nuclear periphery, increasing the association of heterochromatin with the HSV-1 IE promoter, leading to reduced viral gene expression, DNA replication, and proliferation (23). Furthermore, lamin B1 KO MEFs demonstrated reduced HSV-1 proliferation (18). In these studies, the effects of each lamin KO on wild-type HSV-1 nuclear egress efficiency, determined as the rate of virion delivery from the nucleus to the cytoplasm, were not investigated (18, 23). Perhaps, it might be difficult to address the effect of lamins on inhibiting HSV-1 nuclear egress in these MEFs because the lamin KO impairs other functions, including the regulation of HSV-1 gene expression and DNA replication, and maturation of replication compartments.

A previous study reported that lamin A/C and lamin B receptor (LBR) were redundant for regulating the spatial organization of chromatin and that a loss of function of lamin A/C was compensated for by the ectopic expression of LBR (24). The effect of lamins on the spatial organization of chromatin might be involved in the regulation of gene expression in uninfected cells (25 to 27) Furthermore, the spatial organization of host chromatin was associated with that of replication compartments in HSV-1-infected cells (12, 28). Thus, during HSV-1 infection, host chromatin excluded from the replication compartments due to replication compartment expansion is marginalized and compressed, forming a dense layer that surrounds the compartments (12, 28, 29). Ultimately, the chromatin layer is dispersed, allowing viral compartments to reach the nuclear edge (12). Therefore, we postulated that we could investigate the effect of lamin A/C KO on HSV-1 nuclear egress without affecting HSV-1 gene expression and maturation of replication compartments by using cells that constitutively expressed lamin A/C and LBR. This study used HeLa cells constitutively expressing lamin A/C and LBR (30, 31) to investigate the effect of lamin A/C and LBR on HSV-1 nuclear egress and to determine whether there was redundancy between these molecules during HSV-1 infection.

RESULTS

Generation and characterization of lamin A/C KO and/or LBR KO HeLa cells.

HeLa cells express lamin A/C and LBR (30, 31), which was confirmed by immunoblotting (Fig. 1B). The CRISPR/Cas9 system was used to generate lamin A/C KO HeLa cells (LMNA KO cells), LBR KO HeLa cells (LBR KO cells), and lamin A/C and LBR double KO HeLa cells (LMNA/LBR KO cells) (Fig. 1A and B). To examine the off-target effects and complementarity of LBR to lamin A/C and vice versa in LMNA/LBR KO cells, we constructed LMNA/LBR KO-LBR and LMNA/LBR KO-lamin A/C cells in which LBR or lamin A/C were expressed ectopically, respectively (Fig. 1A and B). As expected, lamin A/C was not detected in LMNA KO, LMNA/LBR KO, and LMNA/LBR KO-LBR cells, and LBR was not detected in LBR KO, LMNA/LBR KO, and LMNA/LBR KO-lamin A/C cells (Fig. 1B). Lamin A/C or LBR expression was restored in LMNA/LBR KO-lamin A/C or LMNA/LBR KO-LBR cells, respectively (Fig. 1B). The viability of these newly generated cells was similar to that of the original HeLa cells (Fig. 1C).

FIG 1.

Characterization of LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR-lamin A/C cells. (A) The targeted LMNA and/or LBR mutation sequences and the parental sequences in LMNA KO, LBR KO, and LMNA/LBR KO cells. (B) Lysates of HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were analyzed by immunoblotting with the indicated antibodies. (C) Relative cell viability of HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells. Data are shown as the means ± standard errors of the results of three independent experiments and are expressed relative to the mean value determined for HeLa cells, which was normalized to 100%. n.s., not significant by Tukey’s test.

Effects of lamin A/C and/or LBR on HSV-1 gene expression and DNA replication in HeLa cells.

To examine the effects of lamin A/C KO and/or LBR KO on viral gene expression and DNA replication, and examine the complementary relationship between lamin A/C and LBR in HSV-1-infected HeLa cells, HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, or LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at a multiplicity of infection (MOI) of 10. At 18 h postinfection, the infected cells were harvested, and the mRNA and protein levels of HSV-1 IE, E, and L proteins were analyzed by quantitative reverse transcriptase PCR (qRT-PCR) and immunoblotting, respectively. In addition, the amount of HSV-1 DNA replication in the infected cells was analyzed by qPCR.

Significantly less ICP8 and gB, HSV-1 E and L proteins and their mRNAs were accumulated in LMNA/LBR KO cells compared with HeLa cells (Fig. 2 and 3). In contrast, LMNA KO and LBR KO cells accumulated similar HSV-1 mRNAs and protein to those in HeLa cells (Fig. 2 and 3). The ectopic expression of lamin A/C or LBR in LMNA/LBR KO cells restored the accumulation of ICP8 and gB mRNAs and proteins to levels similar to those in HeLa, LMNA KO, and LBR KO cells in LMNA/LBR KO-lamin A/C or LMNA/LBR KO-LBR cells, respectively (Fig. 2 and 3). The accumulated mRNA and protein levels of ICP27, an HSV-1 IE protein, in these cells were similar (Fig. 2 and 3). In agreement, the levels of accumulated HSV-1 E protein dUTPase and L protein Us11 in LMNA KO, LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were similar to those in HeLa cells (Fig. 3). In contrast, fewer viral proteins were accumulated in LMNA/LBR KO cells than HeLa cells (Fig. 3). The amount of accumulated ICP4, an HSV-1 IE protein, was similar in all cell types (Fig. 3).

FIG 2.

Effect of lamin A/C and/or LBR on the accumulation of HSV-1 mRNAs and proteins. (A) HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10, harvested at 18 h postinfection, and analyzed for the amounts of ICP27, ICP8, and gB mRNAs by quantitative RT-PCR. Each value represents the mean ± standard error of the results from three independent experiments. *, P < 0.05 (by Tukey’s test); n.s., not significant. (B) The amounts of ICP27, ICP8, and gB proteins from the experiment in Fig. 3 were quantitated and normalized to those of β-actin (loading control). Each value represents the mean ± standard error of the results from three independent experiments. *, P < 0.05 (by Tukey’s test); n.s., not significant.

FIG 3.

Effect of lamin A/C and/or LBR on the accumulation of HSV-1 proteins. HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10, harvested at 18 h postinfection, and analyzed by immunoblotting with the indicated antibodies.

HSV-1 genome DNA accumulation in LMNA KO and LBR KO cells was similar to that in HeLa cells, although the levels in LMNA/LBR KO cells were significantly lower than those in HeLa cells (Fig. 4). The ectopic expression of lamin A/C or LBR in LMNA/LBR KO cells restored the accumulation of the HSV-1 genome to levels similar to those in HeLa, LMNA KO, and LBR KO cells in LMNA/LBR KO-lamin A/C or LMNA/LBR KO-LBR cells, respectively.

FIG 4.

Effect of lamin A/C and/or LBR on HSV-1 DNA replication. HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10 and harvested at 18 h postinfection. DNA was extracted from the infected cells, and HSV-1 genomic DNA was quantified by quantitative PCR. Each value is the mean ± standard error of the results from three independent experiments. *, P < 0.05 (by Tukey’s test); n.s., not significant.

Collectively, these results indicated that lamin A/C and LBR were required for the efficient expression of the HSV-1 E and L genes, but not IE genes, and viral DNA replication in HSV-1-infected HeLa cells. Furthermore, lamin A/C or LBR complemented each other during the viral gene expression of the E and L genes, and DNA replication. Therefore, the roles of lamin A/C and LBR on the regulated expression of the HSV-1 E and L genes, and viral DNA replication in HeLa cells, were redundant.

Effects of lamin A/C and/or LBR on the localization of replication compartments and host chromatin in HeLa cells.

To examine the effects of lamin A/C KO and/or LBR KO on the time-spatial organization of the replication compartments and host chromatin, and the complementary relationship between lamin A/C and LBR in HSV-1-infected HeLa cells, we infected HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells with wild-type HSV-1(F) at an MOI of 10. At 9 and 18 h postinfection, the infected cells were fixed and analyzed by confocal microscopy using DAPI (4′,6-diamidino-2-phenylindole) to visualize the cellular DNA (32) and antibodies to ICP4 and the trimethylated form of histone H3 lysine 9 (trimethyl H3K9), markers of replication compartments (33) and heterochromatin (34), respectively. In addition, we used uninfected HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells that were fixed and analyzed by confocal microscopy using DAPI and the antibody to trimethyl H3K9.

In agreement with previous studies (12, 28), replication compartments in HeLa cells detected by the ICP4 antibody annexed dominant portions of the nucleus as the HSV-1 infection progressed and chromatin was compressed and marginalized (Fig. 5). At 9 h postinfection in HeLa cells, multiple replication compartments were formed and occupied areas of the nucleus where chromatin staining was excluded, indicating they were surrounded by marginalized host chromatin. Similar results were noted in all the other cell types (Fig. 5). Although the localization patterns of the host chromatin and replication compartments at 9 h postinfection were similar between the different cell types, quantitative three-dimensional analyses demonstrated that the volume of replication compartments and the ratio of the volume of replication compartments to that of host chromatin in HeLa cells were comparable to those in LMNA KO and LBR KO cells but significantly higher than that in LMNA/LBR KO cells (Fig. 6). At 18 h postinfection in HeLa cells, replication compartments had extended throughout the nucleus and reached the nuclear periphery (Fig. 5 and 7, and S-Movies (a) to (f) in the supplemental material) similar to that observed in LMNA KO and LBR KO cells (Fig. 5 and 7). In contrast, replication compartments in LMNA/LBR KO cells were smaller, did not reach the nuclear periphery, except at the top of the nucleus, and were surrounded by marginalized host chromatin (Fig. 5 and 7, and S-Movies (a) to (f)). Furthermore, the volume of the replication compartments in HeLa cells was similar to that in LMNA KO and LBR KO cells but significantly larger than that in LMNA/LBR KO cells (Fig. 6), and the ratio of replication compartment volume to host chromatin volume in HeLa cells was comparable to that in LMNA KO and LBR KO cells but significantly higher than that in LMNA/LBR KO cells (Fig. 6). The phenotypes observed in HeLa cells were restored in LMNA/LBR KO-LBR or LNMA/LBR KO-lamin A/C cells by the ectopic expression of LBR or lamin A/C, respectively, in LMNA/LBR KO cells at 9 and 18 h postinfection (Fig. 5 to 7, and S-Movies (a) to (f)).

FIG 5.

Effect of lamin A/C and/or LBR on the formation of replication compartments and distribution of host chromatin and heterochromatin during HSV-1 infection. HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10, fixed at 9 and 18 h postinfection, permeabilized, stained with anti-ICP4 and anti-trimethyl H3K9 antibodies and DAPI and examined by confocal microscopy. Uninfected cells were also fixed, permeabilized, and processed as described above. Scale bars = 10 μm.

FIG 6.

Effect of lamin A/C and/or LBR on the volumes of replication compartments and host chromatin during HSV-1 infection. Volumes of ICP4 fluorescent domains (replication compartments) and DAPI fluorescent domains (host chromatin) were quantitated from 3D confocal images of each cell type infected with wild-type HSV-1(F) in the experiments shown in Fig. 5. The ratios of volumes of ICP4 fluorescent domains to those of DAPI fluorescent domains are shown. Each value is the mean ± standard error of data of 10 cells for each cell. *, P < 0.05 (by Tukey’s test); n.s., not significant.

FIG 7.

3D confocal images of the replication compartments and host chromatin in the presence or absence of LBR and/or lamin A/C in HSV-1-infected cells. 3D confocal images of ICP4 fluorescent domains (replication compartments) and DAPI fluorescent domains (host chromatin), and their cross-section views in each of the indicated cell types infected with wild-type HSV-1(F) at 18 h postinfection in the experiments shown in Fig. 5. Scale bar = 5 μm. See also S-Movies (a) to (f) in the supplemental material.

In all uninfected cell types, whole host chromatin detected by DAPI was distributed throughout the nucleus, whereas the localization of host heterochromatin was only detected in limited areas of the nucleus (Fig. 5). In contrast, during the progression of HSV-1 infection, the amount of colocalization between whole host chromatin and host heterochromatin increased, and at 18 h postinfection, whole host chromatin was almost completely colocalized with host heterochromatin in all the cell types (Fig. 5).

At 9 and 18 h postinfection, the volume of host chromatin in all cell types infected with HSV-1 was similar, indicating that the amount of host chromatin was not affected by lamin A/C KO and/or LBR KO during HSV-1 infection (Fig. 6). As reported previously (14, 28, 35), the nuclear volume was increased in HSV-1(F)-infected HeLa cells at 9 and 18 h postinfection (Fig. 8), and similar results were noted in HSV-1(F)-infected LMNA KO and LBR KO cells where the nuclear volumes were comparable with infected HeLa cells (Fig. 8). In contrast, at 9 and 18 h postinfection, the nuclear volume in HSV-1(F)-infected LNMA/LBR KO cells was significantly smaller than in infected HeLa, LMNA KO, and LBR KO cells (Fig. 8). The nuclear volume in HSV-1(F)-infected HeLa cells at 9 and 18 h postinfection were restored in LMNA/LBR KO-LBR or LNMA/LBR KO-lamin A/C cells by the ectopic expression of LBR or lamin A/C, respectively, in LMNA/LBR KO cells (Fig. 8).

FIG 8.

Effect of lamin A/C and/or LBR on nuclear enlargement induced by HSV-1 infection. Nuclear volumes were quantitated from 3D confocal images of each cell type infected with wild-type HSV-1(F) in the experiments shown in Fig. 5. Each value is the mean ± standard error of data of 10 cells for each cell. *, P < 0.05 (by Tukey’s test); n.s., not significant.

Collectively, these results indicated that lamin A/C and LBR were required for the efficient formation of the replication compartments, nuclear enlargement, and proper time-spatial organization of the replication compartments and host chromatin in HSV-1-infected HeLa cells. Furthermore, lamin A/C and LBR complemented each other when regulating the dynamics of the nuclear architecture induced by HSV-1 infection. These results suggested that lamin A/C and LBR were redundant for the regulation of the replication compartments, nuclear enlargement, and time-spatial organization of replication compartments and host chromatin in HSV-1-infected HeLa cells.

Effect of lamin A/C and/or LBR on HSV-1 nuclear egress.

To examine the effects of lamin A/C KO and/or LBR KO on viral nuclear egress, and the complementary relationship between lamin A/C and LBR in HSV-1-infected HeLa cells, we performed three series of experiments.

First, HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-lamin A/C, or LMNA/LBR KO-LBR cells were infected with YK601, a recombinant virus encoding VP26, an HSV-1 capsid and L protein tagged with a monomeric fluorescent protein VenusA206K (VenusA206K-VP26) (36) at a multiplicity of infection (MOI) of 10. Cells were fixed at 24 h postinfection, stained with antibodies to lamin B1, and analyzed with the Airyscan system (Zeiss), a superresolution confocal imaging system (37). It was reported that capsids were visualized by confocal microscopy as fluorescent puncta in cells infected with recombinant HSV-1 encoding VP26 fused to a fluorescent protein (36, 38 to 40). Consistent with these reports, fluorescent puncta were present in the nucleus and cytoplasm of HeLa cells infected with YK601 (VenusA206K-VP26) (Fig. 9). There were more fluorescent VenusA206K-VP26 puncta in the cytoplasm of LMNA KO cells than in the cytoplasm of HeLa cells (Fig. 9), and VenusA206K-VP26 intensities in the cytoplasm of LMNA KO cells were higher than those in the cytoplasm of HeLa cells (Fig. 10A). In agreement with the results above (Fig. 2 and 3), where lamin A/C KO had no obvious effect on the accumulation of HSV-1 gene products, total VenusA206K-VP26 intensities in LNMA KO cells were comparable to those in HeLa cells (Fig. 10B). Therefore, the ratio of VenusA206K-VP26 intensities in the cytoplasm to total VenusA206K-VP26 intensities of LMNA KO cells was significantly higher than that in HeLa cells (Fig. 10C). In contrast, LBR KO cells had phenotypes similar to those in HeLa cells (Fig. 9 and 10). The cytoplasm of LMNA/LBR KO cells contained significantly fewer fluorescent puncta of VenusA206K-VP26 compared with the cytoplasm of HeLa cells (Fig. 9). In agreement with the results above (Fig. 2 and 3), where the simultaneous KO of lamin A/C and LBR impaired the accumulation of the HSV-1 E and L gene products, the total VP26-VenusA206K intensities and VenusA206K-VP26 intensities in the cytoplasm of LNMA/LBR KO cells were significantly lower than those in HeLa cells (Fig. 10A and B). Collectively, the ratio of VP26-VenusA206K intensities in the cytoplasm of LMNA/LBR KO cells to total VP26-VenusA206K intensities in these cells was comparable with that in HeLa cells (Fig. 10C). The phenotypes observed in HeLa cells were restored in LMNA/LBR KO-lamin A/C cells by the ectopic expression of lamin A/C in LMNA/LBR KO cells (Fig. 9 and 10). In contrast, although the total VenusA206K-VP26 intensity was restored in LMNA/LBR KO-LBR cells to a level comparable with that in HeLa cells by the ectopic expression of LBR in LMNA/LBR KO cells, the accumulation level of fluorescent VenusA206K puncta in the cytoplasm, VenusA206K-VP26 intensities in the cytoplasm, and ratio of VP26-VenusA206K intensities in the cytoplasm to total VP26-VenusA206K intensities were significantly higher than in HeLa cells and similar to those in LMNA KO cells (Fig. 9 and 10).

FIG 9.

Effect of lamin A/C and/or LBR on the capsid distribution in HSV-1-infected cells. HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with YK601 (VenusA206K-VP26) at an MOI of 10. At 24 h postinfection, the cells were fixed, permeabilized, stained with anti-lamin B1 antibody, and examined by confocal microscopy. 3D confocal images are shown. Scale bar = 5 μm.

FIG 10.

Quantitation of VenusA206K-VP26 fluorescence in confocal images of HSV-1-infected cells in the presence or absence of LBR and/or lamin A/C. Cytoplasmic (A) and total (B) VenusA206K-VP26 signals in each cell type infected with YK601 (VenusA206K-VP26) in the experiments shown in Fig. 9 were quantitated. The ratios of cytoplasmic VenusA206K-VP26 signals to total VenusA206K-VP26 signals are shown (C). The cytoplasm was segmented based on lamin B1 signals. Each value is the mean ± standard error of data of 10 cells for each cell type. *, P < 0.05 (by Tukey’s test); n.s., not significant.

In the second series of experiments, we analyzed HSV-1 morphogenesis by quantitating the number of virus particles present at different morphogenetic stages in HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells infected with HSV-1(F) at an MOI of 10 for 24 h by electron microscopy. As shown in Fig. 11 and Table 1, 68.0% of the total number of virus particles were inside the INM of HeLa cells, whereas only 48.8% of virus particles were inside the INM of LMNA KO cells. Although only 32.0% of the total number of virus particles were outside the INM (in the perinuclear space, cytoplasm, and extracellular space) in HeLa cells, the fraction of virus particles outside the INM was significantly increased to 51.2% in LMNA-KO cells (Fig. 11 and Table 1). In LMNA-KO cells, PEVs tended to be accumulated in the perinuclear space, especially in membranous structures formed by evaginations of the ONM into the cytoplasm (Table 1). Images of infected LMNA-KO and LMNA/LBR KO-LBR cells containing clusters of PEVs accumulated in the perinuclear space are shown in Fig. 12. In contrast, LBR KO cells had phenotypes similar to those in HeLa cells (Fig. 11 and Table 1). The frequencies of virus particles in different morphogenetic stages in LMNA/LBR KO cells were similar to those in HeLa cells, although the total number of virus particles in LNMA/LBR KO cells was lower than that in HeLa (Fig. 11 and Table 1). The phenotypes observed in HeLa cells were restored in LMNA/LBR KO-lamin A/C cells by the ectopic expression of lamin A/C in LMNA/LBR KO cells (Fig. 11 and Table 1). Although the ectopic expression of LBR in LMNA/LBR KO cells restored the total number of virus particles in LMNA/LBR KO-LBR cells to a level comparable with that in HeLa cells, other phenotypes in LMNA/LBR KO-LBR cells were different from those in HeLa cells but similar to those in LMNA KO cells (Fig. 11 and Table 1). Overall, these results were in accord with those using YK601 (VP26-VenusA206K) for fluorescence microscopy (Fig. 9 and 10).

FIG 11.

Quantification of virus particle distribution in the presence or absence of LBR and/or lamin A/C. The ratio of the number of virus particles outside the INM to the total number of virus particles in each of the cell types infected with wild-type HSV-1(F) in the experiments shown in Fig. 12 was calculated. Each value is the mean ± standard error of data of 10 cells for each cell. *, P < 0.05 (by Tukey’s test); n.s., not significant.

TABLE 1.

Effect of lamin A/C and/or LBR on the distribution of virus particles in infected HeLa cells^a

Cell	Percentage of virus particles in the morphogenetic stage (mean ± SE) (no. of virus particles)						Total (particles/cells)
	Nucleocapsids inside the inner nuclear membrane	Enveloped virions in the perinuclear space	Nucleocapsids in the cytoplasm	Enveloped virions in the cytoplasm	Extracellular enveloped virions	Virus particles outside the inner nuclear membrane
HeLa	68.0 ± 2.7 (807)	3.6 ± 0.9 (43)	6.9 ± 1.1 (82)	6.3 ± 0.7 (75)	15.1 ± 1.2 (179)	32.0 ± 1.5 (379)	1186/10
LMNA KO	48.8 ± 3.4b (652)	5.8 ± 0.6b (77)	9.0 ± 1.04b (120)	13.0 ± 1.3b (174)	23.4 ± 1.8b (313)	51.2 ± 1.7b (684)	1336c/10
LBR KO	67.6 ± 1.9c (823)	2.9 ± 1.9c (35)	5 0.0 ± 0.5c (61)	7.1 ± 0.5c (87)	17.3 ± 0.9c (211)	32.4 ± 0.86c (394)	1217c/10
LMNA/LBR KO	68.7 ± 2.6c (610)	4.3 ± 0.5c (38)	6.2 ± 0.7c (55)	7.7 ± 0.5c (69)	13.1 ± 1.0c (116)	31.3 ± 1.5c (278)	888b/10
LMNA/LBR KO-LBR	49.5 ± 1.7b,d (662)	5.6 ± 0.3b,d (75)	9.2 ± 1.2b,d (123)	11.7 ± 1.2b,d (156)	24.0 ± 1.3b,d (321)	50.5 ± 1.5b,d (675)	1337c/10
LMNA/LBR KO-lamin A/C	67.9 ± 4.8c (822)	3.3 ± 0.4c (40)	4.7 ± 0.8c (57)	6.1 ± 0.5c (74)	17.9 ± 1.1c (217)	32.1 ± 1.5c (388）	1210c/10

^aStatistical analysis was performed by Tukey’s test, and P values of <0.05 were considered significant.

^bStatistically significant differences from HeLa.

^cNo statistically significant differences from HeLa.

^dNo statistically significant differences from LMNA KO.

FIG 12.

Effect of lamin A/C and/or LBR on HSV-1 nuclear egress. HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10. At 24 h postinfection, the infected cells were fixed, embedded, sectioned, stained, and examined by electron microscopy. In LMNA KO and LMNA/LBR KO-LBR cells, the images on the right are magnifications of the corresponding boxed areas on the left. Arrowheads indicate evaginations of the perinuclear enveloped virions. White lines and white dotted lines indicate the INM and ONM, respectively. Nu, nucleus; NM, nuclear membrane; Cy, cytoplasm. Scale bars = 300 nm.

In the third series of experiments, HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, or LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10, harvested at 18 h postinfection, and analyzed by immunoblotting with antibodies to UL34 and UL31, both of which are HSV-1 L proteins. The infected cells were also fixed at 18 h postinfection and stained with antibodies to UL34 and UL31, and the localization of these viral proteins was analyzed by confocal microscopy. In agreement with the results above (Fig. 2 and 3), the accumulated levels of UL34 and UL31 in LMNA KO, LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were similar to those in HeLa cells, whereas fewer viral proteins had accumulated in the LMNA/LBR KO cells (Fig. 13A). These results indicated that lamin A/C single KO or LBR single KO had no effect on the accumulation of UL34 and UL31 proteins, and further support our conclusion that lamin A/C and LBR are required for the efficient expression of HSV-1 L genes. Furthermore, UL34 and UL31 were smoothly distributed around the nuclear rim, and these localization patterns were similar in all types of cells, indicating that lamin A/C and LBR were not required for the proper localization of UL34 and UL31 in HSV-1-infected HeLa cells (Fig. 13B).

FIG 13.

Effect of lamin A/C and/or LBR on the accumulation and localization of UL31 and UL34 in HSV-1-infected cells. (A) HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with wild-type HSV-1(F) at an MOI of 10. At 24 h postinfection, the infected cells were harvested and analyzed by immunoblotting with the indicated antibodies (A) or fixed, permeabilized, stained with the indicated antibodies, and examined by confocal microscopy (B). Scale bars = 10 μm.

Taken together, these results indicated that the lamin A/C single KO, but not the LBR single KO, had an aberrant accumulation of virus particles outside the INM, without affecting the accumulation and localization of UL34 and UL31. In addition, this effect of the lamin A/C single KO on virus particle distribution was antagonized by an additional single LBR KO, and LBR was unable to compensate for the effect of lamin A/C on virus particle distribution. These results suggested that lamin A/C specifically inhibited HSV-1 capsid transport through the INM of HSV-1-infected cells.

Effect of lamin A/C and/or LBR on HSV-1 replication.

We investigated the effects of lamin A/C KO and/or LBR KO on HSV-1 replication, and the complementary relationship between lamin A/C and LBR in HeLa cells, using HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells infected with wild-type HSV-1(F) at an MOI of 10 or 0.01. Then, virus titers were assayed at various times postinfection. Aberrant and significantly increased HSV-1 growth was observed in LMNA KO cells compared with HeLa cells (Fig. 14). In contrast, there was significantly lower HSV-1 growth in LMNA/LBR KO cells compared with HeLa cells, and HSV-1 growth in LBR-KO cells was similar to that in HeLa cells (Fig. 14). Although the ectopic expression of lamin A/C in LMNA/LBR KO cells restored the HSV-1 growth rate in LMNA/LBR KO-lamin A/C cells similar to that in HeLa cells (Fig. 14), the ectopic expression of LBR in LMNA/LBR KO cells increased the HSV-1 growth rate in LMNA/LBR KO-LBR cells beyond that in HeLa cells and similar to that in LMNA KO cells (Fig. 14). These results indicated that (i) the presence of lamin A/C, but not LBR, inhibited HSV-1 replication, (ii) lamin A/C and LBR were both required for efficient HSV-1 proliferation in HeLa cells, and (iii) lamin A/C compensated for the effect of LBR on viral replication, whereas LBR could not compensate for the effect of lamin A/C on viral replication. The effects of lamin A/C KO and/or LBR KO on HSV-1 growth correlated with their effects on HSV-1 gene expression, DNA replication, and virus particle distribution. The aberrant HSV-1 growth in LMNA KO and LMNA/LBR KO-LBR cells correlated with the aberrant accumulation of virus particles outside the INM without affecting viral gene expression and DNA replication. Furthermore, impaired HSV-1 growth in LMNA/LBR KO cells correlated with the downregulation of viral E and L gene expressions and DNA replication without affecting virus particle distribution. HSV-1 growth was not affected in LBR KO and LMNA/LBR KO-lamin A/C cells because the viral gene expression, DNA replication, and virus particle distribution were not affected.

FIG 14.

Effect of lamin A/C and/or LBR on HSV-1 growth. HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells were infected with HSV-1(F) at an MOI of 10 (A) or 0.01 (B). Total virus from the cell culture supernatants and the infected cells was harvested at the indicated times postinfection and assayed on Vero cells. The data are shown as the mean 6 standard error of the results of five independent experiments. *, P < 0.05 (by Tukey’s test); n.s., not significant.

Finally, we examined the effects of lamin A/C KO in HeLa cells on viral replications of UL31-null and UL34-null mutant viruses (YK720 [ΔUL31] and YK723 [ΔUL34], respectively). In contrast to the effect of lamin A/C KO in HeLa cells on the growth of wild-type HSV-1(F) above (Fig. 14), the growth of UL31-null or UL34-null mutant virus in LMNA KO cells was similar to that in HeLa cells (Fig. 15). These results suggested that the aberrantly enhanced replication of the wild-type HSV-1(F) in LMNA KO cells was due to improved nuclear egress.

FIG 15.

Effect of lamin A/C on growth of ΔUL31 and ΔUL34. HeLa and LMNA KO cells were infected with HSV-1(F), YK720 (ΔUL31), or YK723 (ΔUL34) at an MOI of 10. Total virus from the cell culture supernatants and the infected cells was harvested at 36 h postinfection and the titer assayed. The data are shown as the mean ± standard error of the results of three independent experiments. *, P < 0.05 (by Student's t test); n.s., not significant.

DISCUSSION

Previous studies reported the expressions of lamin A/C and LBR in uninfected cells were temporarily coordinated, and dependent on the cell type and stage of development and differentiation (24, 41). The same studies also demonstrated that lamin A/C and LBR were redundant for the regulation of the spatial organization of host heterochromatin required for proper cell differentiation and gene expression (24, 41). In this study, we present evidence that lamin A/C and LBR are redundant for the expression of HSV-1 E and L genes, viral DNA replication, maturation of replication compartments, time-spatial organization of replication compartments and host chromatin, and nuclear enlargement in HSV-1-infected HeLa cells. To the best of our knowledge, this is the first report showing these redundant roles of lamin A/C and LBR during HSV-1 infection as well as the involvement of LBR in the regulation of these viral and cellular features. Of note, the degree of host chromatin marginalization to the nuclear periphery and nuclear enlargement correlated with the size of the replication compartments, expressions of HSV-1 E and L genes, and viral DNA replication during the late stages of HSV-1 infection in HeLa cells. These results suggest that the proper dynamics of nuclear architecture redundantly regulated by lamin A/C and LBR during HSV-1 infection, including host chromatin marginalization to the nuclear periphery and nuclear enlargement, are required for the efficient maturation of replication compartments, and HSV-1 gene expression and DNA replication. Currently, the mechanisms involved in the redundancy of lamin A/C and LBR in the maturation of replication compartments, and HSV-1 gene expression and DNA replication by regulating nuclear dynamics, are unknown. Defects in nuclear enlargement in the absence of both lamin A/C and LBR during the late stages of HSV-1 infection may limit the space needed for the expansion of the replication compartments because the volume of the host chromatin was not affected by the absence of lamin A/C and/or LBR. Therefore, a simple model to explain the observations in this study may be that lamin A/C and LBR have redundant roles associated with the enlargement of the nucleus in HSV-1-infected HeLa cells, possibly in concert with the NEC that was reported to be involved in nuclear enlargement (14), allowing the expansion of the replication compartments with the marginalization of the host chromatin to the nuclear periphery. This expansion of the replication compartment then enabled efficient HSV-1 gene expression and DNA replication. In agreement with this model, it was reported that the ectopic expression of LBR in mouse olfactory neurons, in which endogenous LBR expression is downregulated, increased the nuclear volume, redistributed chromatin, and affected gene expression (41).

The redundant roles of lamin A/C and LBR reported in the current study were partially in agreement with an earlier study (23) where lamin A/C single KO in MEFs impaired maturation of replication compartments, nuclear enlargement, and HSV-1 DNA replication and proliferation as observed with lamin A/C and LBR double KOs in HeLa cells in this study. In contrast, the lamin A/C single KO in MEFs, and lamin A/C and LBR double KOs in HeLa cells, had different phenotypes. In contrast to lamin A/C and LBR double KO in HeLa cells, lamin A/C KO in MEFs had only a little effect on the overall localization of host chromatin and heterochromatin, although colocalization between host heterochromatin and replication compartments was increased, and the expressions of HSV-1 IE genes in addition to E genes were reduced. Whether lamin A/C KO MEFs used in the previous report (23) expressed LBR was not mentioned. However, based on the redundant effects of lamin A/C and LBR clarified in this study, the lamin A/C KO MEFs might not express LBR, or it might be expressed at a very low level. In agreement with this, MEFs (42, 43) available in this laboratory did not express LBR (data not shown). These observations suggest that the phenotypes observed in lamin A/C KO MEFs may be associated with the loss of the redundant functions of lamin A/C and LBR, which might vary during HSV-1 infection in different cell types. However, as described above, LBR expression is temporarily regulated and therefore might be dependent on the passage number of the MEFs. In accord with this, the expression of LBR was detected in a fraction of MEFs (44, 45). These observations raised an alternative possibility that the phenotypes observed in the lamin A/C KO MEFs (23) might be related to the loss-of-function(s) unique to lamin A/C. Furthermore, the temporarily regulated expression of LBR and the redundant roles of lamin A/C and LBR in HSV-1 infection clarified in this study might explain the discrepancy between the two earlier studies using the same lamin A/C KO MEFs: lamin A/C KO reduced HSV-1 replication (23) or had no effect (18).

Investigating the effects of lamins on HSV-1 nuclear egress was hampered by their multifunctional nature. Here, we investigated the effects of lamin A/C on HSV-1 nuclear egress using HeLa cells where the loss of most lamin A/C functions was compensated for by endogenous LBR. Consequently, we showed that lamin A/C KO in HeLa cells had an aberrant accumulation of virus particles outside the INM, lower frequencies of virus particles inside the INM, and aberrant viral replication, but without affecting viral gene expression and DNA replication, time-spatial organization of replication compartments and host chromatin, and nuclear enlargement. Furthermore, we also demonstrated that lamin A/C KO in HeLa cells had no obvious effects on replications of mutant viruses lacking either UL34 or UL31, which is a critical regulator for viral nuclear egress. These results indicated that the absence of lamin A/C in HeLa cells allowed a higher-than-normal transport of virus particles through the INM, leading to aberrant accumulation of virus particles outside the INM and viral replication. Thus, this study provides direct evidence that lamin A/C inhibits the primary envelopment step during HSV-1 nuclear egress, probably by inhibiting nucleocapsid docking at the INM and the securement of an area sufficient for INM deformation.

Based on the observations with the lamin A/C single KO HeLa cells, the barrier effect of lamin A/C should also be missing in lamin A/C and LBR double KO HeLa cells. However, the aberrant accumulation of virus particles outside the INM was not observed in lamin A/C and LBR double KO HeLa cells. This suggested that an additional obstacle other than lamin A/C inhibited HSV-1 transport through and/or to the INM specifically in lamin A/C and LBR double KO HeLa cells and that the effect of the lamin A/C KO on HSV-1 nuclear egress was antagonized by this additional obstacle. Of note, replication compartments did not reach the nuclear periphery and were surrounded by a layer of host chromatin in the lamin A/C and LBR double KO HeLa cells during the late stage of HSV-1 infection, suggesting the host chromatin layer might inhibit capsid trafficking to the INM, as has long been anticipated (7, 12, 46, 47). Thus, there seem to be two obstacles to HSV-1 capsid transport to and through the INM in the host cell nucleus: lamins and the host chromatin layer surrounding the replication compartments. HSV-1 appears to have evolved various mechanisms, including the modification of lamins, regulation of the nuclear size, and time-spatial organization of the host chromatin and replication compartments, to overcome these two nuclear obstacles. In this study, we were able to reveal the barrier effect of lamin A/C against HSV-1 nuclear egress in lamin A/C single KO HeLa cells in which LBR compensated for the function of lamin A/C to attenuate the effect of the additional obstacle. We should note, however, that there is an alternative possibility that the aberrant accumulation of virus particles outside the INM was not observed in lamin A/C and LBR double KO HeLa cells due to the decreased expression of viral factors functioned in viral nuclear egress such as UL34, UL31, and Us3, which may impair efficiency of viral nuclear egress by itself, in addition to failing to clear host chromatin from the nuclear periphery.

MATERIALS AND METHODS

Cells and viruses.

HeLa and Plat-GP cells were described previously (48 to 50). Wild-type HSV-1(F), UL31-null mutant virus YK720 (ΔUL31), UL34-null mutant virus YK723 (ΔUL34), and YK601, a recombinant virus encoding VP26, a capsid protein fused to VenusA206K fluorescent protein, were described previously (36, 48, 51, 52).

Plasmids.

pMXs-LBR, pMXs-lamin A, and pMXs-lamin C were constructed by cloning the LBR, lamin A, or lamin C open reading frame (ORF) sequences amplified by PCR from cDNA synthesized from the total RNA of HeLa cells, respectively, into pMxs-puro, as described previously (53). To construct pX458-LBR and pX458-LMNA, sense and antisense oligonucleotides were designed for insertion into the BbsI site in the pX458 bicistronic expression vector, which expresses GFP, Cas9, and a synthetic single-guide RNA (Addgene), as follows: pX458-LBR 5′-CACCGGTGAAGTGGTAAGAGGTCGA-3′ and 5′-AAACTCGACCTCTTACCACTTCACC-3′, and pX458-LMNA 5′-CACCGCCGGCCCCACGAGGAACGCC-3′ and 5′-AAACGGCGTTCCTCGTGGGGCCGGC-3′. DNA oligonucleotides were annealed and incorporated into the pX458 vector linearized with the BbsI restriction enzyme. pMAL-Us11:1-240 was constructed by cloning the HSV-1 Us11 domain encoding codons 1 to 240 amplified by PCR from the HSV-1(F) genome into pMAL-c (New England BioLabs) in frame with maltose binding protein (MBP).

Establishment of LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells.

HeLa cells were transfected with pX458-LBR or pX458-LMNA using a NEPA21 electroporator (NEPA Gene). GFP positive cells were sorted at 48 h posttransfection using FACSMelody (BD Bioscience), plated into 96-well flat-bottom plates, and designated LBR KO cells or LMNA KO cells, respectively. LMNA/LBR KO cells were generated as described above by transfecting pX458-LBR into LMNA KO cells. To determine the genotypes of each allele in the LMNA KO, LBR KO, and LMNA/LBR KO cells, genomic DNA from each cell type was amplified by PCR and sequenced directly. The sequencing of PCR products showed mixed patterns of sequences, and therefore PCR products were cloned into plasmids and their sequences were determined. We obtained several patterns of sequences, which represented the LMNA sequences of the 3 LMNA alleles from LMNA KO and LMNA/LBR KO cells, and LBR sequences of the 2 or 3 LBR alleles from LBR KO cells or LMNA/LBR KO cells, respectively, and did not include the patterns of wild-type sequences (Fig. 1A).

LMNA/LBR KO cells were then transduced with supernatants from Plat-GP cells cotransfected with pMDG in combination with pMXs-LBR or pMXs-lamin A and pMXs-lamin C, and selected with 1 μg/mL puromycin. These cells were then diluted and seeded onto plates to form single colonies, which were picked for further analysis to identify LMNA/LBR KO-LBR cells and LMNA/LBR KO-lamin A/C cells.

Production and purification of MBP fusion proteins.

MBP fusion proteins (MBP-Us11_1-240) were expressed in Escherichia coli transformed with MBP-Us11_1-240 and purified as described previously (54).

Antibodies.

Commercial antibodies used in this study were mouse monoclonal antibodies to gB (H1817; Virusys), ICP8 (10A3; Millipore), ICP27 (H1113; Abcam), Lamin A/C (636; Santa Cruz Biotechnology), and β-actin (AC15; Sigma); commercial rabbit monoclonal antibody to LBR (E398L; Abcam); and commercial rabbit polyclonal antibodies to Histone H3 (tri methyl K9, EPR16601; Abcam) and Lamin B1 (ab16048-100; Abcam). Mouse polyclonal antibodies to ICP4 and UL31 and rabbit polyclonal antibodies to UL34 and vdUTPase were described previously (35, 52, 54). To generate a rabbit polyclonal antibody to Us11, rabbits were immunized with purified MBP-Us11_1-240, following the standard protocol of Eurofins Genomics (Tokyo, Japan).

Immunoblotting.

Immunoblotting was performed as described previously (55). The amount of protein present in immunoblotting bands was quantitated using ChemiDoc MP (Bio-Rad) with ImageJ software according to the manufacturer’s instructions and normalized to that of β-actin proteins and then to the sum of the signals across multiple experiments, as described previously (56).

qRT-PCR for quantitation of HSV-1 mRNAs.

Total RNA was isolated from infected cells using a SuperPrep cell lysis kit for qPCR (Toyobo) according to the manufacturer's instructions. Then, cDNA was synthesized from the isolated RNA as described above. The amount of cDNA of specific genes was quantitated using the Universal ProbeLibrary (Roche) with TaqMan Master (Roche) and the LightCycler 96 system (Roche) according to the manufacturer’s instructions. Gene-specific primers and universal probes were designed using ProbeFinder software (Roche). The primer and probe sequences for ICP27 were 5′-TCCGACAGCGATCTGGAC-3′, 5′-TCCGACGAGGAACACTCC-3′, and Universal ProbeLibrary probe 56; those for ICP8 were 5′-ACAGCTGCAGATCGAGGACT-3′, 5′-CCATCATCTCCTCGCTTAGG-3′, and Universal ProbeLibrary probe 65; those for gB were 5′-GTCAGCACCTTCATCGACCT-3′, 5′-CAGGGGGACAAACTCGTG-3′, and Universal ProbeLibrary probe 52; and those for 18S rRNA were 5′-GCAATTATTCCCCATGAACG-3′, 5′-GGGACTTAATCAACGCAAGC-3′, and Universal ProbeLibrary probe 48. The expressions of ICP27, ICP8, and gB mRNAs were normalized to the amount of 18S rRNA expression. The relative amount of each gene expression was calculated using the comparative cycle threshold (2^−ΔΔCT) method (57).

qPCR for quantitation.

To quantify HSV-1 genomic DNAs, DNA was extracted from HSV-1-infected cells using the DNeasy kit (Qiagen). Quantitative PCR was performed using the SYBR green and the LightCycler 96 system (Roche) according to the manufacturer’s instructions. The primer sequences for the ICP4 promoter were 5′-GCCGGGGCGCTGCTTGTTCTCC-3′ and 5′-CGTCCGCCGTCGCAGCCGTATC-3′, and those for GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were 5′-CAGGCGCCCAATACGACCAAATC-3′ and 5′-TTCGACAGTCAGCCGCATCTTCTT-3′. The amount of ICP4 DNA was normalized to the amount of GAPDH DNA. The relative amount of each gene expression was calculated using the comparative cycle threshold (2^−ΔΔCT) method (57).

Assay for cell viability.

The viability of HeLa, LMNA KO, LBR KO, LMNA/LBR KO, LMNA/LBR KO-LBR, and LMNA/LBR KO-lamin A/C cells was assayed using a Cell Counting Kit-8 (Dojindo) (58, 59). Cells were seeded in a 96-well culture plate at a density of 5 × 10³ cells/well. After 48 h, the cell Counting Kit 8 (CCK-8) reagent was added to the cells and incubated for 1 h. Then, the optical density in 450 nm was measured by Perkin Elmer EnSpire multimode plate reader.

Fluorescence.

Fluorescence was performed as described previously (53), and images were analyzed using Imaris software version 9.2.1 (Bitplane). Minimum cross-entropy segmentation of the stained chromatin signal in the nucleus was detected by DAPI and by filling possible holes within the segmented geometry. Volumes of the nuclei and fluorescent domains were calculated by a 3D object counter (60).

Electron microscopy.

HeLa cells or their derivatives infected with wild-type HSV-1(F) at an MOI of 10 for 24 h were examined by ultrathin-section electron microscopy as described previously (59). The number of virus particles at different morphogenetic stages was quantitated in randomly chosen cells.

Statistical analysis.

To compare two groups, statistical analysis was performed using the unpaired Student's t test. Tukey’s test was used for multiple comparisons. A P value of >0.05 was considered not significant (n.s.). All statistical analyses were performed using GraphPad Prism 8 (GraphPad Software, San Diego, CA). No methods were used to determine whether the data met the assumptions of the statistical approach.