Herpesviruses consistently disrupt nuclear lamina in fluorescent cell model

A cell-based reporter developed in primary human fibroblasts revealed a consistent herpesviral strategy for opening the nuclear envelope: infection by representatives of all three major herpesvirus subfamilies produced localized areas where the nuclear lamina had been depleted. The brief report compared herpes simplex virus type 1, varicella-zoster virus, human cytomegalovirus, Epstein–Barr virus and murine herpesvirus 68. In the experimental model, lamin A/C was tagged with red fluorescent protein, making changes in the normally rigid nuclear meshwork visible by confocal microscopy. The researchers reported that the frequency of cells with these lamina-depleted areas, abbreviated LDAs, ranged from 68.0% to 96.0% across the infected-cell conditions, with every virus-specific effect reaching statistical significance relative to the relevant uninfected controls. This was a laboratory comparison of selected viruses in particular cell systems, not a clinical study of human disease.

The biological problem addressed by the model is the passage of large herpesviral capsids through the nuclear envelope. These capsids measure about 125 nanometres in diameter, whereas nuclear pore complexes are approximately 39 nanometres across and therefore cannot accommodate direct nucleocytoplasmic transport of the particles. Herpesviruses instead assemble a core nuclear egress complex at the inner nuclear membrane. This complex recruits viral and cellular regulatory proteins that locally destabilize the nuclear lamina, giving capsids access to the membrane. The capsids then bud into the space between the inner and outer nuclear membranes before undergoing de-envelopment and release into the cytoplasm. Because efficient capsid egress is a rate-limiting step in productive replication, the integrity of this pathway provides a measurable cellular indicator of viral activity.

According to the study, the conserved regulatory sequence begins when a viral kinase phosphorylates lamin A/C at serine 22. That modification creates a recognition site for the host peptidyl-prolyl cis/trans isomerase Pin1. Pin1 binds phosphorylated lamin A/C and promotes a cis/trans conformational change that contributes to local disassembly of the lamina. The resulting gaps in the fluorescent nuclear rim identify LDAs associated with herpesviral nuclear egress. Previous work with human and animal cytomegaloviruses had linked this process to productive egress, while the present experiments broadened the comparison to representative alpha-, beta- and gamma-herpesviruses. The study therefore tested whether a shared cellular phenotype could be quantified across distinct viruses, host-cell contexts and selected forms of regulatory interference.

Biochemical analysis showed increased phosphorylation of lamin A/C at serine 22 in cells infected with each of the five viruses tested. Total lamin A/C remained comparatively stable, while the phosphorylated fraction increased clearly above mock controls. The response did not always rise steadily as the multiplicity of infection increased, which the authors interpreted as possible saturation. Most infections were performed in engineered primary human foreskin fibroblasts, whereas EBV was tested in SVK-CR2 epithelial cells because that line is permissive for the virus. MHV-68, a murine gamma-herpesvirus that can infect the fibroblasts but supports only a low level of productive replication, nevertheless produced detectable serine-22 phosphorylation and the associated nuclear phenotype. The researchers also detected viral infection markers by immunoblotting, using ICP0 for HSV-1, IE1p72 for HCMV and fluorescent reporter signals for VZV, MHV-68 and EBV.

Microscopy supported the biochemical result. HSV-1, VZV, HCMV, MHV-68 and EBV all generated the same broad phenotype: breaks in what otherwise appeared as a continuous nuclear rim, accompanied by localized depletion of the lamin meshwork. For quantitative analysis, researchers counted 50 cells in triplicate and reported mean values with standard deviations, using Student’s t-test for comparisons. In HCMV-infected cells, phosphorylation-specific immunostaining was strong, and treatment with lambda phosphatase removed the nuclear signal, supporting the specificity of the assay for phosphorylated lamin A/C. Earlier experiments with modified lamin reporters also found that a version lacking the phosphorylatable serine 22 residue showed reduced LDA formation after cytomegalovirus infection, whereas a phosphorylation-mimicking version produced an intermediate phenotype that was partly independent of infection. Those earlier experiments were cited as supporting evidence rather than newly performed here.

The study then tested whether viral kinase activity is required for the observable change. Researchers infected reporter fibroblasts with wild-type HCMV, a mutant lacking the entire UL97 open reading frame, or a UL97 mutant deficient in the cyclin-binding interface needed for full kinase activity. Wild-type infection increased lamin A/C serine-22 phosphorylation, but both mutants showed substantially lower signals. Microscopically, cells infected with either mutant retained a nuclear lamina structure that was indistinguishable from uninfected cells and were scored as negative for the LDA phenotype. Quantitative evaluation confirmed significant differences between wild-type pUL97 kinase activity and the mutant conditions. These comparisons support a direct role for the HCMV pUL97 kinase in phosphorylating lamin A/C and promoting localized lamina depletion during nuclear egress. Because the mutants showed delayed replication kinetics, the investigators prepared their virus stocks through prolonged pre-culture before carrying out the comparative infections at broadly comparable multiplicities of infection.

Small-molecule experiments produced a complementary pattern. The reporter fibroblasts were infected with VZV, HCMV or MHV-68 and treated with arsenic trioxide, a host-directed inhibitor of Pin1, or P-020, an experimental inhibitor directed against the viral nuclear egress complex. At twice their respective half-maximal effective antiviral concentrations, both compounds significantly reduced the frequency of LDAs compared with untreated infected cultures. Western blot analysis of HCMV-infected cells found that P-020 had little detectable effect on the abundance of the viral kinase, core nuclear egress proteins or other viral products tested. By contrast, brincidofovir, a developmental antiviral whose established activity involves inhibition of herpesviral DNA polymerase, did not comparably reduce virus-induced LDA formation. The compounds were refreshed daily in the reporter experiments, apart from the MHV-68 cultures, which had low-level replication and received continuous treatment without daily refreshment.

The compounds also differed in their measured antiviral potency. Brincidofovir showed half-maximal effective concentrations in the nanomolar range, arsenic trioxide in the low-micromolar range and P-020 in the intermediate-micromolar range for the viruses tested. The study therefore links LDA suppression with activity against the host-virus interface controlling nuclear egress, rather than treating the phenotype as a universal consequence of any drug that blocks viral replication. This distinction matters because the results concern a cellular structural readout under defined laboratory conditions, not a direct measurement of newly released virions in every experimental comparison. The paper also notes that successful egress may involve host kinases in addition to the viral kinases examined here, leaving the relative contributions of cellular kinases, including protein kinase C isoforms and cyclin-dependent kinases 1 and 2, unresolved.

The authors describe the fluorescent LDA assay as one option among several methods used to investigate herpesviral nuclear egress. Existing approaches include quantification of cell-associated viral DNA, electron microscopy of capsids, assays of nuclear rim formation, complementation of defective nuclear egress complexes, protein-interaction tests and detection of cytoplasmic viral assembly compartments. Cellular imaging assays such as LDA measurement can directly visualize egress-related changes, but they require specialized microscopy and may be technically demanding. More scalable molecular assays can process larger numbers of samples but may show greater inherent variability. The choice of nuclear egress measurement consequently depends on the scientific question and assay conditions. The new report also emphasizes that phosphorylation of lamin A/C at serine 22 and LDA formation are quantifiable indicators of egress activity, but only parts of the complete mechanism, which also involves the nuclear egress complex, viral and cellular kinases, Pin1 and host membrane changes.

Within the scope of the experiments, the findings show that an engineered lamin A/C–red fluorescent protein model can detect a shared, mechanistically linked feature of herpesvirus infection and distinguish inhibitors acting on nuclear egress regulation from an antiviral with a different primary mechanism. The comparison used selected virus strains, primary human foreskin fibroblasts or SVK-CR2 epithelial cells, defined multiplicities of infection and fixed collection times, and several constructs carried fluorescent markers. LDAs were observed with representatives of the alpha-, beta- and gamma-herpesvirus subfamilies, while the detailed perturbation experiments focused primarily on HCMV kinase mutants and inhibitor treatments involving VZV, HCMV and MHV-68. The results are mechanistic cellular findings and do not establish clinical efficacy or patient benefit. They support lamin A/C serine-22 phosphorylation and localized lamina depletion as practical experimental readouts for studying a conserved stage of herpesviral nuclear egress.

Subject of Research: Quantifying herpesvirus-induced nuclear lamina depletion with an autofluorescent reporter cell model

Article Title: Herpesviruses show consistency in their nuclear egress-associated induction of lamina-depleted areas as measured in an autofluorescent reporter cell model

Article References: Dhotre, K., Weghorn, L., Schütz, M., & Marschall, M. (2026). Herpesviruses show consistency in their nuclear egress-associated induction of lamina-depleted areas as measured in an autofluorescent reporter cell model. Virology Journal, 23(1), Article 242. https://doi.org/10.1186/s12985-026-03334-8

Image Credits: AI Generated

DOI: 10.1186/s12985-026-03334-8

Keywords: herpesviruses, viral nuclear egress, nuclear lamina, lamin A/C, serine-22 phosphorylation, Pin1, cytomegalovirus, primary human fibroblasts, fluorescence microscopy, antiviral inhibitors, show, consistency