The MHV-68 nuclear egress complex supports C-capsid selective capsid egress.

Herpesvirus capsids assemble within the host nucleus and must traverse the nuclear envelope to undergo final envelopment in the cytoplasm. This exit strategy is orchestrated by the nuclear egress complex (NEC), a conserved heterodimeric protein complex that forms lattices that remodel the inner nuclear membrane. While the NEC is critical for efficient replication across α-, β-, and γ-herpesviruses, residual infectivity has been noted in NEC-deficient mutants of the ɑ-herpesvirus Pseudorabies virus. Here, we investigated the NEC's role in the γ-herpesvirus murine γ-herpesvirus 68 (MHV-68) by generating a mutant virus (ΔC-ORF69) lacking the majority of the nucleoplasmic NEC component, ORF69. Unlike prior studies utilizing transfection-based systems, we demonstrate that NEC-deficient MHV-68 produces infectious progeny in non-complementing cells, albeit with a significant replication defect. Quantitative electron microscopy confirmed that while nuclear capsid assembly remained regular, the canonical export mechanism was abolished. Whereas the parental virus predominantly exported mature, enveloped, genome-filled C-capsids out of the nucleus, the ΔC-ORF69 mutant released all major capsid forms (A, B, and C) into the cytoplasm and extracellular space, associated with loss of nuclear integrity. These findings suggest that, in addition to mediating efficient capsid translocation across the nuclear envelope, the MHV-68 NEC imposes C-capsid selectivity during nuclear egress, thereby acting as a quality control checkpoint. Inhibition of cyclin-dependent kinases reduced viral spread in both parental and ΔC-ORF69 viruses, indicating that cyclin-dependent kinase activity contributes to productive infection and may influence the residual spread observed in ΔC-ORF69, consistent with a potential contribution to late-stage nuclear envelope integrity loss.IMPORTANCEHuman γ-herpesviruses, such as Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus, are significant pathogens that cause lifelong infections and malignancies. A critical bottleneck in their life cycle is the translocation of viral capsids from the nucleus to the cytoplasm, a process driven by the viral nuclear egress complex (NEC). By characterizing a specific NEC mutant in the murine model MHV-68, we uncovered a fundamental, previously underappreciated function of this complex. While the NEC is often primarily viewed as transport machinery, our data demonstrate that it functions as a critical specificity determinant, selectively allowing only mature, DNA-filled capsids to exit the nucleus. In the absence of a functional NEC, this selectivity is lost, leading to the non-selective leakage of all major capsid forms via nuclear envelope disruption.