Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction.

Herpesvirus nucleocapsids leave the nucleus by a vesicle-mediated translocation mediated by the viral nuclear egress complex (NEC). The NEC is composed of two conserved viral proteins, designated pUL34 and pUL31 in the alphaherpesvirus pseudorabies virus (PrV). It is required for efficient nuclear egress and is sufficient for vesicle formation and scission from the inner nuclear membrane (INM). Structure-based mutagenesis identified a lysine at position 242 (K242) in pUL31, located in the most membrane distal part of the NEC, to be crucial for efficient nucleocapsid incorporation into budding vesicles. Replacing the lysine by alanine (K242A) resulted in accumulations of empty vesicles in the perinuclear space, despite the presence of excess nucleocapsids in the nucleus. However, it remained unclear whether the defect in capsid incorporation was due to interference with a direct, electrostatic interaction between the capsid and the NEC or structural restrictions. To test this, we replaced K242 with several amino acids, thereby modifying the charge, size, and side chain orientation. In addition, virus recombinants expressing pUL31-K242A were passaged and screened for second-site mutations. Compensatory mutations at different locations in pUL31 or pUL34 were identified, pointing to an inherent flexibility of the NEC. In summary, our data suggest that the amino acid at position 242 does not directly interact with the nucleocapsid but that rearrangements in the NEC coat are required for efficient nucleocapsid envelopment at the INM.IMPORTANCE Herpesviruses encode an exceptional vesicle formation and scission machinery, which operates at the inner nuclear membrane, translocating the viral nucleocapsid from the nucleus into the perinuclear space. The conserved herpesviral nuclear egress complex (NEC) orchestrates this process. High-resolution imaging approaches as well as the recently solved crystal structures of the NEC provided deep insight into the molecular details of vesicle formation and scission. Nevertheless, the molecular mechanism of nucleocapsid incorporation remained unclear. In accordance with structure-based predictions, a basic amino acid could be pinpointed in the most membrane-distal domain of the NEC (pUL31-K242), indicating that capsid incorporation might depend on a direct electrostatic interaction. Our follow-up study, described here, however, shows that the positive charge is not relevant but that the overall structure matters.

Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress.

Nuclear egress of herpesvirus capsids is mediated by the conserved nuclear egress complex (NEC), composed of the membrane-anchored pUL34 and its nucleoplasmic interaction partner, pUL31. The recently solved crystal structures of the NECs from different herpesviruses show a high structural similarity, with the pUL34 homologs building a platform recruiting pUL31 to the inner nuclear membrane. Both proteins possess a central globular fold, while the conserved N-terminal portion of pUL31 forms an extension reaching around the core of pUL34. However, the extreme N terminus of the pUL31 homologs, which is highly variable in length and amino acid composition, had to be removed for crystallization. Several pUL31 homologs contain a classical nuclear localization signal (NLS) within this part mediating efficient nuclear import. In addition, membrane-binding activity, blocking premature interaction with pUL34, nucleocapsid trafficking, and regulation of NEC assembly and disassembly via phosphorylation were assigned to the extreme pUL31 N terminus. To test the functional importance in the alphaherpesvirus pseudorabies virus (PrV) pUL31, N-terminal truncations and site-specific mutations were generated, and the resulting proteins were tested for intracellular localization, interaction with pUL34, and functional complementation of PrV-ΔUL31. Our data show that neither the bipartite NLS nor the predicted phosphorylation sites are essential for pUL31 function during nuclear egress. Moreover, nearly the complete variable N-terminal part was dispensable for function as long as a stretch of basic amino acids was retained. Phosphorylation of this domain controls efficient nucleocapsid release from the perinuclear space.IMPORTANCE Nuclear egress of herpesvirus capsids is a unique vesicle-mediated nucleocytoplasmic transport. Crystal structures of the heterodimeric NECs from different herpesviruses provided important details of this viral nuclear membrane deformation and scission machinery but excluded the highly variable N terminus of the pUL31 component. We present here a detailed mutagenesis study of this important portion of pUL31 and show that basic amino acid residues within this domain play an essential role for proper targeting, complex formation, and function during nuclear egress, while phosphorylation modulates efficient release from the perinuclear space. Thus, our data complement previous structure-function assignments of the nucleocapsid-interacting component of the NEC.

Lysine 242 within Helix 10 of the Pseudorabies Virus Nuclear Egress Complex pUL31 Component Is Critical for Primary Envelopment of Nucleocapsids.

Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane (INM), as well as from synthetic lipid bilayers. The recently solved crystal structures for the NECs from different herpesviruses revealed molecular details of this membrane deformation and scission machinery uncovering the interfaces involved in complex and coat formation. However, the interaction domain with the nucleocapsid remained undefined. Since the NEC assembles a curved hexagonal coat on the nucleoplasmic side of the INM consisting of tightly interwoven pUL31/pUL34 heterodimers arranged in hexamers, only the membrane-distal end of the NEC formed by pUL31 residues appears to be accessible for interaction with the nucleocapsid cargo. To identify the amino acids involved in capsid incorporation, we mutated the corresponding regions in the alphaherpesvirus pseudorabies virus (PrV). Site-specifically mutated pUL31 homologs were tested for localization, interaction with pUL34, and complementation of PrV-ΔUL31. We identified a conserved lysine residue at amino acid position 242 in PrV pUL31 located in the alpha-helical domain H10 exposed on the membrane-distal end of the NEC as a key residue for nucleocapsid incorporation into the nascent primary particle.IMPORTANCE Vesicular transport through the nuclear envelope is a focus of research but is still not well understood. Herpesviruses pioneered this mechanism for translocation of the newly assembled nucleocapsid from the nucleus into the cytosol via vesicles derived from the inner nuclear membrane which fuse in a well-tuned process with the outer nuclear membrane to release their content. The structure of the viral nuclear membrane budding and scission machinery has been solved recently, providing in-depth molecular details. However, how cargo is incorporated remained unclear. We identified a conserved lysine residue in the membrane-distal portion of the nuclear egress complex required for capsid uptake into inner nuclear membrane-derived vesicles.