Mutational functional analysis of the pseudorabies virus nuclear egress complex – nucleocapsid interaction

Herpesvirus nucleocapsids leave the nucleus by a vesicle-mediated translocation coordinated by the viral nuclear egress complex (NEC). The NEC, composed of two conserved viral proteins, designated as pUL34 and pUL31 in the alphaherpesvirus pseudorabies virus (PrV) is required for efficient nuclear egress and 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 as 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 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 substituted K242 by several amino acids thereby modifying 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.