Identification of Conserved Amino Acids in pUL34 Which Are Critical for Function of the Pseudorabies Virus Nuclear Egress Complex

Nuclear egress of herpesvirus capsids is mediated by a conserved heterodimeric complex of two viral proteins, designated pUL34 and pUL31 in herpes simplex virus and pseudorabies virus (PrV). pUL34, a tail-anchored membrane protein, is targeted to the nuclear envelope and recruits pUL31 to the inner nuclear membrane (INM) to provide the docking and envelopment machinery for the nascent capsid. While the less conserved C-terminal part of pUL34 is required for correct positioning of the nuclear egress complex (NEC) at the INM, the conserved N-terminal part functions as a docking site for pUL31. Since no crystal struc-ture of NEC is available yet, structure-function studies depend onmutational analyses, with several approaches already being performed for different herpesvirus NECs. Here, we extended our studies on PrV pUL34 and identified two asparagine residues (N75, N103) and a dileucine motif (LL166/167), adjacent to an endoplasmic reticulum retention signal, which are absolutely re-quired for NEC function.While the pUL34–N75A substitutionmutant is unable to interact with pUL31, the pUL34–N103Amu-tant is nonfunctional, despite continuing complex formation. Surprisingly, mutant pUL34–G77A, which does not efficiently recruit pUL31 to the nuclear rim after cotransfection, nonetheless complements a UL34 deletion mutant, indicating that the NECmay be stabilized by additional viral factors during infection. IMPORTANCE In the absence of a crystal structure of the nuclear egress complex (NEC) required for herpesvirus maturation, site-directed mu