Function of the non-conserved 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 showed 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.