The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-⌬NLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. IMPORTANCEThe molecular details of nuclear egress of herpesvirus capsids are still enigmatic. Although the key players, homologs of herpes simplex virus pUL34 and pUL31, which interact and form the heterodimeric nuclear egress complex, are well known, the molecular basis of this interaction and the successive budding, vesicle formation, and scission from the INM, as well as capsid release into the cytoplasm, remain largely obscure. Here we show that classical cellular targeting signals for nuclear import and export are important for proper localization and function of the NEC, thus regulating herpesvirus nuclear egress.

The nuclear egress complex (NEC) is required for efficient translocation of newly synthesized herpesvirus nucleocapsids from the nucleus to the cytosol. It consists of the type II membrane protein pUL34 which interacts with pUL31 at the inner nuclear membrane (INM). To map regions within pUL34 required for nuclear membrane targeting and pUL31 interaction, we constructed deletion/substitution mutations. Previously, we showed that 50 C-terminal amino acids (aa) of pseudorabies virus (PrV) pUL34, including the transmembrane domain, could be functionally replaced by cellular lamina-associated polypeptide 2␤ (Lap2␤) sequences. In contrast, replacement of the C-terminal 100 aa abrogated complementation but not pUL31 interaction. To further delineate essential sequences within this region, C-terminal pUL34 truncations of 60, 70, 80, 85, and 90 aa fused to Lap2␤ sequences were generated. While truncations up to 85 aa were functional, deletion of the C-terminal 90 aa abrogated function, which indicates that the important region is located between aa 171 and 176. Amino acids 173 to 175 represent RQR, a motif suggested to mediate INM targeting. Mutagenesis to RQG revealed that the mutant protein exhibited pronounced Golgi localization, but a fraction still reached the INM. Deletion mutations in the N-terminal domain of pUL34 demonstrated that absence of the first 4 aa was tolerated, while removal of 9 or more residues resulted in a nonfunctional protein. In addition, mutation of three conserved cysteines did not abrogate pUL34 function, whereas alteration of a conserved glutamine/tyrosine sequence yielded a nonfunctional protein.H erpesvirus capsids are assembled and viral genomes are packaged in the host cell nucleus while further virion maturation occurs in the cytosol. For transit from the nucleus to the cytoplasm, herpesvirus nucleocapsids bud at the inner nuclear membrane (INM), thereby acquiring a primary envelope which subsequently fuses with the outer nuclear membrane (ONM) to release the nucleocapsids into the cytosol. This process can be regarded as a vesicle (primary envelope)-mediated transport of cargo (nucleocapsids) through the nuclear envelope. Although mechanistic details of this process are not yet known, conserved herpesviral proteins homologous to herpes simplex virus 1 (HSV-1) pUL31 and pUL34 are required for efficient translocation (reviewed in references 1 to 3).The pUL34 homologs constitute type II, tail-anchored membrane proteins which are efficiently targeted to the nuclear envelope. The pUL34 interaction partner pUL31 is diffusely distributed in the nucleoplasm in the absence of pUL34 but relocates to the INM in its presence, where both proteins form the heterodimeric nuclear egress complex (NEC). This complex recruits cellular and viral kinases to phosphorylate the nuclear lamina, a tight network of intermediate filaments underneath the INM, to partly dissolve or soften the lamina, and to permit access of nucleocapsids to the budding sites (reviewed in references 1, 3, and 4). Expression of only...

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