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Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34.

ABSTRACT: Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-?UL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.

SUBMITTER: Schuster F

PROVIDER: S-EPMC3302415 | biostudies-literature | 2012 Feb

REPOSITORIES: biostudies-literature

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Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34.

Schuster Franziska F Klupp Barbara G BG Granzow Harald H Mettenleiter Thomas C TC

Journal of virology 20111207 4

Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic ...[more]

PMID: 22156520

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Project description: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-?UL31. 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.

Project description: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.The 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.

Project description: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.

Dataset Information

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34.

Publications

Structural determinants for nuclear envelope localization and function of pseudorabies virus pUL34.

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