Dataset Information

Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress.

ABSTRACT: 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.

SUBMITTER: Klupp BG

PROVIDER: S-EPMC6052286 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress.

Klupp Barbara G BG Hellberg Teresa T Rönfeldt Sebastian S Franzke Kati K Fuchs Walter W Mettenleiter Thomas C TC

Journal of virology 20180717 15

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 ex ...[more]

PMID: 29793954

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Project description:Progeny capsids of herpesviruses leave the nucleus by budding through the nuclear envelope. Two viral proteins, the membrane protein pUL34 and the nucleo-phosphoprotein pUL31 form the nuclear egress complex that is required for capsid egress out of the nucleus. All pUL31 orthologs are composed of a diverse N-terminal domain with 1 to 3 basic patches and a conserved C-terminal domain. To decipher the functions of the N-terminal domain, we have generated several Herpes simplex virus mutants and show here that the N-terminal domain of pUL31 is essential with basic patches being critical for viral propagation. pUL31 and pUL34 entered the nucleus independently of each other via separate routes and the N-terminal domain of pUL31 was required to prevent their premature interaction in the cytoplasm. Unexpectedly, a classical bipartite nuclear localization signal embedded in this domain was not required for nuclear import of pUL31. In the nucleus, pUL31 associated with the nuclear envelope and newly formed capsids. Viral mutants lacking the N-terminal domain or with its basic patches neutralized still associated with nucleocapsids but were unable to translocate them to the nuclear envelope. Replacing the authentic basic patches with a novel artificial one resulted in HSV1(17+)Lox-UL31-hbpmp1mp2, that was viable but delayed in nuclear egress and compromised in viral production. Thus, while the C-terminal domain of pUL31 is sufficient for the interaction with nucleocapsids, the N-terminal domain was essential for capsid translocation to sites of nuclear egress and a coordinated interaction with pUL34. Our data indicate an orchestrated sequence of events with pUL31 binding to nucleocapsids and escorting them to the inner nuclear envelope. We propose a common mechanism for herpesviral nuclear egress: pUL31 is required for intranuclear translocation of nucleocapsids and subsequent interaction with pUL34 thereby coupling capsid maturation with primary envelopment.

Project description:UnlabelledThe 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-ΔNLS 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.

Project description:Newly assembled herpesvirus nucleocapsids are translocated from the nucleus to the cytosol by a vesicle-mediated process engaging the nuclear membranes. This transport is governed by the conserved nuclear egress complex (NEC), consisting of the alphaherpesviral pUL34 and pUL31 homologs. The NEC is not only required for efficient nuclear egress but also sufficient for vesicle formation from the inner nuclear membrane (INM), as well as from synthetic lipid bilayers. The recently solved crystal structures for the NECs from different herpesviruses revealed molecular details of this membrane deformation and scission machinery uncovering the interfaces involved in complex and coat formation. However, the interaction domain with the nucleocapsid remained undefined. Since the NEC assembles a curved hexagonal coat on the nucleoplasmic side of the INM consisting of tightly interwoven pUL31/pUL34 heterodimers arranged in hexamers, only the membrane-distal end of the NEC formed by pUL31 residues appears to be accessible for interaction with the nucleocapsid cargo. To identify the amino acids involved in capsid incorporation, we mutated the corresponding regions in the alphaherpesvirus pseudorabies virus (PrV). Site-specifically mutated pUL31 homologs were tested for localization, interaction with pUL34, and complementation of PrV-ΔUL31. We identified a conserved lysine residue at amino acid position 242 in PrV pUL31 located in the alpha-helical domain H10 exposed on the membrane-distal end of the NEC as a key residue for nucleocapsid incorporation into the nascent primary particle.IMPORTANCE Vesicular transport through the nuclear envelope is a focus of research but is still not well understood. Herpesviruses pioneered this mechanism for translocation of the newly assembled nucleocapsid from the nucleus into the cytosol via vesicles derived from the inner nuclear membrane which fuse in a well-tuned process with the outer nuclear membrane to release their content. The structure of the viral nuclear membrane budding and scission machinery has been solved recently, providing in-depth molecular details. However, how cargo is incorporated remained unclear. We identified a conserved lysine residue in the membrane-distal portion of the nuclear egress complex required for capsid uptake into inner nuclear membrane-derived vesicles.

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.

Project description:Protein kinases homologous to the US3 gene product (pUS3) of herpes simplex virus (HSV) are conserved throughout the alphaherpesviruses but are absent from betaherpesviruses and gammaherpesviruses. pUS3 homologs are multifunctional and are involved in many processes, including modification of the cytoskeleton, inhibition of apoptosis, and immune evasion. pUS3 also plays a role in efficient nuclear egress of alphaherpesvirus nucleocapsids. In the absence of pUS3, primary enveloped virions accumulate in the perinuclear space (PNS) in large invaginations of the inner nuclear membrane (INM), pointing to a modulatory function for pUS3 during deenvelopment. The HSV and pseudorabies virus (PrV) US3 genes are transcribed into two mRNAs encoding two pUS3 isoforms, which have different aminoterminal sequences and abundances. To test whether the two isoforms in PrV serve different functions, we constructed mutant viruses expressing exclusively either the larger minor or the smaller major isoform, a mutant virus with decreased expression of the smaller isoform, or a mutant with impaired kinase function. Respective virus mutants were investigated in several cell lines. Our results show that absence of the larger pUS3 isoform has no detectable effect on viral replication in cell culture, while full expression of the smaller isoform and intact kinase activity is required for efficient nuclear egress. Absence of pUS3 resulted in only minor titer reduction in most cell lines tested but disclosed a more severe defect in Madin-Darby bovine kidney cells. However, accumulations of primary virions in the PNS do not account for the observed titer reduction in PrV.IMPORTANCE A plethora of substrates and functions have been assigned to the alphaherpesviral pUS3 kinase, including a role in nuclear egress. In PrV, two different pUS3 isoforms are expressed, which differ in size, abundance, and intracellular localization. Their respective role in replication is unknown, however. Here, we show that efficient nuclear egress of PrV requires the smaller isoform and intact kinase activity, whereas absence of the larger isoform has no significant effect on viral replication. Thus, there is a clear distinction in function between the two US3 gene products of PrV.

Dataset Information

Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress.

Publications

Function of the Nonconserved N-Terminal Domain of Pseudorabies Virus pUL31 in Nuclear Egress.

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