scholarly journals Function of Torsin AAA+ ATPases in Pseudorabies Virus Nuclear Egress

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 738
Author(s):  
Julia E. Hölper ◽  
Barbara G. Klupp ◽  
G. W. Gant Luxton ◽  
Kati Franzke ◽  
Thomas C. Mettenleiter
Keyword(s):  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Gene Knockout ◽  
Early Time ◽  
Wild Type ◽  
Mutant Proteins ◽  
Nuclear Egress ◽  
Cytoplasmic Transport ◽  
Virion Envelope ◽  
Aaa Atpases

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.

scholarly journals Role of Vesicle-Associated Membrane Protein-Associated Proteins (VAP) A and VAPB in Nuclear Egress of the Alphaherpesvirus Pseudorabies Virus

Viruses ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1117
Author(s):  
Anna D. Dorsch ◽  
Julia E. Hölper ◽  
Kati Franzke ◽  
Luca M. Zaeck ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Rabbit Kidney ◽  
Double Knockout ◽  
Virus Propagation ◽  
Herpes Simplex Virus 1 ◽  
Nuclear Egress ◽  
Virion Envelope ◽  
Simplex Virus

The molecular mechanism affecting translocation of newly synthesized herpesvirus nucleocapsids from the nucleus into the cytoplasm is still not fully understood. The viral nuclear egress complex (NEC) mediates budding at and scission from the inner nuclear membrane, but the NEC is not sufficient for efficient fusion of the primary virion envelope with the outer nuclear membrane. Since no other viral protein was found to be essential for this process, it was suggested that a cellular machinery is recruited by viral proteins. However, knowledge on fusion mechanisms involving the nuclear membranes is rare. Recently, vesicle-associated membrane protein-associated protein B (VAPB) was shown to play a role in nuclear egress of herpes simplex virus 1 (HSV-1). To test this for the related alphaherpesvirus pseudorabies virus (PrV), we mutated genes encoding VAPB and VAPA by CRISPR/Cas9-based genome editing in our standard rabbit kidney cells (RK13), either individually or in combination. Single as well as double knockout cells were tested for virus propagation and for defects in nuclear egress. However, no deficiency in virus replication nor any effect on nuclear egress was obvious suggesting that VAPB and VAPA do not play a significant role in this process during PrV infection in RK13 cells.

scholarly journals Lysine 242 within Helix 10 of the Pseudorabies Virus Nuclear Egress Complex pUL31 Component Is Critical for Primary Envelopment of Nucleocapsids

2017 ◽  
Vol 91 (22) ◽  
Author(s):  
Sebastian Rönfeldt ◽  
Barbara G. Klupp ◽  
Kati Franzke ◽  
Thomas C. Mettenleiter
Keyword(s):  
Lipid Bilayers ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Amino Acid Position ◽  
Distal Portion ◽  
Lysine Residue ◽  
Interaction Domain ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Membrane Budding

ABSTRACT 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.

scholarly journals Integrity of the Linker of Nucleoskeleton and Cytoskeleton Is Required for Efficient Herpesvirus Nuclear Egress

2017 ◽  
Vol 91 (19) ◽  
Author(s):  
Barbara G. Klupp ◽  
Teresa Hellberg ◽  
Harald Granzow ◽  
Kati Franzke ◽  
Beatriz Dominguez Gonzalez ◽  
...  
Keyword(s):  
Nuclear Membrane ◽  
Dominant Negative ◽  
Progeny Virus ◽  
Linc Complex ◽  
Outer Nuclear Membrane ◽  
Inner Nuclear Membrane ◽  
Nuclear Membranes ◽  
Nuclear Egress ◽  
Virion Envelope ◽  
In Cells

ABSTRACT Herpesvirus capsids assemble in the nucleus, while final virion maturation proceeds in the cytoplasm. This requires that newly formed nucleocapsids cross the nuclear envelope (NE), which occurs by budding at the inner nuclear membrane (INM), release of the primary enveloped virion into the perinuclear space (PNS), and subsequent rapid fusion with the outer nuclear membrane (ONM). During this process, the NE remains intact, even at late stages of infection. In addition, the spacing between the INM and ONM is maintained, as is that between the primary virion envelope and nuclear membranes. The linker of nucleoskeleton and cytoskeleton (LINC) complex consists of INM proteins with a luminal SUN (Sad1/UNC-84 homology) domain connected to ONM proteins with a KASH (Klarsicht, ANC-1, SYNE homology) domain and is thought to be responsible for spacing the nuclear membranes. To investigate the role of the LINC complex during herpesvirus infection, we generated cell lines constitutively expressing dominant negative (dn) forms of SUN1 and SUN2. Ultrastructural analyses revealed a significant expansion of the PNS and the contiguous intracytoplasmic lumen, most likely representing endoplasmic reticulum (ER), especially in cells expressing dn-SUN2. After infection, primary virions accumulated in these expanded luminal regions, also very distant from the nucleus. The importance of the LINC complex was also confirmed by reduced progeny virus titers in cells expressing dn-SUN2. These data show that the intact LINC complex is required for efficient nuclear egress of herpesviruses, likely acting to promote fusion of primary enveloped virions with the ONM. IMPORTANCE While the viral factors for primary envelopment of nucleocapsids at the inner nuclear membrane are known to the point of high-resolution structures, the roles of cellular components and regulators remain enigmatic. Furthermore, the machinery responsible for fusion with the outer nuclear membrane is unsolved. We show here that dominant negative SUN2 interferes with efficient herpesvirus nuclear egress, apparently by interfering with fusion between the primary virion envelope and outer nuclear membrane. This identifies a new cellular component important for viral egress and implicates LINC complex integrity in nonconventional nuclear membrane trafficking.

scholarly journals Pathogenic FAM83G palmoplantar keratoderma mutations inhibit the PAWS1:CK1α association and attenuate Wnt signalling.

2020 ◽  
Vol 4 ◽  
pp. 133
Author(s):  
Kevin Z.L. Wu ◽  
Rebecca A. Jones ◽  
Theresa Tachie-Menson ◽  
Thomas J. Macartney ◽  
Nicola T. Wood ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Wnt Signalling ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Palmoplantar Keratoderma ◽  
Mutant Proteins ◽  
Recessive Mutations ◽  
Type Protein ◽  
Wild Type Protein ◽  
Equivalent Residue

Background: Two recessive mutations in the FAM83G gene, causing A34E and R52P amino acid substitutions in the DUF1669 domain of the PAWS1 protein, are associated with palmoplantar keratoderma (PPK) in humans and dogs respectively. We have previously reported that PAWS1 associates with the Ser/Thr protein kinase CK1α through the DUF1669 domain to mediate canonical Wnt signalling. Methods: Co-immunoprecipitation was used to investigate possible changes to PAWS1 interactors caused by the mutations. We also compared the stability of wild-type and mutant PAWS1 in cycloheximide-treated cells. Effects on Wnt signalling were determined using the TOPflash luciferase reporter assay in U2OS cells expressing PAWS1 mutant proteins. The ability of PAWS1 to induce axis duplication in Xenopus embryos was also tested. Finally, we knocked-in the A34E mutation at the native gene locus and measured Wnt-induced AXIN2 gene expression by RT-qPCR. Results: We show that these PAWS1A34E and PAWS1R52P mutants fail to interact with CK1α but, like the wild-type protein, do interact with CD2AP and SMAD1. Like cells carrying a PAWS1F296A mutation, which also abolishes CK1α binding, cells carrying the A34E and R52P mutants respond poorly to Wnt signalling to an extent resembling that observed in FAM83G gene knockout cells. Consistent with this observation, these mutants, in contrast to the wild-type protein, fail to induce axis duplication in Xenopus embryos. We also found that the A34E and R52P mutant proteins are less abundant than the native protein and appear to be less stable, both when overexpressed in FAM83G-knockout cells and when knocked-in at the native FAM83G locus. Ala34 of PAWS1 is conserved in all FAM83 proteins and mutating the equivalent residue in FAM83H (A31E) also abolishes interaction with CK1 isoforms. Conclusions: We propose that mutations in PAWS1 cause PPK pathogenesis through disruption of the CK1α interaction and attenuation of Wnt signalling.

scholarly journals Pathogenic FAM83G palmoplantar keratoderma mutations inhibit the PAWS1:CK1α association and attenuate Wnt signalling.

2019 ◽  
Vol 4 ◽  
pp. 133 ◽  
Author(s):  
Kevin Z.L. Wu ◽  
Rebecca A. Jones ◽  
Theresa Tachie-Menson ◽  
Thomas J. Macartney ◽  
Nicola T. Wood ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Wnt Signalling ◽  
Luciferase Reporter ◽  
Wild Type ◽  
Palmoplantar Keratoderma ◽  
Mutant Proteins ◽  
Recessive Mutations ◽  
Type Protein ◽  
Wild Type Protein ◽  
Equivalent Residue

Background: Two recessive mutations in the FAM83G gene, causing A34E and R52P amino acid substitutions in the DUF1669 domain of the PAWS1 protein, are associated with palmoplantar keratoderma (PPK) in humans and dogs respectively. We have previously reported that PAWS1 associates with the Ser/Thr protein kinase CK1α through the DUF1669 domain to mediate canonical Wnt signalling. Methods: Co-immunoprecipitation was used to investigate possible changes to PAWS1 interactors caused by the mutations. We also compared the stability of wild-type and mutant PAWS1 in cycloheximide-treated cells. Effects on Wnt signalling were determined using the TOPflash luciferase reporter assay in U2OS cells expressing PAWS1 mutant proteins. The ability of PAWS1 to induce axis duplication in Xenopus embryos was also tested. Finally, we knocked-in the A34E mutation at the native gene locus and measured Wnt-induced AXIN2 gene expression by RT-qPCR. Results: We show that these PAWS1A34E and PAWS1R52P mutants fail to interact with CK1α but, like the wild-type protein, do interact with CD2AP and SMAD1. Like cells carrying a PAWS1F296A mutation, which also abolishes CK1α binding, cells carrying the A34E and R52P mutants respond poorly to Wnt signalling to an extent resembling that observed in FAM83G gene knockout cells. Consistent with this observation, these mutants, in contrast to the wild-type protein, fail to induce axis duplication in Xenopus embryos. We also found that the A34E and R52P mutant proteins are less abundant than the native protein and appear to be less stable, both when overexpressed in FAM83G-knockout cells and when knocked-in at the native FAM83G locus. Ala34 of PAWS1 is conserved in all FAM83 proteins and mutating the equivalent residue in FAM83H (A31E) also abolishes interaction with CK1 isoforms. Conclusions: We propose that mutations in PAWS1 cause PPK pathogenesis through disruption of the CK1α interaction and attenuation of Wnt signalling.

scholarly journals Pseudorabies Virus UL37 Gene Product Is Involved in Secondary Envelopment

2001 ◽  
Vol 75 (19) ◽  
pp. 8927-8936 ◽  
Author(s):  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Egbert Mundt ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Ordered Structure ◽  
Wild Type ◽  
Tegument Proteins ◽  
Outer Leaflet ◽  
Perinuclear Space ◽  
Viral Glycoproteins ◽  
Capsid Maturation

ABSTRACT Herpesvirus envelopment is a two-step process which includes acquisition of a primary envelope resulting from budding of intranuclear capsids through the inner nuclear membrane. Fusion with the outer leaflet of the nuclear membrane releases nucleocapsids into the cytoplasm, which then gain their final envelope by budding intotrans-Golgi vesicles. It has been shown that the UL34 gene product is required for primary envelopment of the alphaherpesvirus pseudorabies virus (PrV) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 74:10063–10073, 2000). For secondary envelopment, several virus-encoded PrV proteins are necessary, including glycoproteins E, I, and M (A. R. Brack, J. M. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364–5372, 1999). We show here that the product of the UL37 gene of PrV, which is a constituent of mature virions, is involved in secondary envelopment. Replication of a UL37 deletion mutant, PrV-ΔUL37, was impaired in normal cells; this defect could be complemented on cells stably expressing UL37. Ultrastructural analysis demonstrated that intranuclear capsid maturation and budding of capsids into and release from the perinuclear space were unimpaired. However, secondary envelopment was drastically reduced. Instead, apparently DNA-filled capsids accumulated in the cytoplasm in large aggregates similar to those observed in the absence of glycoproteins E/I and M but lacking the surrounding electron-dense tegument material. Although displaying an ordered structure, capsids did not contact each other directly. We postulate that the UL37 protein is necessary for correct addition of other tegument proteins, which are required for secondary envelopment. In the absence of the UL37 protein, capsids interact with each other through unknown components but do not acquire the electron-dense tegument which is normally found around wild-type capsids during and after secondary envelopment. Thus, apposition of the UL37 protein to cytoplasmic capsids may be crucial for the addition of other tegument proteins, which in turn are able to interact with viral glycoproteins to mediate secondary envelopment.

scholarly journals Infectivity of a Pseudorabies Virus Mutant Lacking Attachment Glycoproteins C and D

1998 ◽  
Vol 72 (9) ◽  
pp. 7341-7348 ◽  
Author(s):  
Axel Karger ◽  
Jerg Schmidt ◽  
Thomas C. Mettenleiter
Keyword(s):  
Receptor Binding ◽  
Pseudorabies Virus ◽  
Cell Clone ◽  
Plaque Formation ◽  
Wild Type ◽  
Virion Protein ◽  
Gene Encoding ◽  
Mdbk Cells ◽  
Virion Envelope ◽  
Virus Mutant

ABSTRACT Initiation of herpesvirus infection requires attachment of virions to the host cell followed by fusion of virion envelope and cellular cytoplasmic membrane during penetration. In several alphaherpesviruses, glycoprotein C (gC) is the primary attachment protein, interacting with cell-surface heparan sulfate proteoglycans. Secondary binding is mediated by gD, which, normally, is also required for penetration. Recently, we described the isolation of a gD-negative infectious pseudorabies virus (PrV) mutant, PrV gD− Pass (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). In PrV gD− Pass, attachment and penetration occur in the absence of gD. To assess the importance of specific attachment for infectivity of PrV gD− Pass, the gene encoding gC was deleted, resulting in mutant PrV gCD− Pass. Deletion of both known attachment proteins reduced specific infectivity compared to wild-type PrV by more than 10,000-fold. Surprisingly, the virus mutant still retained significant infectivity and could be propagated on normal noncomplementing cells, indicating the presence of another receptor-binding virion protein. Selection of bovine kidney (MDBK) cells resistant to infection by PrV gCD− Pass resulted in the isolation of a cell clone, designated NB, which was susceptible to infection by wild-type PrV but refractory to infection by either PrV gCD− Pass or PrV gD− Pass, a defect which could partially be overcome by polyethylene glycol (PEG)-induced membrane fusion. However, even after PEG-induced infection plaque formation of PrV gCD− Pass or PrV gD− Pass did not ensue in NB cells. Also, phenotypic gD complementation of PrV gCD− Pass or PrV gD−Pass rescued the defect in infection of NB cells but did not restore plaque formation. Glycosaminoglycan analyses of MDBK and NB cells yielded identical results, and NB cells were normally susceptible to infection by other alphaherpesviruses as well as vesicular stomatitis virus. Infectious center assays after PEG-induced infection of NB cells with PrV gD− Pass on MDBK cells indicated efficient exit of virions from infected NB cells. Together, our data suggest the presence of another receptor and receptor-binding virion protein which can mediate PrV entry and cell-to-cell spread in MDBK cells.

scholarly journals Phosphorylation of the UL31 Protein of Herpes Simplex Virus 1 by the US3-Encoded Kinase Regulates Localization of the Nuclear Envelopment Complex and Egress of Nucleocapsids

2009 ◽  
Vol 83 (10) ◽  
pp. 5181-5191 ◽  
Author(s):  
Fan Mou ◽  
Elizabeth Wills ◽  
Joel D. Baines
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Normal Function ◽  
Viral Envelope ◽  
Productive Infection ◽  
Herpes Simplex Virus 1 ◽  
Nuclear Egress ◽  
Virion Envelope ◽  
Simplex Virus

ABSTRACT Herpes simplex virus 1 nucleocapsids bud through the inner nuclear membrane (INM) into the perinuclear space to obtain a primary viral envelope. This process requires a protein complex at the INM composed of the UL31 and UL34 gene products. While it is clear that the viral kinase encoded by the US3 gene regulates the localization of pUL31/pUL34 within the INM, the molecular mechanism by which this is accomplished remains enigmatic. Here, we have determined the following. (i) The N terminus of pUL31 is indispensable for the protein's normal function and contains up to six serines that are phosphorylated by the US3 kinase during infection. (ii) Phosphorylation at these six serines was not essential for a productive infection but was required for optimal viral growth kinetics. (iii) In the presence of active US3 kinase, changing the serines to alanine caused the pUL31/pUL34 complex to aggregate at the nuclear rim and caused some virions to accumulate aberrantly in herniations of the nuclear membrane, much as in cells infected with a US3 kinase-dead mutant. (iv) The replacement of the six serines of pUL31 with glutamic acid largely restored the smooth distribution of pUL34/pUL31 at the nuclear membrane and precluded the accumulation of virions in herniations whether or not US3 kinase was active but also precluded the optimal primary envelopment of nucleocapsids. These observations indicate that the phosphorylation of pUL31 by pUS3 represents an important regulatory event in the virion egress pathway that can account for much of pUS3's role in nuclear egress. The data also suggest that the dynamics of pUL31 phosphorylation modulate both the primary envelopment and the subsequent fusion of the nascent virion envelope with the outer nuclear membrane.

scholarly journals The Pseudorabies Virus US3 Protein Is a Component of Primary and of Mature Virions

2004 ◽  
Vol 78 (3) ◽  
pp. 1314-1323 ◽  
Author(s):  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Immunoelectron Microscopy ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Gene Products ◽  
Tegument Proteins ◽  
Outer Leaflet ◽  
Nuclear Egress ◽  
Intracytoplasmic Inclusions

ABSTRACT Herpesviruses acquire a primary envelope by budding of capsids at the inner leaflet of the nuclear membrane. They then traverse into the cytoplasm after fusion of the primary envelope with the outer leaflet of the nuclear membrane. In the alphaherpesvirus pseudorabies virus (PrV), the latter process is impaired when the US3 protein is absent. Acquisition of final tegument and envelope occurs in the cytoplasm. Besides the capsid components, only the UL31 and UL34 gene products of PrV have unequivocally been shown to be part of primary enveloped virions, whereas they lack several tegument proteins present in mature virions (reviewed by T. C. Mettenleiter, J. Virol. 76:1537-1547, 2002). Using immunoelectron microscopy, we show that the US3 protein is present in primary enveloped as well as in mature virions. It is also detectable in intracytoplasmic inclusions produced in the absence of other viral tegument components or envelope-associated glycoproteins. In particular, inclusions formed in the absence of the inner tegument protein UL37 contained the US3 protein. Thus, the US3 protein is a tegument component of both forms of enveloped alphaherpes virions. We hypothesize that US3 protein in primary virions modulates deenvelopment at the outer leaflet of the nuclear membrane and is either lost from primary virions during nuclear egress and subsequently reacquired early during tegumentation or is retained during transit of the nucleocapsid through the nuclear membrane.

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

2018 ◽  
Vol 92 (15) ◽  
Author(s):  
Barbara G. Klupp ◽  
Teresa Hellberg ◽  
Sebastian Rönfeldt ◽  
Kati Franzke ◽  
Walter Fuchs ◽  
...  
Keyword(s):  
Amino Acid ◽  
Crystal Structures ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Intracellular Localization ◽  
Basic Amino Acid ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Egress ◽  
Perinuclear Space ◽  
N Terminus

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-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. 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.

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