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 Mutational Functional Analysis of the Pseudorabies Virus Nuclear Egress Complex-Nucleocapsid Interaction

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Sebastian Rönfeldt ◽  
Kati Franzke ◽  
Julia E. Hölper ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Amino Acid ◽  
Electrostatic Interaction ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Basic Amino Acid ◽  
Vesicle Formation ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Perinuclear Space ◽  
Resolution Imaging

ABSTRACT 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.

scholarly journals The HSV1 Tail-Anchored Membrane Protein pUL34 Contains a Basic Motif That Supports Active Transport to the Inner Nuclear Membrane Prior to Formation of the Nuclear Egress Complex

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1544
Author(s):  
Christina Funk ◽  
Débora Marques da Silveira e Santos ◽  
Melanie Ott ◽  
Verena Raschbichler ◽  
Susanne M. Bailer
Keyword(s):  
Nuclear Membrane ◽  
Insertion Site ◽  
Nuclear Periphery ◽  
Pore Channel ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Viral Mutant ◽  
Membrane Budding ◽  
Simplex Virus ◽  
Host Nucleus

Herpes simplex virus type 1 nucleocapsids are released from the host nucleus by a budding process through the nuclear envelope called nuclear egress. Two viral proteins, the integral membrane proteins pUL34 and pUL31, form the nuclear egress complex at the inner nuclear membrane, which is critical for this process. The nuclear import of both proteins ensues separately from each other: pUL31 is actively imported through the central pore channel, while pUL34 is transported along the peripheral pore membrane. With this study, we identified a functional bipartite NLS between residues 178 and 194 of pUL34. pUL34 lacking its NLS is mislocalized to the TGN but retargeted to the ER upon insertion of the authentic NLS or a mimic NLS, independent of the insertion site. If co-expressed with pUL31, either of the pUL34-NLS variants is efficiently, although not completely, targeted to the nuclear rim where co-localization with pUL31 and membrane budding seem to occur, comparable to the wild-type. The viral mutant HSV1(17+)Lox-UL34-NLS mt is modestly attenuated but viable and associated with localization of pUL34-NLS mt to both the nuclear periphery and cytoplasm. We propose that targeting of pUL34 to the INM is facilitated by, but not dependent on, the presence of an NLS, thereby supporting NEC formation and viral replication.

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 Structural Basis for Capsid Recruitment and Coat Formation during HSV-1 Nuclear Egress

Proceedings ◽  
2020 ◽  
Vol 50 (1) ◽  
pp. 101
Author(s):  
Elizabeth Draganova ◽  
Jiayan Zhang ◽  
Hong Zhou ◽  
Ekaterina Heldwein
Keyword(s):  
Capsid Protein ◽  
Nuclear Membrane ◽  
Structural Basis ◽  
Hexagonal Array ◽  
Herpesvirus Infection ◽  
Viral Capsids ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Proper Curvature ◽  
Hsv 1

During herpesvirus infection, nascent viral capsids egress the nucleus into the cytoplasm by an unusual mechanism whereby capsids bud at the inner nuclear membrane. This process is mediated by the conserved heterodimeric nuclear egress complex (NEC), anchored to the inner nuclear membrane, that deforms the membrane around the capsid by forming a hexagonal array. However, how the NEC coat interacts with the capsid and how proper curvature of the coat is achieved to enable budding are yet unclear. Here, we show that the binding of a capsid protein, UL25, promotes the formation of a pentagonal rather than hexagonal NEC arrangement. Our results suggest that during nuclear budding interactions between the UL25 bound to the pentagonal capsid vertices and the NEC introduce pentagonal insertions into the hexagonal NEC array to yield an NEC coat of the appropriate size and curvature, leading to the productive budding and egress of UL25-decorated capsids.

scholarly journals Herpes Simplex Virus Infection Induces Phosphorylation and Delocalization of Emerin, a Key Inner Nuclear Membrane Protein

2007 ◽  
Vol 81 (9) ◽  
pp. 4429-4437 ◽  
Author(s):  
James B. Morris ◽  
Helmut Hofemeister ◽  
Peter O'Hare
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Inner Nuclear Membrane ◽  
Nuclear Egress ◽  
Phosphatase Treatment ◽  
Dna Alterations ◽  
Infected Cells ◽  
Simplex Virus ◽  
Inner Nuclear Membrane Protein

ABSTRACT The inner nuclear membrane (INM) contains specialized membrane proteins that selectively interact with nuclear components including the lamina, chromatin, and DNA. Alterations in the organization of and interactions with INM and lamina components are likely to play important roles in herpesvirus replication and, in particular, exit from the nucleus. Emerin, a member of the LEM domain class of INM proteins, binds a number of nuclear components including lamins, the DNA-bridging protein BAF, and F-actin and is thought to be involved in maintaining nuclear integrity. Here we report that emerin is quantitatively modified during herpes simplex virus (HSV) infection. Modification begins early in infection, involves multiple steps, and is reversed by phosphatase treatment. Emerin phosphorylation during infection involves one or more cellular kinases but can also be influenced by the US3 viral kinase, a protein whose function is known to be involved in HSV nuclear egress. The results from biochemical extraction analyses and from immunofluorescence of the detergent-resistant population demonstrate that emerin association with the INM significantly reduced during infection. We propose that the induction of emerin phosphorylation in infected cells may be involved in nuclear egress and uncoupling interactions with targets such as the lamina, chromatin, or cytoskeletal components.

scholarly journals Inner-Nuclear-Membrane-Associated Degradation Employs Dfm1-Independent Retrotranslocation and Alleviates Misfolded Transmembrane-Protein Toxicity

2021 ◽  
pp. mbc.E20-11-0720
Author(s):  
Matthew P. Flagg ◽  
Margaret A. Wangeline ◽  
Sarah R. Holland ◽  
Sascha H. Duttke ◽  
Christopher Benner ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Nuclear Membrane ◽  
Transmembrane Protein ◽  
Transmembrane Proteins ◽  
Extraction Process ◽  
26S Proteasome ◽  
Energetic Cost ◽  
Inner Nuclear Membrane ◽  
Transmembrane Segments ◽  
Protein Toxicity

Prior to their delivery to and degradation by the 26S proteasome, misfolded transmembrane proteins of the ER and inner-nuclear membrane must be extracted from lipid bilayers. This extraction process, known as retrotranslocation, requires both quality-control E3 ubiquitin ligases and dislocation factors that diminish the energetic cost of dislodging the transmembrane segments of a protein. Recently, we showed that retrotranslocation of all ER transmembrane proteins requires the Dfm1 rhomboid pseudoprotease. However, we did not investigate whether Dfm1 also mediated retrotranslocation of transmembrane substrates in the inner-nuclear membrane (INM), which is contiguous with the ER but functionally separated from it by nucleoporins. Here, we show that canonical retrotranslocation occurs during INM-associated degradation (INMAD) but proceeds independently of Dfm1. Despite this independence, ERAD-M and INMAD cooperate to mitigate proteotoxicity. We show a novel misfolded-transmembrane-protein toxicity that elicits genetic suppression, demonstrating the cell's ability to tolerate a toxic burden of misfolded transmembrane proteins without functional INMAD or ERAD-M. This strikingly contrasted the suppression of the dfm1Δ null, which leads to the resumption of ERAD-M through HRD-complex remodeling. Thus, we conclude that INM retrotranslocation proceeds through a novel, private channel, which can be studied by virtue of its role in alleviating membrane-associated proteotoxicity.

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 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 Partial Functional Complementation of a Pseudorabies Virus UL25 Deletion Mutant by Herpes Simplex Virus Type 1 pUL25 Indicates Overlapping Functions of Alphaherpesvirus pUL25 Proteins

2008 ◽  
Vol 82 (12) ◽  
pp. 5725-5734 ◽  
Author(s):  
Jana Kuhn ◽  
Tobias Leege ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Nuclear Membrane ◽  
Deletion Mutant ◽  
Pseudorabies Virus ◽  
Current Evidence ◽  
Inner Nuclear Membrane ◽  
Functional Homology ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Homologs of the UL25 gene product of herpes simplex virus 1 (HSV-1) are highly conserved among the Herpesviridae. However, their exact function during viral replication is unknown. Current evidence suggests that in the alphaherpesvirus pseudorabies virus (PrV) the capsid-associated pUL25 plays a role in primary envelopment of DNA-containing mature capsids at the inner nuclear membrane. In the absence of pUL25, capsids were found in close association with the inner nuclear membrane, but nuclear egress was not observed (B. G. Klupp, H. Granzow, G. M. Keil, and T. C. Mettenleiter, J. Virol. 80:6235-6246, 2006). In contrast, HSV-1 pUL25 has been assigned a role in stable packaging of viral genomes (N. Stow, J. Virol. 75:10755-10765, 2001). Despite these apparently divergent functions, we wanted to assess whether the high sequence homology translates into functional homology. Therefore, we first analyzed a newly constructed HSV-1 UL25 deletion mutant in our assay system and observed a similar phenotype as in PrV. In the nuclei of infected cells, numerous electron-dense C capsids were detected, whereas primary envelopment of these capsids did not ensue. In agreement with results from PrV, vesicles were observed in the perinuclear space. Since these data indicated functional homology, we analyzed the ability of pUL25 of HSV-1 to complement a PrV UL25 deletion mutant and vice versa. Whereas a HSV-1 pUL25-expressing cell line partially complemented the pUL25 defect in PrV, reciprocal complementation of a HSV-1 UL25 deletion mutant by PrV pUL25 was not observed. Thus, our data demonstrate overlapping, although not identical functions of these two conserved herpesvirus proteins, and point to a conserved functional role in herpes virion formation.

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