scholarly journals The dynamic nature of the conserved tegument protein UL37 of herpesviruses

2018 ◽  
Vol 293 (41) ◽  
pp. 15827-15839 ◽  
Author(s):  
Andrea L. Koenigsberg ◽  
Ekaterina E. Heldwein
Keyword(s):  
Viral Replication ◽  
Pseudorabies Virus ◽  
Structural Information ◽  
Surface Region ◽  
Helical Structure ◽  
Tegument Protein ◽  
Dynamic Nature ◽  
Multiple Partners ◽  
Capsid Shell ◽  
Folded Core

In all herpesviruses, the space between the capsid shell and the lipid envelope is occupied by the unique tegument layer composed of proteins that, in addition to structural roles, play many other roles in the viral replication. UL37 is a highly conserved tegument protein that has activities ranging from virion morphogenesis to directional capsid trafficking to manipulation of the host innate immune response and binds multiple partners. The N-terminal half of UL37 (UL37N) has a compact bean-shaped α-helical structure that contains a surface region essential for neuroinvasion. However, no biochemical or structural information is currently available for the C-terminal half of UL37 (UL37C) that mediates most of its interactions with multiple binding partners. Here, we show that the C-terminal half of UL37 from pseudorabies virus UL37C is a conformationally flexible monomer composed of an elongated folded core and an unstructured C-terminal tail. This elongated structure, along with that of its binding partner UL36, explains the nature of filamentous tegument structures bridging the capsid and the envelope. We propose that the dynamic nature of UL37 underlies its ability to perform diverse roles during viral replication.

scholarly journals Identification of Functional Domains within the Essential Large Tegument Protein pUL36 of Pseudorabies Virus

2007 ◽  
Vol 81 (24) ◽  
pp. 13403-13411 ◽  
Author(s):  
Sindy Böttcher ◽  
Harald Granzow ◽  
Christina Maresch ◽  
Britta Möhl ◽  
Barbara G. Klupp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Virus Replication ◽  
Pseudorabies Virus ◽  
Leucine Zipper ◽  
Tegument Protein ◽  
Infection Model ◽  
Survival Times ◽  
Internal Deletion ◽  
Mouse Infection Model

ABSTRACT Proteins of the capsid proximal tegument are involved in the transport of incoming capsids to the nucleus and secondary envelopment after nuclear egress. Homologs of the essential large capsid proximal tegument protein pUL36 are conserved within the Herpesviridae. They interact with another tegument component, pUL37, and contain a deubiquitinating activity in their N termini which, however, is not essential for virus replication. Whereas an internal deletion of 709 amino acids (aa) within the C-terminal half of the alphaherpesvirus pseudorabies virus (PrV) pUL36 does not impair its function (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006), deletion of the very C terminus does (J. Lee, G. Luxton, and G. A. Smith, J. Virol. 80:12086-12094, 2006). For further characterization we deleted several predicted functional and structural motifs within PrV pUL36 and analyzed the resulting phenotypes in cell culture and a mouse infection model. Extension of the internal deletion to encompass aa 2087 to 2981 exerted only minor effects on virus replication but resulted in prolonged mean survival times of infected mice. Any additional extension did not yield viable virus. Deletion of an N-terminal region containing the deubiquitinating activity (aa 22 to 248) only slightly impaired viral replication in cell culture but slowed neuroinvasion in our mouse model, whereas a strong impairment of viral replication was observed after simultaneous removal of both nonessential domains. Absence of a region containing two predicted leucine zipper motifs (aa 748 to 991) also strongly impaired virus replication and spread. Thus, we identify several domains within the PrV UL36 protein, which, though not essential, are nevertheless important for virus replication.

scholarly journals Crystal Structure of the N-Terminal Half of the Traffic Controller UL37 from Herpes Simplex Virus 1

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Andrea L. Koenigsberg ◽  
Ekaterina E. Heldwein
Keyword(s):  
Crystal Structure ◽  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Pseudorabies Virus ◽  
Surface Region ◽  
Tegument Protein ◽  
Herpes Simplex Virus 1 ◽  
Tegument Proteins ◽  
Simplex Virus ◽  
Hsv 1

ABSTRACT Inner tegument protein UL37 is conserved among all three subfamilies of herpesviruses. Studies of UL37 homologs from two alphaherpesviruses, herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV), have suggested that UL37 plays an essential albeit poorly defined role in intracellular capsid trafficking. At the same time, HSV and PRV homologs cannot be swapped, which suggests that in addition to a conserved function, UL37 homologs also have divergent virus-specific functions. Accurate dissection of UL37 functions requires detailed maps in the form of atomic-resolution structures. Previously, we reported the crystal structure of the N-terminal half of UL37 (UL37N) from PRV. Here, we report the crystal structure of HSV-1 UL37N. Comparison of the two structures reveals that UL37 homologs differ in their overall shapes, distributions of surface charges, and locations of projecting loops. In contrast, the previously identified R2 surface region is structurally conserved. We propose that within the N-terminal half of UL37, functional conservation is centered within the R2 surface region, whereas divergent structural elements pinpoint regions mediating virus-specific functions and may engage different binding partners. Together, the two structures can now serve as templates for a structure-guided exploration of both conserved and virus-specific functions of UL37. IMPORTANCE The ability to move efficiently within host cell cytoplasm is essential for replication in all viruses. It is especially important in the neuroinvasive alphaherpesviruses, such as human herpes simplex virus 1 (HSV-1), HSV-2, and veterinarian pseudorabies virus (PRV), that infect the peripheral nervous system and have to travel long distances along axons. Capsid movement in these viruses is controlled by capsid-associated tegument proteins, yet their specific roles have not yet been defined. Systematic exploration of the roles of tegument proteins in capsid trafficking requires detailed navigational charts in the form of their three-dimensional structures. Here, we determined the crystal structure of the N-terminal half of a conserved tegument protein, UL37, from HSV-1. This structure, along with our previously reported structure of the UL37 homolog from PRV, provides a much needed 3-dimensional template for the dissection of both conserved and virus-specific functions of UL37 in intracellular capsid trafficking.

scholarly journals Essential Function of the Pseudorabies Virus UL36 Gene Product Is Independent of Its Interaction with the UL37 Protein

2004 ◽  
Vol 78 (21) ◽  
pp. 11879-11889 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Pseudorabies Virus ◽  
Rabbit Kidney ◽  
Tegument Protein ◽  
Wild Type ◽  
Coding Region ◽  
Ordered Structures ◽  
Ca 50 ◽  
Essential Function ◽  
Full Length Clone

ABSTRACT The large tegument protein encoded by the UL36 gene of pseudorabies virus (PrV) physically interacts with the product of the adjacent UL37 gene (B. G. Klupp, W. Fuchs, H. Granzow, R. Nixdorf, and T. C. Mettenleiter, J. Virol. 76:3065-3071, 2002). To analyze UL36 function, two PrV recombinants were generated by mutagenesis of an infectious PrV full-length clone in Escherichia coli: PrV-ΔUL36F exhibited a deletion of virtually the complete UL36 coding region, whereas PrV-UL36BSF contained two in-frame deletions of 238 codons spanning the predicted UL37 binding domain. Coimmunoprecipitation experiments confirmed that the mutated gene product of PrV-UL36BSF did not interact with the UL37 protein. Like the previously described PrV-ΔUL37 (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 75:8927-8936, 2001) but in contrast to PrV-ΔUL36F, PrV-UL36BSF was able to replicate in rabbit kidney (RK13) cells, although maximum virus titers were reduced ca. 50-fold and plaque diameters were reduced by ca. 45% compared to wild-type PrV. PrV-ΔUL36F was able to productively replicate after repair of the deleted gene or in a trans-complementing cell line. Electron microscopy of infected RK13 cells revealed that PrV-UL36BSF and phenotypically complemented PrV-ΔUL36F were capable of nucleocapsid formation and egress from the nucleus by primary envelopment and deenvelopment at the nuclear membrane. However, reenvelopment of nucleocapsids in the cytoplasm was blocked. Only virus-like particles without capsids were released efficiently from cells. Interestingly, cytoplasmic nucleocapsids of PrV-UL36BSF but not of PrV-ΔUL36F were found in large ordered structures similar to those which had previously been observed with PrV-ΔUL37. In summary, our results demonstrate that the interaction between the UL36 and UL37 proteins is important but not strictly essential for the formation of secondary enveloped, infectious PrV particles. Furthermore, UL36 possesses an essential function during virus replication which is independent of its ability to bind the UL37 protein.

scholarly journals Actin Is a Component of the Compensation Mechanism in Pseudorabies Virus Virions Lacking the Major Tegument Protein VP22

2005 ◽  
Vol 79 (13) ◽  
pp. 8614-8619 ◽  
Author(s):  
T. del Rio ◽  
C. J. DeCoste ◽  
L.W. Enquist
Keyword(s):  
Epithelial Cell ◽  
Cell Lines ◽  
Fusion Proteins ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Compensation Mechanism ◽  
Wild Type ◽  
Epithelial Cell Lines ◽  
Del Rio ◽  
Protein Vp22

ABSTRACT Despite being a major component of the pseudorabies virus tegument, VP22 is not required for PRV replication, virulence, or neuroinvasion (T. del Rio, H. C. Werner, and L. W. Enquist, J. Virol. 76:774-782, 2002). In the absence of VP22, tegument assembly compensates in a limited fashion with increased incorporation of cellular actin. Infection of epithelial cell lines expressing fluorescent actin fusion proteins resulted in the incorporation of filamentous and nonfilamentous actin into individual virions that were predominately light, noninfectious particles. We conclude that cellular actin is incorporated in the tegument of wild-type virions and is part of a compensation mechanism for VP22-null virions.

scholarly journals The Carboxyl Terminus of Tegument Protein pUL21 Contributes to Pseudorabies Virus Neuroinvasion

2019 ◽  
Vol 93 (7) ◽  
Author(s):  
Kai Yan ◽  
Jie Liu ◽  
Xiang Guan ◽  
Yi-Xin Yin ◽  
Hui Peng ◽  
...  
Keyword(s):  
Axonal Transport ◽  
Pseudorabies Virus ◽  
Motor Proteins ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Bimolecular Fluorescence Complementation ◽  
Tegument Protein ◽  
Carboxyl Terminus ◽  
Anterograde Axonal Transport

ABSTRACTFollowing its entry into cells, pseudorabies virus (PRV) utilizes microtubules to deliver its nucleocapsid to the nucleus. Previous studies have shown that PRV VP1/2 is an effector of dynein-mediated capsid transport. However, the mechanism of PRV for recruiting microtubule motor proteins for successful neuroinvasion and neurovirulence is not well understood. Here, we provide evidence that PRV pUL21 is an inner tegument protein. We tested its interaction with the cytoplasmic light chains using a bimolecular fluorescence complementation (BiFC) assay and observed that PRV pUL21 interacts with Roadblock-1. This interaction was confirmed by coimmunoprecipitation (co-IP) assays. We also determined the efficiency of retrograde and anterograde axonal transport of PRV strains in explanted neurons using a microfluidic chamber system and investigated pUL21’s contribution to PRV neuroinvasionin vivo. Further data showed that the carboxyl terminus of pUL21 is essential for its interaction with Roadblock-1, and this domain contributes to PRV retrograde axonal transportin vitroandin vivo. Our findings suggest that the carboxyl terminus of pUL21 contributes to PRV neuroinvasion.IMPORTANCEHerpesviruses are a group of DNA viruses that infect both humans and animals. Alphaherpesviruses are distinguished by their ability to establish latent infection in peripheral neurons. After entering neurons, the herpesvirus capsid interacts with cellular motor proteins and undergoes retrograde transport on axon microtubules. This elaborate process is vital to the herpesvirus lifecycle, but the underlying mechanism remains poorly understood. Here, we determined that pUL21 is an inner tegument protein of pseudorabies virus (PRV) and that it interacts with the cytoplasmic dynein light chain Roadblock-1. We also observed that pUL21 promotes retrograde transport of PRV in neuronal cells. Furthermore, our findings confirm that pUL21 contributes to PRV neuroinvasionin vivo. Importantly, the carboxyl terminus of pUL21 is responsible for interaction with Roadblock-1, and this domain contributes to PRV neuroinvasion. This study offers fresh insights into alphaherpesvirus neuroinvasion and the interaction between virus and host during PRV infection.

scholarly journals Random Transposon-Mediated Mutagenesis of the Essential Large Tegument Protein pUL36 of Pseudorabies Virus

2010 ◽  
Vol 84 (16) ◽  
pp. 8153-8162 ◽  
Author(s):  
Britta S. Möhl ◽  
Sindy Böttcher ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pseudorabies Virus ◽  
Plaque Assay ◽  
Tegument Protein ◽  
Nuclear Pore ◽  
Viral Spread ◽  
One Step ◽  
Step Growth ◽  
Insertion Mutants

ABSTRACT Homologs of the pseudorabies virus (PrV) essential large tegument protein pUL36 are conserved throughout the Herpesviridae. pUL36 functions during transport of the nucleocapsid to and docking at the nuclear pore as well as during virion formation after nuclear egress in the cytoplasm. Deletion analyses revealed several nonessential regions within the 3,084-amino-acid PrV pUL36 (S. Böttcher, B. G. Klupp, H. Granzow, W. Fuchs, K. Michael, and T. C. Mettenleiter, J. Virol. 80:9910-9915, 2006; S. Böttcher, H. Granzow, C. Maresch, B. Möhl, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 81:13403-13411, 2007), while the C-terminal 62 amino acids are essential for virus replication (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). To identify additional functional domains, we performed random mutagenesis of PrV pUL36 by transposon-mediated insertion of a 15-bp linker. By this approach, 26 pUL36 insertion mutants were selected and tested in transient transfection assays for their ability to complement one-step growth and/or viral spread of a PrV UL36 null mutant. Ten insertion mutants in the N-terminal half and 10 in the C terminus complemented both, whereas six insertion mutants clustering in the center of the protein did not complement in either assay. Interestingly, several insertions within conserved parts yielded positive complementation, including those located within the essential C-terminal 62 amino acids. For 15 mutants that mediated productive replication, stable virus recombinants were isolated and further characterized by plaque assay, in vitro growth analysis, and electron microscopy. Except for three mutant viruses, most insertion mutants replicated like wild-type PrV. Two insertion mutants, at amino acids (aa) 597 and 689, were impaired in one-step growth and viral spread and exhibited a defect in virion maturation in the cytoplasm. In contrast, one functional insertion (aa 1800) in a region which otherwise yielded only nonfunctional insertion mutants was impaired in viral spread but not in one-step growth without a distinctive ultrastructural phenotype. In summary, these studies extend and refine previous analyses of PrV pUL36 and demonstrate the different sensitivities of different regions of the protein to functional loss by insertion.

scholarly journals Primary Envelopment of Pseudorabies Virus at the Nuclear Membrane Requires the UL34 Gene Product

2000 ◽  
Vol 74 (21) ◽  
pp. 10063-10073 ◽  
Author(s):  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Thomas C. Mettenleiter
Keyword(s):  
Gene Product ◽  
Nuclear Membrane ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Outer Nuclear Membrane ◽  
Virus Particles ◽  
Enveloped Virus ◽  
Perinuclear Space ◽  
Infected Cells ◽  
Mature Virus

ABSTRACT Primary envelopment of several herpesviruses has been shown to occur by budding of intranuclear capsids through the inner nuclear membrane. By subsequent fusion of the primary envelope with the outer nuclear membrane, capsids are released into the cytoplasm and gain their final envelope by budding into vesicles in thetrans-Golgi area. We show here that the product of the UL34 gene of pseudorabies virus, an alphaherpesvirus of swine, is localized in transfected and infected cells in the nuclear membrane. It is also detected in the envelope of virions in the perinuclear space but is undetectable in intracytoplasmic and extracellular enveloped virus particles. Conversely, the tegument protein UL49 is present in mature virus particles and absent from perinuclear virions. In the absence of the UL34 protein, acquisition of the primary envelope is blocked and neither virus particles in the perinuclear space nor intracytoplasmic capsids or virions are observed. However, light particles which label with the anti-UL49 serum are formed in the cytoplasm. We conclude that the UL34 protein is required for primary envelopment, that the primary envelope is biochemically different from the final envelope in that it contains the UL34 protein, and that perinuclear virions lack the tegument protein UL49, which is present in mature virions. Thus, we provide additional evidence for a two-step envelopment process in herpesviruses.

scholarly journals Structural dissection of alkaline-denatured pepsin

2004 ◽  
Vol 18 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Yuji O. Kamatari ◽  
Christopher M. Dobson ◽  
Takashi Konno
Keyword(s):  
Limited Proteolysis ◽  
Helical Structure ◽  
Cd Spectroscopy ◽  
Core Region ◽  
Detailed Knowledge ◽  
Native State ◽  
Denatured State ◽  
Residual Structure ◽  
Alkaline Denaturation ◽  
Folded Core

Pepsin, a gastric aspartic proteinase, is a zymogen‒derived protein that undergoes irreversible alkaline denaturation at pH 6–7. Detailed knowledge of the structure of the alkaline‒denatured state is an important step in understanding the mechanism of the formation of the active enzyme. It has been established in a number of studies that the alkaline‒denatured state of pepsin (the IPstate) is composed of a compact C‒terminal lobe and a largely unstructured N‒terminal lobe. In the present study, we have investigated the residual structure in the IPstate in more detail, using limited proteolysis to isolate and characterize a tightly folded core region from this partially denatured pepsin. The isolated core region corresponds to the 141 C‒terminal residues of the pepsin molecule, which in the fully native state forms one of the two lobes of the structure. A comparative study using NMR and CD spectroscopy has revealed, however, that the N‒terminal lobe contributes a substantial amount of additional residual structure to the IPstate of pepsin. CD spectra indicate in addition that significant non‒native α-helical structure is present in the C‒terminal lobe of the structure when the N‒terminal lobe of pepsin is either unfolded or removed by proteolysis. This study demonstrates that the structure of pepsin in the IPstate is significantly more complex than that of a fully folded C‒terminal lobe connected to an unstructured N‒terminal lobe. The “misfolding” in this state could inhibit the proper refolding of the protein when returned to conditions that stabilize the native state.

scholarly journals Composition of Pseudorabies Virus Particles Lacking Tegument Protein US3, UL47, or UL49 or Envelope Glycoprotein E

2006 ◽  
Vol 80 (3) ◽  
pp. 1332-1339 ◽  
Author(s):  
Kathrin Michael ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Axel Karger
Keyword(s):  
Protein Interactions ◽  
Pseudorabies Virus ◽  
Cellular Protein ◽  
Tegument Protein ◽  
Virion Protein ◽  
Two Dimensional Gel Electrophoresis ◽  
Glycoprotein E ◽  
Tegument Proteins ◽  
Glycoprotein H ◽  
The Impact

ABSTRACT Proteins located in the tegument layer of herpesvirus particles play important roles in the replicative cycle at both early and late times after infection. As major constituents of the virion, they execute important functions in particular during formation of progeny virions. These functions have mostly been elucidated by construction and analysis of mutant viruses deleted in single or multiple tegument protein-encoding genes (reviewed in the work of T. C. Mettenleiter, Virus Res. 106:167-180, 2004). However, since tegument proteins have been shown to be involved in numerous protein-protein interactions, the impact of single protein deletions on the composition of the virus particle is unknown, but they could impair correct interpretation of the results. To analyze how the absence of single virion constituents influences virion composition, we established a procedure to assay relative amounts of virion structural proteins in deletion mutants of the alphaherpesvirus Pseudorabies virus (PrV) in comparison to wild-type particles. The assay is based on the mass spectrometric quantitation of virion protein-derived peptides carrying stable isotope mass tags. After deletion of the US3, UL47, UL49, or glycoprotein E gene, relative amounts of a capsid protein (UL38), a capsid-associated protein (UL25), several tegument proteins (UL36 and UL47, if present), and glycoprotein H were unaffected, whereas the content of other tegument proteins (UL46, UL48, and UL49, if present) varied significantly. In the case of the UL48 gene product, a specific increase in incorporation of a smaller isoform was observed after deletion of the UL47 or UL49 gene, whereas a larger isoform remained unaffected. The cellular protein actin was enriched in virions of mutants deficient in any of the tegument proteins UL47, UL49, or US3. By two-dimensional gel electrophoresis multiple isoforms of host cell-derived heat shock protein 70 and annexins A1 and A2 were also identified as structural components of PrV virions.

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