Relevance of the Interaction between Alphaherpesvirus UL3.5 and UL48 Proteins for Virion Maturation and Neuroinvasion

ABSTRACT The UL3.5 and UL48 genes, which are conserved in most alphaherpesvirus genomes, are important for maturation of pseudorabies virus (PrV) particles in the cytoplasm of infected cells (W. Fuchs, B. G. Klupp, H. J. Rziha, and T. C. Mettenleiter, J. Virol. 70:3517-3527, 1996; W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002). In bovine herpesvirus 1 (BoHV-1), the homologous gene products pUL3.5 and pUL48 have been demonstrated to interact physically (N. Lam and G. Letchworth, J. Virol. 74:2876-2884, 2000). Moreover, BoHV-1 pUL3.5 partially complemented a pUL3.5 defect in PrV (W. Fuchs, H. Granzow, and T. C. Mettenleiter, J. Virol. 71:8886-8892, 1997). By using coimmunoprecipitation and yeast two-hybrid studies, we observed a similar interaction between pUL3.5 and pUL48 of PrV, as well as a heterologous interaction between the PrV and BoHV-1 gene products. The relevant domain could be confined to the first 43 amino acids of PrV pUL3.5. Unlike its BoHV-1 homologue, PrV pUL3.5 is processed by proteolytic cleavage, and only an abundant 14-kDa fragment consisting of amino acids 1 to ≥116 could be detected by peptide mass fingerprint analysis of purified wild-type PrV particles, which also contain the pUL48 tegument component. To determine the biological relevance of the protein-protein interaction, pUL3.5-, pUL48-, and double-negative PrV mutants were analyzed in parallel. All deletion mutants were replication competent but exhibited significantly reduced plaque sizes and virus titers in cultured rabbit kidney cells compared to wild-type and rescued viruses, which correlated with a delayed neuroinvasion in intranasally infected mice. Remarkably, the defects of the double-negative mutant were similar to those of pUL48-negative virus. Electron microscopy of cells infected with either deletion mutant revealed the retention of naked nucleocapsids in the cytoplasm and the absence of mature virus particles. In summary, our studies for the first time demonstrate the relevance of the pUL3.5-pUL48 interaction for secondary envelopment of an alphaherpesvirus, give a molecular basis for the observed trans-complementation between the PrV and BHV-1 pUL3.5 homologs, yield conclusive evidence for the incorporation of a proteolytically processed pUL3.5 into PrV virions, and demonstrate the importance of both proteins for neuroinvasion and neurovirulence of PrV.

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

Journal of Virology ◽

10.1128/jvi.78.21.11879-11889.2004 ◽

2004 ◽

Vol 78 (21) ◽

pp. 11879-11889 ◽

Cited By ~ 89

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.

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Pseudorabies Virus Expressing Bovine Herpesvirus 1 Glycoprotein B Exhibits Altered Neurotropism and Increased Neurovirulence

Journal of Virology ◽

10.1128/jvi.74.2.817-827.2000 ◽

2000 ◽

Vol 74 (2) ◽

pp. 817-827 ◽

Cited By ~ 15

Author(s):

Volker Gerdts ◽

Jörg Beyer ◽

Béla Lomniczi ◽

Thomas C. Mettenleiter

Keyword(s):

Respiratory Symptoms ◽

Pseudorabies Virus ◽

Bovine Herpesvirus ◽

Glycoprotein B ◽

Target Cells ◽

Homologous Gene ◽

Wild Type ◽

Bovine Herpesvirus 1 ◽

Herpesvirus 1

ABSTRACT Herpesvirus glycoproteins play dominant roles in the initiation of infection of target cells in culture and thus may also influence viral tropism in vivo. Whereas the relative contribution of several nonessential glycoproteins to neurovirulence and neurotropism ofPseudorabies virus (PrV), an alphaherpesvirus which causes Aujeszky's disease in pigs, has recently been uncovered in studies using viral deletion mutants, the importance of essential glycoproteins is more difficult to assess. We isolated an infectious PrV mutant, PrV-9112C2, which lacks the gene encoding the essential PrV glycoprotein B (gB) but stably carries in its genome and expresses the homologous gene of bovine herpesvirus 1 (BHV-1) (A. Kopp and T. C. Mettenleiter, J. Virol. 66:2754–2762, 1992). Apart from exhibiting a slight delay in penetration kinetics, PrV-9112C2 was similar in its growth characteristics in cell culture to wild-type PrV. To analyze the effect of the exchange of these homologous glycoproteins in PrV's natural host, swine, 4-week-old piglets were intranasally infected with 106 PFU of either wild-type PrV strain Kaplan (PrV-Ka), PrV-9112C2, or PrV-9112C2R, in which the PrV gB gene was reinserted instead of the BHV-1 gB gene. Animals infected with PrV-Ka and PrV-9112C2R showed a similar course of disease, i.e., high fever, marked respiratory symptoms but minimal neurological disorders, and excretion of high amounts of virus. All animals survived the infection. In contrast, animals infected with PrV-9112C2 showed no respiratory symptoms and developed only mild fever. However, on day 5 after infection, all piglets developed severe central nervous system (CNS) symptoms leading to death within 48 to 72 h. Detailed histological analyses showed that PrV-9112C2R infected all regions of the nasal mucosa and subsequently spread to the CNS preferentially by the trigeminal route. In contrast, PrV-9112C2 primarily infected the olfactory epithelium and spread via the olfactory route. In the CNS, more viral antigen and significantly more pronounced histological changes resulting in more severe encephalitis were found after PrV-9112C2 infection. Thus, our results demonstrate that replacement of PrV gB by the homologous BHV-1 glycoprotein resulted in a dramatic increase in neurovirulence combined with an alteration in the route of neuroinvasion, indicating that the essential gB is involved in determining neurotropism and neurovirulence of PrV.

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Pseudorabies Virus Glycoprotein M Inhibits Membrane Fusion

Journal of Virology ◽

10.1128/jvi.74.15.6760-6768.2000 ◽

2000 ◽

Vol 74 (15) ◽

pp. 6760-6768 ◽

Cited By ~ 108

Author(s):

Barbara G. Klupp ◽

Ralf Nixdorf ◽

Thomas C. Mettenleiter

Keyword(s):

Amino Acids ◽

Membrane Fusion ◽

Pseudorabies Virus ◽

Inhibitory Effect ◽

Rabbit Kidney ◽

General Mechanism ◽

Homologous Proteins ◽

Infectious Laryngotracheitis Virus ◽

Syncytial Virus ◽

Hsv 1

ABSTRACT A transient transfection-fusion assay was established to investigate membrane fusion mediated by pseudorabies virus (PrV) glycoproteins. Plasmids expressing PrV glycoproteins under control of the immediate-early 1 promoter-enhancer of human cytomegalovirus were transfected into rabbit kidney cells, and the extent of cell fusion was quantitated 27 to 42 h after transfection. Cotransfection of plasmids encoding PrV glycoproteins B (gB), gD, gH, and gL resulted in formation of polykaryocytes, as has been shown for homologous proteins of herpes simplex virus type 1 (HSV-1) (A. Turner, B. Bruun, T. Minson, and H. Browne, J. Virol. 72:873–875, 1998). However, in contrast to HSV-1, fusion was also observed when the gD-encoding plasmid was omitted, which indicates that PrV gB, gH, and gL are sufficient to mediate fusion. Fusogenic activity was enhanced when a carboxy-terminally truncated version of gB (gB-008) lacking the C-terminal 29 amino acids was used instead of wild-type gB. With gB-008, only gH was required in addition for fusion. A very rapid and extended fusion was observed after cotransfection of plasmids encoding gB-008 and gDH, a hybrid protein consisting of the N-terminal 271 amino acids of gD fused to the 590 C-terminal amino acids of gH. This protein has been shown to substitute for gH, gD, and gL function in the respective viral mutants (B. G. Klupp and T. C. Mettenleiter, J. Virol. 73:3014–3022, 1999). Cotransfection of plasmids encoding PrV gC, gE, gI, gK, and UL20 with gB-008 and gDH had no effect on fusion. However, inclusion of a gM-expressing plasmid strongly reduced the extent of fusion. An inhibitory effect was also observed after inclusion of plasmids encoding gM homologs of equine herpesvirus 1 or infectious laryngotracheitis virus but only in conjunction with expression of the gM complex partner, the gN homolog. Inhibition by PrV gM was not limited to PrV glycoprotein-mediated fusion but also affected fusion induced by the F protein of bovine respiratory syncytial virus, indicating a general mechanism of fusion inhibition by gM.

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Cloning and overexpression of the xylose isomerase gene from Burkholderia sacchari and production of polyhydroxybutyrate from xylose

Canadian Journal of Microbiology ◽

10.1139/w09-055 ◽

2009 ◽

Vol 55 (8) ◽

pp. 1012-1015 ◽

Cited By ~ 8

Author(s):

Mateus Schreiner Garcez Lopes ◽

José Gregório Cabrera Gomez ◽

Luiziana Ferreira Silva

Keyword(s):

Amino Acids ◽

Type Strain ◽

Wild Type Strain ◽

Xylose Isomerase ◽

Homologous Gene ◽

Broad Host Range ◽

Xylose Utilization ◽

Wild Type ◽

Broad Host Range Plasmid ◽

In The Wild

A different organization for the xyl operon was found in different genomes of Burkholderia and Pseudomomas species. Degenerated primers were designed based on Burkholderia genomes and used to amplify the xylose isomerase gene (xylA) from Burkholderia sacchari IPT101. The gene encoded a protein of 329 amino acids, which showed the highest similarity (90%) to the homologous gene of Burkholderia dolosa . It was cloned in the broad host range plasmid pBBR1MCS-2, which partially restored growth and polyhydroxybutyrate production capability in xylose to a B. sacchari xyl– mutant. When xylA was overexpressed in the wild-type strain, it was not able to increase growth and polyhydroxybutyrate production, suggesting that XylA activity is not limiting for xylose utilization in B. sacchari.

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A Chicken Embryo Eye Model for the Analysis of Alphaherpesvirus Neuronal Spread and Virulence

Journal of Virology ◽

10.1128/jvi.72.6.4580-4588.1998 ◽

1998 ◽

Vol 72 (6) ◽

pp. 4580-4588 ◽

Cited By ~ 29

Author(s):

Bruce W. Banfield ◽

G. S. Yap ◽

A. C. Knapp ◽

L. W. Enquist

Keyword(s):

Deletion Mutant ◽

Pseudorabies Virus ◽

Infectious Virus ◽

Vitreous Humor ◽

Pathological Response ◽

Embryonic Brain ◽

Gene Products ◽

Wild Type ◽

Eye Model ◽

The Brain

ABSTRACT We describe use of developing chicken embryos as a model to study neuronal spread and virulence of pseudorabies virus (PRV). At embryonic day 12, β-galactosidase-expressing PRV strains were injected into the vitreous humor of one eye, and virus replication and spread from the eye to the brain were measured by β-galactosidase activity and the recovery of infectious virus from tissues. The wild-type PRV strain, Becker, replicated in the eye and then spread to the brain, causing extensive pathology characterized by edema, hemorrhage, and necrosis that localized to virally infected tissue. The attenuated vaccine strain, Bartha, replicated in the eye and spread throughout specific regions of the brain, producing little to no overt pathology. Becker mutants lacking membrane proteins gE or gI replicated in the eye and were able to spread to the brain efficiently. The pathology associated with replication of these mutants in the brain was intermediate to that induced by Becker or Bartha. Mixed infection of a gE deletion mutant and a gI deletion mutant restored the pathogenic phenotype to wild-type levels. These data indicate that the replication of virus in embryonic brain tissue is not sufficient to induce the characteristic pathological response and that the gE and gI gene products actively affect pathological responses in the developing chicken brain.

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The UL7 Gene of Pseudorabies Virus Encodes a Nonessential Structural Protein Which Is Involved in Virion Formation and Egress

Journal of Virology ◽

10.1128/jvi.79.17.11291-11299.2005 ◽

2005 ◽

Vol 79 (17) ◽

pp. 11291-11299 ◽

Cited By ~ 20

Author(s):

Walter Fuchs ◽

Harald Granzow ◽

Robert Klopfleisch ◽

Barbara G. Klupp ◽

Daniela Rosenkranz ◽

...

Keyword(s):

Pseudorabies Virus ◽

Rabbit Antiserum ◽

Wild Type Virus ◽

Rabbit Kidney ◽

Structural Protein ◽

Wild Type ◽

Survival Times ◽

Virion Protein ◽

Infected Cells

ABSTRACT Homologues of the UL7 gene of herpes simplex virus type 1 are conserved in alpha-, beta-, and gammaherpesviruses. However, little is known about their functions. Using a monospecific rabbit antiserum raised against a bacterial fusion protein, we identified the UL7 gene product of the neurotropic alphaherpesvirus pseudorabies virus (PrV). In Western blot analyses of infected cells and purified PrV particles the serum specifically detected a 29-kDa protein, which matches the calculated mass of the 266-amino-acid translation product of PrV UL7. For functional analysis, UL7 was deleted by mutagenesis of an infectious full-length clone of the PrV genome in Escherichia coli. The obtained recombinant PrV-ΔUL7F was replication competent in rabbit kidney cells, but maximum virus titers were decreased nearly 10-fold and plaque diameters were reduced by ca. 60% compared to wild-type PrV. Electron microscopy of infected cells revealed that in the absence of UL7, formation and nuclear egress of nucleocapsids were not affected, whereas secondary envelopment of cytoplasmic nucleocapsids appeared to be delayed and release of mature virions was less efficient. The observed replication defects were corrected by repair of the viral UL7 gene or by propagation of PrV-ΔUL7F in UL7-expressing cells. PrV-ΔUL7F was moderately attenuated in mice. Compared to wild-type virus, mean survival times were prolonged from 2 to 3 days after intranasal infection. However, neuroinvasion and transneuronal spread of PrV were not abolished in the absence of UL7. Thus, UL7 encodes a virion protein of PrV, which plays a role during virion maturation and egress both in vitro and in vivo.

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Identification of a baculovirus polyhedron formation mutant with a novel phenotype

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100166440 ◽

1996 ◽

Vol 54 ◽

pp. 796-797

Author(s):

James M. Slavicek ◽

Melissa J. Mercer ◽

Mary Ellen Kelly

Keyword(s):

Viral Gene ◽

Gene Products ◽

Type Number ◽

Infection Cycle ◽

Wild Type ◽

Protein Matrix ◽

Insect Midgut ◽

Viral Particles ◽

Budded Virus ◽

Viral Form

Nucleopolyhedroviruses (NPV, family Baculoviridae) produce two morphological forms, a budded virus form and a viral form that is occluded into a paracrystalline protein matrix. This structure is termed a polyhedron and is composed primarily of the protein polyhedrin. Insects are infected by NPVs after ingestion of the polyhedron and release of the occluded virions through dissolution of the polyhedron in the alkaline environment of the insect midgut. Early after infection the budded virus form is produced. It buds through the plasma membrane and then infects other cells. Later in the infection cycle the occluded form of the virus is generated (reviewed by Blissard and Rohrmann, 1990).The processes of polyhedron formation and virion occlusion are likely to involve a number of viral gene products. However, only two genes, the polyhedrin gene and 25K FP gene, have been identified to date that are necessary for the wild type number of polyhedra to be formed and viral particles occluded.

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