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 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 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 The Streptococcus mutans vicX Gene Product Modulates gtfB/C Expression, Biofilm Formation, Genetic Competence, and Oxidative Stress Tolerance

2006 ◽  
Vol 189 (4) ◽  
pp. 1451-1458 ◽  
Author(s):  
M. Dilani Senadheera ◽  
Andrew W. C. Lee ◽  
David C. I. Hung ◽  
Grace A. Spatafora ◽  
Steven D. Goodman ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Stress Tolerance ◽  
Gene Product ◽  
Streptococcus Mutans ◽  
Parent Strain ◽  
Adhesion Assay ◽  
Wild Type ◽  
Coding Region ◽  
Oxidative Stress Tolerance ◽  
Cell Clusters

ABSTRACT Streptococcus mutans is considered one of the primary etiologic agents of dental caries. Previously, we characterized the VicRK two-component signal transduction system, which regulates multiple virulence factors of S. mutans. In this study, we focused on the vicX gene of the vicRKX tricistronic operon. To characterize vicX, we constructed a nonpolar deletion mutation in the vicX coding region in S. mutans UA159. The growth kinetics of the mutant (designated SmuvicX) showed that the doubling time was longer and that there was considerable sensitivity to paraquat-induced oxidative stress. Supplementing a culture of the wild-type UA159 strain with paraquat significantly increased the expression of vicX (P < 0.05, as determined by analysis of variance [ANOVA]), confirming the role of this gene in oxidative stress tolerance in S. mutans. Examination of mutant biofilms revealed architecturally altered cell clusters that were seemingly denser than the wild-type cell clusters. Interestingly, vicX-deficient cells grown in a glucose-supplemented medium exhibited significantly increased glucosyltransferase B/C (gtfB/C) expression compared with the expression in the wild type (P < 0.05, as determined by ANOVA). Moreover, a sucrose-dependent adhesion assay performed using an S. mutans GS5-derived vicX null mutant demonstrated that the adhesiveness of this mutant was enhanced compared with that of the parent strain and isogenic mutants of the parent strain lacking gtfB and/or gtfC. Also, disruption of vicX reduced the genetic transformability of the mutant approximately 10-fold compared with that of the parent strain (P < 0.05, as determined by ANOVA). Collectively, these findings provide insight into important phenotypes controlled by the vicX gene product that can impact S. mutans pathogenicity.

scholarly journals Physical Interaction between Envelope Glycoproteins E and M of Pseudorabies Virus and the Major Tegument Protein UL49

2002 ◽  
Vol 76 (16) ◽  
pp. 8208-8217 ◽  
Author(s):  
Walter Fuchs ◽  
Barbara G. Klupp ◽  
Harald Granzow ◽  
Christoph Hengartner ◽  
Alexandra Brack ◽  
...  
Keyword(s):  
Gene Product ◽  
Pseudorabies Virus ◽  
Tegument Protein ◽  
Physical Interaction ◽  
Virus Maturation ◽  
Virus Particles ◽  
Tegument Proteins ◽  
Infected Cells ◽  
Simplex Virus

ABSTRACT Envelope glycoprotein M (gM) and the complex formed by glycoproteins E (gE) and I (gI) are involved in the secondary envelopment of pseudorabies virus (PrV) particles in the cytoplasm of infected cells. In the absence of the gE-gI complex and gM, envelopment is blocked and capsids surrounded by tegument proteins accumulate in the cytoplasm (A. R. Brack, J. Dijkstra, H. Granzow, B. G. Klupp, and T. C. Mettenleiter, J. Virol. 73:5364-5372, 1999). Here we demonstrate by yeast two-hybrid analyses that the cytoplasmic domains of gE and gM specifically interact with the C-terminal part of the UL49 gene product of PrV, which represents a major tegument protein and which is homologous to VP22 of herpes simplex virus type 1. However, deletion of the UL49 gene from PrV had only minor effects on viral replication, and ultrastructural analyses of infected cells confirmed that virus maturation and egress, including secondary envelopment in the cytoplasm, were not detectably affected by the absence of UL49. Moreover, the UL49 gene product was shown to be dispensable for virion localization of gE and gM, and mutants lacking either gE or gM incorporated the UL49 protein efficiently into virus particles. In contrast, a PrV mutant with deletions of gE-gI and gM failed to incorporate the UL49 protein despite apparently unaltered intracytoplasmic UL49 expression. In summary, we describe specific interactions between herpesvirus envelope and tegument proteins which may play a role in secondary envelopment during herpesvirus virion maturation.

scholarly journals Efficient Incorporation of Tegument Proteins pUL46, pUL49, and pUS3 into Pseudorabies Virus Particles Depends on the Presence of pUL21

2006 ◽  
Vol 81 (2) ◽  
pp. 1048-1051 ◽  
Author(s):  
Kathrin Michael ◽  
Barbara G. Klupp ◽  
Axel Karger ◽  
Thomas C. Mettenleiter
Keyword(s):  
Virus Particle ◽  
Protein Interactions ◽  
Pseudorabies Virus ◽  
Structural Proteins ◽  
Tegument Protein ◽  
Wild Type ◽  
Protein Protein Interactions ◽  
Virus Particles ◽  
Tegument Proteins ◽  
Drastic Decrease

ABSTRACT The mature virion of the alphaherpesvirus pseudorabies virus (PrV) contains a minimum of 31 structural proteins which are recruited into the virus particle by a network of protein-protein interactions which is only incompletely understood. We show here that deletion of the tegument protein pUL21 resulted in a drastic decrease in the incorporation of the pUL46, pUL49, and pUS3 tegument components into mature virions. Moreover, the attenuated PrV strain Bartha (PrV-Ba), which, among other defects, carries mutations in pUL21, also fails to package pUL46, pUL49, and pUS3 efficiently. By the reconstitution of wild-type pUL21 expression to PrV-Ba and the transfer of mutated PrV-Ba pUL21 into wild-type PrV, we demonstrate that this phenotype is due to the mutated pUL21.

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

2005 ◽  
Vol 79 (17) ◽  
pp. 11291-11299 ◽  
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.

scholarly journals Mutagenesis of the Active-Site Cysteine in the Ubiquitin-Specific Protease Contained in Large Tegument Protein pUL36 of Pseudorabies Virus Impairs Viral Replication In Vitro and Neuroinvasion In Vivo

2008 ◽  
Vol 82 (12) ◽  
pp. 6009-6016 ◽  
Author(s):  
Sindy Böttcher ◽  
Christina Maresch ◽  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Jens P. Teifke ◽  
...  
Keyword(s):  
Active Site ◽  
Pseudorabies Virus ◽  
Wild Type Virus ◽  
Tegument Protein ◽  
Wild Type ◽  
Active Site Cysteine ◽  
Specific Protease ◽  
Ubiquitin Specific Protease

ABSTRACT Herpesviruses specify a ubiquitin-specific protease activity located within their largest tegument protein. Although its biological role is still largely unclear, mutation within the active site abolished deubiquitinating (DUB) activity and decreased virus replication in vitro and in vivo. To further elucidate the role of DUB activity for herpesvirus replication, the conserved active-site cysteine at amino acid position 26 within pUL36 of Pseudorabies virus (PrV) (Suid herpesvirus 1), a neurotropic alphaherpesvirus, was mutated to serine. Whereas one-step growth kinetics of the resulting mutant virus PrV-UL36(C26S) were moderately reduced, plaque size was decreased to 62% of that of the wild-type virus. Ultrastructural analysis revealed large accumulations of unenveloped nucleocapsids in the cytoplasm, but incorporation of the tegument protein pUL37 was not abolished. After intranasal infection with PrV-UL36(C26S) mice showed survival times two times longer than those of mice infected with wild-type or rescued virus. Thus, the DUB activity is important for PrV replication in vitro and for neuroinvasion in mice.

scholarly journals Structure and transcription of the singed locus of Drosophila melanogaster.

Genetics ◽  
1991 ◽  
Vol 129 (4) ◽  
pp. 1073-1084 ◽  
Author(s):  
J Paterson ◽  
K O'Hare
Keyword(s):  
Drosophila Melanogaster ◽  
Gene Product ◽  
Time Course ◽  
Single Gene ◽  
Wild Type ◽  
Coding Region ◽  
Adult Females ◽  
Zygotic Transcription ◽  
Early Embryos ◽  
Female Germline

Abstract Developmental and genetic studies of the singed gene of Drosophila melanogaster indicate that the gene has a role in somatic cells during the formation of adult bristles and hairs, and in the female germline during oogenesis. During metamorphosis a single 3.6-kilobase (kb) RNA is made, and this RNA is also present in adults and early embryos. Early embryos and adult females have additional 3.3- and 3.0-kb RNAs. The RNAs differ only in the length of the 3' untranslated region and a single gene product of 57 kilodaltons is predicted. Analysis of RNA from females lacking ovaries suggests that the 3.3- and 3.0-kb RNAs are made only in ovaries. The absence of the 3.3- and 3.0-kb RNAs in pupae and the time course of their appearance in adult females after eclosion suggests that transcription of singed in the ovary is from middle to late stages of oogenesis. Analysis of RNA in embryos from the reciprocal crosses between wild type and singed-3 showed that all three RNAs are maternally inherited with very little zygotic transcription in embryos. The mutation singed-3 appears to separate the two requirements for singed function as it has an extreme effect upon bristle development, but does not obviously affect oogenesis. In singed-3, there is a deletion at the 5' end of the gene, but the coding region is intact. Transcription in singed-3 is from a cryptic promoter in the upstream flanking sequences which is sufficiently active during oogenesis for fertility, but less active than the wild-type promoter during metamorphosis. The role of the single singed gene product may be in the asymmetric organization and/or movement of cytoplasmic components.

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

2007 ◽  
Vol 81 (17) ◽  
pp. 9307-9318 ◽  
Author(s):  
Walter Fuchs ◽  
Harald Granzow ◽  
Barbara G. Klupp ◽  
Axel Karger ◽  
Kathrin Michael ◽  
...  
Keyword(s):  
Amino Acids ◽  
Pseudorabies Virus ◽  
Conclusive Evidence ◽  
Rabbit Kidney ◽  
Homologous Gene ◽  
Double Negative ◽  
Gene Products ◽  
Wild Type ◽  
Protein Protein Interaction ◽  
Peptide Mass

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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