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 Simultaneous Deletion of Pseudorabies Virus Tegument Protein UL11 and Glycoprotein M Severely Impairs Secondary Envelopment

2004 ◽  
Vol 78 (6) ◽  
pp. 3024-3034 ◽  
Author(s):  
Martina Kopp ◽  
Harald Granzow ◽  
Walter Fuchs ◽  
Barbara Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Pseudorabies Virus ◽  
Plaque Formation ◽  
Tegument Protein ◽  
Electron Microscopic ◽  
Glycoprotein E ◽  
Intracytoplasmic Inclusions ◽  
One Step ◽  
Infected Cells ◽  
Step Growth ◽  
Growth Phenotype

ABSTRACT The pseudorabies virus (PrV) proteins UL11, glycoprotein E (gE), and gM are involved in secondary envelopment of tegumented nucleocapsids in the cytoplasm. To assess the relative contributions of these proteins to the envelopment process, virus mutants with deletions of either UL11, gM, or gE as well as two newly constructed mutant viruses with simultaneous deletions of UL11 and gE or of UL11 and gM were analyzed in cell culture for their growth phenotype. We show here that simultaneous deletion of UL11 and gE reduced plaque size in an additive manner over the reduction observed by deletion of only UL11 or gE. However, one-step growth was not further impaired beyond the level of the UL11 deletion mutant. Moreover, in electron microscopic analyses PrV-ΔUL11/gE exhibited a phenotype similar to that of the UL11 mutant virus. In contrast, plaque formation was virtually abolished by the simultaneous absence of UL11 and gM, and one-step growth was significantly reduced. Electron microscopy showed the presence of huge intracytoplasmic inclusions in PrV-ΔUL11/gM-infected cells, with a size reaching 3 μm and containing nucleocapsids embedded in tegument. We hypothesize that UL11 and gM are involved in different steps during secondary envelopment and that simultaneous deletion of both interrupts both processes, resulting in the observed drastic impairment of secondary envelopment.

scholarly journals Repair of the UL21 Locus in Pseudorabies Virus Bartha Enhances the Kinetics of Retrograde, Transneuronal Infection In Vitro and In Vivo

2008 ◽  
Vol 83 (3) ◽  
pp. 1173-1183 ◽  
Author(s):  
D. Curanović ◽  
M. G. Lyman ◽  
C. Bou-Abboud ◽  
J. P. Card ◽  
L. W. Enquist
Keyword(s):  
Pseudorabies Virus ◽  
Particle Production ◽  
Neural Circuit ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Infectious Particle ◽  
Viral Spread ◽  
Kinetics Of

ABSTRACT The attenuated pseudorabies virus (PRV) strain Bartha contains several characterized mutations that affect its virulence and ability to spread through neural circuits. This strain contains a small genomic deletion that abrogates anterograde spread and is widely used as a retrograde-restricted neural circuit tracer. Previous studies showed that the retrograde-directed spread of PRV Bartha is slower than that of wild-type PRV. We used compartmented neuronal cultures to characterize the retrograde defect and identify the genetic basis of the phenotype. PRV Bartha is not impaired in retrograde axonal transport, but transneuronal spread among neurons is diminished. Repair of the UL21 locus with wild-type sequence restored efficient transneuronal spread both in vitro and in vivo. It is likely that mutations in the Bartha UL21 gene confer defects that affect infectious particle production, causing a delay in spread to presynaptic neurons and amplification of infection. These events manifest as slower kinetics of retrograde viral spread in a neural circuit.

scholarly journals Generation and characterization of variants of NWS/G70C influenza virus after in vitro passage in 4-amino-Neu5Ac2en and 4-guanidino-Neu5Ac2en.

1996 ◽  
Vol 40 (1) ◽  
pp. 40-46 ◽  
Author(s):  
J L McKimm-Breschkin ◽  
T J Blick ◽  
A Sahasrabudhe ◽  
T Tiong ◽  
D Marshall ◽  
...  
Keyword(s):  
Amino Acids ◽  
Influenza Virus ◽  
Receptor Binding ◽  
Liquid Culture ◽  
Plaque Assay ◽  
Enoic Acid ◽  
Cross Resistance ◽  
Receptor Binding Site ◽  
Significant Difference

The compounds 4-amino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-amino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid) and 4-guanidino-Neu5Ac2en (5-acetylamino-2,6-anhydro-4-guanidino-3,4,5- trideoxy-D-glycerol-D-galacto-non-2-enoic acid), which selectively inhibit the influenza virus neuraminidase, have been tested in vitro for their ability to generate drug-resistant variants. NWS/G70C virus (H1N9) was cultured in each drug by limiting-dilution passaging. After five or six passages in either compound, there emerged viruses which had a reduced sensitivity to the inhibitors in cell culture. Variant viruses were up to 1,000-fold less sensitive in plaque assays, liquid culture, and a hemagglutination-elution assay. In addition, cross-resistance to both compounds was seen in all three assays. Some isolates demonstrated drug dependence with an increase in both size and number of plaques in a plaque assay and an increase in virus yield in liquid culture in the presence of inhibitors. No significant difference in neuraminidase enzyme activity was detected in vitro, and no sequence changes in the conserved sites of the neuraminidase were found. However, changes in conserved amino acids in the hemagglutinin were detected. These amino acids were associated with either the hemagglutinin receptor binding site, Thr-155, or the left edge of the receptor binding pocket, Val-223 and Arg-229. Hence, mutations at these sites could be expected to affect the affinity or specificity of the hemagglutinin binding. Compensating mutations resulting in a weakly binding hemagglutinin thus seem to be circumventing the inhibition of the neuraminidase by allowing the virus to be released from cells with less dependence on the neuraminidase.

STUDIES OF INFECTIOUS BOVINE RHINOTRACHEITIS VIRUS: 1. PLAQUE ASSAY AND SOME CHARACTERISTICS IN BOVINE KIDNEY CELLS

1963 ◽  
Vol 9 (4) ◽  
pp. 567-576 ◽  
Author(s):  
Leslie R. Sabina ◽  
Raymond C. Parker
Keyword(s):  
Infectious Virus ◽  
Plaque Assay ◽  
Growth Curves ◽  
Kidney Cells ◽  
Infectious Bovine Rhinotracheitis ◽  
High Input ◽  
Bovine Kidney ◽  
One Step ◽  
Step Growth ◽  
Infectious Bovine Rhinotracheitis Virus

A reproducible plaquing procedure for infectious bovine rhinotracheitis virus (IBRV) in an established bovine kidney cell line is reported. The validity of this system for quantitative analysis has been established by conventional methods.After infection at different multiplicities, one-step growth curves have shown that the eclipse period for IBRV lasts approximately 4 hours and that the infectious virus increases at a logarithmic rate for 12 to 14 hours. The virus yield with the low and high input is 30 PFU and 210 PFU per cell, respectively. Only 1 to 9% of the total virus is released at 24 hours postinfection. The data presented indicate the half-life of IBRV at 37 °C and 42 °C to be 16 and 3.5 hours, respectively. A comparison of hyperimmune bovine and rabbit sera has shown that 92% of the infective particles are neutralized within 30 minutes.

scholarly journals VP8, the Major Tegument Protein of Bovine Herpesvirus 1, Interacts with Cellular STAT1 and Inhibits Interferon Beta Signaling

2016 ◽  
Vol 90 (10) ◽  
pp. 4889-4904 ◽  
Author(s):  
Sharmin Afroz ◽  
Robert Brownlie ◽  
Michel Fodje ◽  
Sylvia van Drunen Littel-van den Hurk
Keyword(s):  
Amino Acids ◽  
Interferon Beta ◽  
De Novo ◽  
Nuclear Accumulation ◽  
Tegument Protein ◽  
Bovine Herpesvirus 1 ◽  
Infected Cells ◽  
Herpesvirus 1

ABSTRACTTheUL47gene product, VP8, is the most abundant tegument protein of bovine herpesvirus 1 (BoHV-1). Previously, we demonstrated that aUL47-deleted BoHV-1 mutant (BoHV1-ΔUL47) exhibits 100-fold-reduced virulencein vitroand is avirulentin vivo. In this study, we demonstrated that VP8 expression or BoHV-1 infection inhibits interferon beta (IFN-β) signaling by using an IFN-α/β-responsive plasmid in a luciferase assay. As transducer and activator of transcription (STAT) is an essential component in the IFN-signaling pathways, the effect of VP8 on STAT was investigated. An interaction between VP8 and STAT1 was established by coimmunoprecipitation assays in both VP8-transfected and BoHV-1-infected cells. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for its interaction with STAT1. The expression of VP8 did not induce STAT1 ubiquitination or degradation. Moreover, VP8 did not reduce STAT1 tyrosine phosphorylation to downregulate IFN-β signaling. However, the expression of VP8 or a version of VP8 (amino acids 219 to 741) that contains the STAT1-interacting domains but not the nuclear localization signal prevented nuclear accumulation of STAT1. Inhibition of nuclear accumulation of STAT1 also occurred during BoHV-1 infection, while nuclear translocation of STAT1 was observed in BoHV1-ΔUL47-infected cells. During BoHV-1 infection, VP8 was detected in the cytoplasm at 2 h postinfection without anyde novoprotein synthesis, at which time STAT1 was already retained in the cytoplasm. These results suggest that viral VP8 downregulates IFN-β signaling early during infection, thus playing a role in overcoming the antiviral response of BoHV-1-infected cells.IMPORTANCESince VP8 is the most abundant protein in BoHV-1 virions and thus may be released in large amounts into the host cell immediately upon infection, we proposed that it might have a function in the establishment of conditions suitable for viral replication. Indeed, while nonessentialin vitro, it is critical for BoHV-1 replicationin vivo. In this study, we determined that VP8 plays a role in downregulation of the antiviral host response by inhibiting IFN-β signaling. VP8 interacted with and prevented nuclear accumulation of STAT1 at 2 h postinfection in the absence ofde novoviral protein synthesis. Two domains of VP8, amino acids 259 to 482 and 632 to 686, were found to be responsible for this interaction. These results provide a new functional role for VP8 in BoHV-1 infection and a potential explanation for the lack of viral replication of theUL47deletion mutant in cattle.

A BACTERIOPHAGE THAT ATTACKS NUMEROUS PHYTOPATHOGENIC XANTHOMONAS SPECIES

1958 ◽  
Vol 4 (5) ◽  
pp. 493-497 ◽  
Author(s):  
M. D. Sutton ◽  
H. Katznelson ◽  
C. Quadling
Keyword(s):  
Burst Size ◽  
Lytic Phage ◽  
Plant Materials ◽  
Single Burst ◽  
Xanthomonas Species ◽  
One Step ◽  
Average Burst Size ◽  
Step Growth ◽  
Rot Disease

This paper reports the isolation of a lytic phage that attacks in vitro numerous phytopathogenic Xanthomonas species, including X. campestris (Pammel) Dowson, the cause of black rot disease of crucifers. Although 'one-step' growth experiments suggested an average burst size of ca. four for this phage-host system, 'single burst' experiments indicated a burst size of ca. one hundred phage particles per bacterium. The particles have typical phage morphology, as determined by electron microscopy. This phage gave satisfactory results when used in the rapid plaque count test for detection of phage-sensitive bacteria in plant materials.

scholarly journals ONE-STEP GROWTH CURVE OF WESTERN EQUINE ENCEPHALOMYELITIS VIRUS ON CHICKEN EMBRYO CELLS GROWN IN VITRO AND ANALYSIS OF VIRUS YIELDS FROM SINGLE CELLS

1954 ◽  
Vol 99 (2) ◽  
pp. 183-199 ◽  
Author(s):  
R. Dulbecco ◽  
Marguerite Vogt
Keyword(s):  
Chicken Embryo ◽  
Cell Layer ◽  
Growth Curves ◽  
Virus Growth ◽  
Embryo Cells ◽  
Cell Layers ◽  
A Cell ◽  
One Step ◽  
Step Growth

The rate of adsorption of WEE virus onto chicken embryo cells in vitro was determined both on a cell layer and on a cell suspension. One-step growth curves were determined in cell suspensions and on cell layers. The latent period varied between 2 and 3½ hours; it was shorter on cell layers and decreased with higher multiplicity of infection. The shortest period is probably the real latent period. The growth curves of the virus showed an initial exponential rise and reached a maximal constant value after 6 to 8 hours. The maximum virus yield per cell varied between 200 and 1000 on the cell layer, and between 100 and 200 in suspended cells. The yield of single infected cells was determined. An analysis of the distributions of the individual yields obtained after various periods of virus growth led to two main conclusions: (1) that virus is released from the same cell over a long period of time; (2) that one phase of the intracellular virus growth is exponential.

scholarly journals Phenotypic Characterization and Whole-Genome Analysis of a Novel Bacteriophage HCF1 Infecting Citrobacter amalonaticus and C. freundii

2021 ◽  
Vol 12 ◽  
Author(s):  
Prince Kumar ◽  
Mukesh K. Meghvansi ◽  
Dev V. Kamboj
Keyword(s):  
Environmental Stress ◽  
Host Range ◽  
Growth Curve ◽  
Phenotypic Characterization ◽  
Query Coverage ◽  
One Step ◽  
Step Growth ◽  
Tract Infections ◽  
Citrobacter Amalonaticus

Citrobacter species often occur in sewage, food, soil, wastewater, and in the intestinal tract of animals and humans. Citrobacter spp. cause urinary tract infections (UTIs) and infantile meningitis in humans. Due to the presence of plasmid-encoded resistance genes, Citrobacter spp. are often resistant to many antibiotics. In this study, Citrobacter virus HCF1, a novel virulent bacteriophage capable of killing Citrobacter amalonaticus and Citrobacter freundii, was isolated from the sewage water. The isolated bacteriophage was characterized with respect to transmission electron microscopy, one-step growth curve, host range, in vitro efficacy, storage stability, and environmental stress tolerance. The one-step growth curve analysis revealed that the latent period of HCF1 was 30 min and the estimated burst size was 121 plaque-forming units (PFU) per bacterial cell. Host range testing indicated that the HCF1 was specific to the Citrobacter genus. In vitro efficacy assay in the effluent of an anaerobic biodigester showed that the HCF1 completely eliminated the host within 4 and 5 h at MOI:100 and MOI:10, respectively, thereby indicating its potential for combating C. amalonaticus infections. The isolated bacteriophage is considerably stable and tolerant to environmental stress. Furthermore, the complete genome of HCF1 was sequenced using Oxford Nanopore sequencing and the data were subjected to detailed bioinformatic analyses. NCBI-BLASTn analysis revealed that the HCF1 genome had a query coverage of 15–21% and a maximum similarity of 77.27–78.49% with 11 bacteriophages of the Drexlerviridae family. Detailed bioinformatic analysis of the genome profile suggests that HCF1 is a novel T1svirus belonging to the Tempevirinae subfamily of the Drexlerviridae family.

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 Intracellular Localization of the Pseudorabies Virus Large Tegument Protein pUL36

2009 ◽  
Vol 83 (19) ◽  
pp. 9641-9651 ◽  
Author(s):  
Britta S. Möhl ◽  
Sindy Böttcher ◽  
Harald Granzow ◽  
Jana Kuhn ◽  
Barbara G. Klupp ◽  
...  
Keyword(s):  
Transient Expression ◽  
Immunoelectron Microscopy ◽  
Pseudorabies Virus ◽  
Intracellular Localization ◽  
Tegument Protein ◽  
Nuclear Pore ◽  
Intracellular Location ◽  
Nuclear Localization Signals ◽  
Virus Particles ◽  
Mature Virus

ABSTRACT Homologs of the essential large tegument protein pUL36 of herpes simplex virus 1 are conserved throughout the Herpesviridae, complex with pUL37, and form part of the capsid-associated “inner” tegument. pUL36 is crucial for transport of the incoming capsid to and docking at the nuclear pore early after infection as well as for virion maturation in the cytoplasm. Its extreme C terminus is essential for pUL36 function interacting with pUL25 on nucleocapsids to start tegumentation (K. Coller, J. Lee, A. Ueda, and G. Smith, J. Virol. 81:11790-11797, 2007). However, controversy exists about the cellular compartment in which pUL36 is added to the nascent virus particle. We generated monospecific rabbit antisera against four different regions spanning most of pUL36 of the alphaherpesvirus pseudorabies virus (PrV). By immunofluorescence and immunoelectron microscopy, we then analyzed the intracellular location of pUL36 after transient expression and during PrV infection. While reactivities of all four sera were comparable, none of them showed specific intranuclear staining during PrV infection. In immunoelectron microscopy, neither of the sera stained primary enveloped virions in the perinuclear cleft, whereas extracellular mature virus particles were extensively labeled. However, transient expression of pUL36 alone resulted in partial localization to the nucleus, presumably mediated by nuclear localization signals (NLS) whose functionality was demonstrated by fusion of the putative NLS to green fluorescent protein (GFP) and GFP-tagged pUL25. Since PrV pUL36 can enter the nucleus when expressed in isolation, the NLS may be masked during infection. Thus, our studies show that during PrV infection pUL36 is not detectable in the nucleus or on primary enveloped virions, correlating with the notion that the tegument of mature virus particles, including pUL36, is acquired in the cytosol.

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