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 Glycoprotein D-Independent Infectivity of Pseudorabies Virus Results in an Alteration of In Vivo Host Range and Correlates with Mutations in Glycoproteins B and H

2001 ◽  
Vol 75 (21) ◽  
pp. 10054-10064 ◽  
Author(s):  
Jerg Schmidt ◽  
Volker Gerdts ◽  
Jörg Beyer ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter
Keyword(s):  
Host Range ◽  
Pseudorabies Virus ◽  
Natural Host ◽  
Target Cells ◽  
Glycoprotein D ◽  
Wild Type ◽  
Cellular Receptors ◽  
Receptor Usage

ABSTRACT Infection of cells by herpesviruses is initiated by the interaction of viral envelope glycoproteins with cellular receptors. In the alphaherpesvirus pseudorabies virus (PrV), the causative agent of Aujeszky's disease in pigs, the essential glycoprotein D (gD) mediates secondary attachment of virions to target cells by binding to newly identified cellular receptors (R. J. Geraghty, C. Krummenacher, G. H. Cohen, R. J. Eisenberg, and P. G. Spear, Science 280:1618–1620, 1998). However, in the presence of compensatory mutations, infection can also occur in the absence of gD, as evidenced by the isolation in cell culture of an infectious gD-negative PrV mutant (PrV-gD− Pass) (J. Schmidt, B. G. Klupp, A. Karger, and T. C. Mettenleiter, J. Virol. 71:17–24, 1997). PrV-gD− Pass is replication competent with an only moderate reduction in specific infectivity but appears to bind to receptors different from those recognized by wild-type PrV (A. Karger, J. Schmidt, and T. C. Mettenleiter, J. Virol. 72:7341–7348, 1998). To analyze whether this alteration in receptor usage in vitro influences infection in vivo, the model host mouse and the natural host pig were intranasally infected with PrV-gD− Pass and were compared to animals infected by wild-type PrV. For mice, a comparable progress of disease was observed, and all animals infected with mutant virus died, although they exhibited a slight delay in the onset of symptoms and, correspondingly, a longer time to death. In contrast, whereas wild-type PrV-infected pigs showed clinical signs and histological and histopathological findings typical of PrV infection, no signs of disease were observed after infection with PrV-gD− Pass. Moreover, in these animals, virus-infected cells were not detectable by immunohistochemical staining of different organ samples and no virus could be isolated from nasal swabs. Mutations in glycoproteins B and H were found to correlate with, and probably contribute to, gD-independent infectivity. In conclusion, although PrV-gD− Pass is virulent in mice, it is apparently unable to infect the natural host, the pig. This altered host range in vivo correlates with a difference of receptor usage in vitro and demonstrates for the first time the importance of gD receptors in alphaherpesvirus infection of an animal host.

scholarly journals Investigation of the in vitro and in vivo activity of the type IV pilus extension ATPase BfpD

2020 ◽  
Author(s):  
Jinlei Zhao ◽  
Shahista Nisa ◽  
Michael S. Donnenberg
Keyword(s):  
Atpase Activity ◽  
Loss Of Function ◽  
Wild Type ◽  
Multifunctional Protein ◽  
Mutant Proteins ◽  
E Coli ◽  
Type Iv ◽  
Kinetics Of

AbstractType IV pili (T4Ps) are multifunctional protein fibers found in many bacteria and archaea. All T4P systems have an extension ATPase, which provides the energy required to push structural subunits out of the membrane. We previously reported that the BfpD T4P ATPase from enteropathogenic E. coli (EPEC) has the expected hexameric structure and ATPase activity, the latter enhanced by the presence of the N-terminal cytoplasmic domains of its partner proteins BfpC and BfpE. In this study, we further investigated the kinetics of the BfpD ATPase. Despite high purity of the proteins, the reported enhanced ATPase activity was found to be from (an) ATPase(s) contaminating the N-BfpC preparation. Furthermore, although two mutations in highly conserved bfpD sites led to loss of function in vivo, the purified mutant proteins retained some ATPase activity, albeit less than the wild-type protein. Therefore, the observed ATPase activity of BfpD was also affected by (a) contaminating ATPase(s). Expression of the mutant bfpD alleles did not interfere with BfpD function in bacteria that also expressed wild-type BfpD. However, a similar mutation of bfpF, which encodes the retraction ATPase, blocked the function of wild-type BfpF when both were present. These results highlight similarities and differences in function and activity of T4P extension and retraction ATPases in EPEC.

scholarly journals Human Immunodeficiency Virus Bearing a Disrupted Central DNA Flap Is Pathogenic In Vivo

2007 ◽  
Vol 81 (11) ◽  
pp. 6146-6150 ◽  
Author(s):  
Matthew D. Marsden ◽  
Jerome A. Zack
Keyword(s):  
Human Immunodeficiency Virus ◽  
Growth Characteristics ◽  
Deficient Mutant ◽  
Wild Type ◽  
Hiv Replication ◽  
Immunodeficiency Virus ◽  
In Vivo Growth ◽  
Kinetics Of

ABSTRACT The central DNA flap is an important component of lentiviral vectors, but its significance in the context of wild-type human immunodeficiency virus (HIV) is currently unclear. To address this issue, we have compared the in vitro infection kinetics of NL4-3 with those of a flap-deficient mutant and evaluated the in vivo growth characteristics of these viruses by using the SCID-hu mouse model of HIV infection. Flap-deficient virus was only modestly attenuated in vitro, as assessed by single-round and spreading infection assays, and exhibited levels of replication and pathogenesis close to those of the wild-type in vivo. Hence, an intact central flap is not essential for HIV replication.

scholarly journals Suboptimal Enhancer Sequences Are Required for Efficient Bovine Leukemia Virus Propagation In Vivo: Implications for Viral Latency

2001 ◽  
Vol 75 (15) ◽  
pp. 6977-6988 ◽  
Author(s):  
C. Merezak ◽  
C. Pierreux ◽  
E. Adam ◽  
F. Lemaigre ◽  
G. G. Rousseau ◽  
...  
Keyword(s):  
Bovine Leukemia Virus ◽  
Leukemia Virus ◽  
Viral Proteins ◽  
Transient Transfection ◽  
Wild Type ◽  
Viral Spread ◽  
Viral Expression ◽  
E Box

ABSTRACT Repression of viral expression is a major strategy developed by retroviruses to escape from the host immune response. The absence of viral proteins (or derived peptides) at the surface of an infected cell does not permit the establishment of an efficient immune attack. Such a strategy appears to have been adopted by animal oncoviruses such as bovine leukemia virus (BLV) and human T-cell leukemia virus (HTLV). In BLV-infected animals, only a small fraction of the infected lymphocytes (between 1 in 5,000 and 1 in 50,000) express large amounts of viral proteins; the vast majority of the proviruses are repressed at the transcriptional level. Induction of BLV transcription involves the interaction of the virus-encoded Tax protein with the CREB/ATF factors; the resulting complex is able to interact with three 21-bp Tax-responsive elements (TxRE) located in the 5′ long terminal repeat (5′ LTR). These TxRE contain cyclic AMP-responsive elements (CRE), but, remarkably, the “TGACGTCA” consensus is never strictly conserved in any viral strain (e.g.,AGACGTCA, TGACGGCA, TGACCTCA). To assess the role of these suboptimal CREs, we introduced a perfect consensus sequence within the TxRE and showed by gel retardation assays that the binding efficiency of the CREB/ATF proteins was increased. However,trans-activation of a luciferase-based reporter by Tax was not affected in transient transfection assays. Still, in the absence of Tax, the basal promoter activity of the mutated LTR was increased as much as 20-fold. In contrast, mutation of other regulatory elements within the LTR (the E box, NF-κB, and glucocorticoid- or interferon-responsive sites [GRE or IRF]) did not induce a similar alteration of the basal transcription levels. To evaluate the biological relevance of these observations made in vitro, the mutations were introduced into an infectious BLV molecular clone. After injection into sheep, it appeared that all the recombinants were infectious in vivo and did not revert into a wild-type virus. All of them, except one, propagated at wild-type levels, indicating that viral spread was not affected by the mutation. The sole exception was the CRE mutant; proviral loads were drastically reduced in sheep infected with this type of virus. We conclude that a series of sites (NF-κB, IRF, GRE, and the E box) are not required for efficient viral spread in the sheep model, although mutation of some of these motifs might induce a minor phenotype during transient transfection assays in vitro. Remarkably, a provirus (pBLV-Δ21-bp) harboring only two TxRE was infectious and propagated at wild-type levels. And, most importantly, reconstitution of a consensus CRE, within the 21-bp enhancers increases binding of CREB/ATF proteins but abrogates basal repression of LTR-directed transcription in vitro. Suboptimal CREs are, however, essential for efficient viral spread within infected sheep, although these sites are dispensable for infectivity. These results suggest an evolutionary selection of suboptimal CREs that repress viral expression with escape from the host immune response. These observations, which were obtained in an animal model for HTLV-1, are of interest for oncovirus-induced pathogenesis in humans.

Fibronectin binding proteins contribute to the adherence of Staphylococcus aureus to intact endothelium in vivo

2006 ◽  
Vol 96 (08) ◽  
pp. 183-189 ◽  
Author(s):  
Bhanu Sinha ◽  
Klaus Preissner ◽  
Michael Menger ◽  
Sylvain Kerdudou ◽  
Matthias Laschke ◽  
...  
Keyword(s):  
Vessel Wall ◽  
Matrix Proteins ◽  
Adherence Rate ◽  
Wild Type ◽  
Hamster Model ◽  
Interaction Kinetics ◽  
Kinetics Of ◽  
Primary Interaction

SummaryStaphylococcal adhesins mediate attachment to matrix proteins and endothelial cells in vitro, yet, their role in primary adherence to the physiologic vessel wall has not been studied in vivo, and complex endocarditis models yielded ambiguous results. Recently, we developed a hamster model to study interaction kinetics of S. aureus with intact microvasculature using intravital fluorescence microscopy (Laschke et al. J Infect Dis 2005; 191: 435-43) providing the basis for this study. S. aureus Cowan 1 wild type (WT) log phase cells adhered to postcapillary venules to a significantly larger extent compared to stationary phase staphylococci, a finding in congruence with the fact that the staphylococcal adhesin repertoire largely depends on the growth phase. In comparison, the adherence rate of the fnbA deleted mutant (DU5895) to the vessel wall was significantly reduced to approximately 40% of WT. These DU5895 attachment rates were similar to those of an S. carnosus strain (TM300). In contrast, upon heterologous complementation of TM300 with either fnbA and fnbB, adherence of these transformants to the microvasculature increased, an increase found to be significant for fnbA transformant single cocci and clusters at 30 and 60 min when compared to S. carnosus TM300 WT. In conclusion, these results demonstrate that staphylococcal FnBPs significantly contribute to primary interaction with intact endothelium under physiologic conditions. Accordingly, this attribution of staphylococcal FnBPs providea rationale for novel intervention strategies such as the use of anti-FnBP antibodies in endovascular S. aureus disease.

scholarly journals Innate STAT1-Dependent Genomic Response of Neurons to the Antiviral Cytokine Alpha Interferon

2005 ◽  
Vol 79 (13) ◽  
pp. 8295-8302 ◽  
Author(s):  
Jianping Wang ◽  
Iain L. Campbell
Keyword(s):  
Viral Infection ◽  
Lymphocytic Choriomeningitis ◽  
Gene Chip ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Rnase Protection ◽  
The Brain ◽  
Genomic Response

ABSTRACT Alpha/beta interferons (IFNs-α/β) are cytokines that play an essential role in the host defense against viral infection. Our previous studies have shown that the key IFN signaling molecule STAT1 is highly elevated and activated in central nervous system neurons during viral infection and in transgenic mice with astrocyte production of IFN-α (glial fibrillary acidic protein [GFAP]-IFN-α), suggesting that neurons are a very responsive target cell population for IFNs. To elucidate the genomic response of neurons to IFN-α, we undertook studies both in vitro and in vivo. Gene chip analysis was applied to RNA from IFN-α-treated or untreated primary cortical neuronal cultures derived from embryonic day 15 fetal wild-type or STAT1 knockout (KO) mice. The expression of 51 known and 5 unknown genes was increased significantly by more than twofold after exposure of wild-type but not STAT1 KO neurons to IFN-α. Some more highly expressed genes included IFN-induced 15-kDa protein, ubiquitin-specific protease 18, glucocorticoid attenuated response genes, IFN-induced GTPases, and the chemokine CXCL10. For several of these genes, the gene chip findings were confirmed by RNase protection assays. In addition, examination of the expression of some of these selected genes revealed that they were increased in neurons in the brain of either GFAP-IFN-α mice or mice infected with lymphocytic choriomeningitis virus. In conclusion, our study revealed a robust STAT1-dependent genomic response of neurons to IFN-α, highlighting an innate potential of these cells to defend against viral infection in the brain.

scholarly journals Regulation of Cholinergic Phenotype in Developing Neurons

2008 ◽  
Vol 99 (5) ◽  
pp. 2443-2455 ◽  
Author(s):  
Xinhuai Liu ◽  
Ion R. Popescu ◽  
Janna V. Denisova ◽  
Rachael L. Neve ◽  
Roderick A. Corriveau ◽  
...  
Keyword(s):  
Nmda Receptors ◽  
Neural Circuit ◽  
Synaptic Activity ◽  
Neuronal Cultures ◽  
Extrinsic Factors ◽  
Phenotypic Properties ◽  
Cholinergic Phenotype ◽  
Developing Neurons

Specification of neurotransmitter phenotype is critical for neural circuit development and is influenced by intrinsic and extrinsic factors. Recent findings in rat hypothalamus in vitro suggest the role of neurotransmitter glutamate in the regulation of cholinergic phenotype. Here we extended our previous studies on the mechanisms of glutamate-dependent regulation of cholinergic phenotypic properties in hypothalamic neurons. Using immunocytochemistry, electrophysiology, and calcium imaging, we demonstrate that hypothalamic expression of choline acetyltransferase (the cholinergic marker) and responsiveness of neurons to acetylcholine (ACh) receptor agonists increase during chronic administration of an N-methyl-d-aspartate receptor (NMDAR) blocker, MK-801, in developing rats in vivo and genetic and pharmacological inactivation of NMDARs in mouse and rat developing neuronal cultures. In hypothalamic cultures, an inactivation of NMDA receptors also induces ACh-dependent synaptic activity, as do inactivations of PKA, ERK/MAPK, CREB, and NF-κB, which are known to be regulated by NMDA receptors. Interestingly, the increase in cholinergic properties in developing neurons that is induced by NMDAR blockade is prevented by the blockade of ACh receptors, suggesting that function of ACh receptor is required for the cholinergic up-regulation. Using dual recording of monosynaptic excitatory postsynaptic currents, we further demonstrate that chronic inactivation of ionotropic glutamate receptors induces the cholinergic phenotype in a subset of glutamatergic neurons. The phenotypic switch is partial as ACh and glutamate are coreleased. The results suggest that developing neurons may not only coexpress multiple transmitter phenotypes, but can also change the phenotypes following changes in signaling in neuronal circuits.

scholarly journals IFN-γ stimulated murine and human neurons mount anti-parasitic defenses against the intracellular parasite Toxoplasma gondii

2021 ◽  
Author(s):  
Sambamurthy Chandrasekaran ◽  
Joshua A Kochanowsky ◽  
Emily F Merritt ◽  
Anita A Koshy
Keyword(s):  
Toxoplasma Gondii ◽  
Defense Mechanisms ◽  
Neuronal Cultures ◽  
Wild Type ◽  
Intracellular Parasites ◽  
Human Neurons ◽  
Transgenic Parasites ◽  
Ifn Γ

Dogma holds that Toxoplasma gondii persists in neurons because neurons cannot clear intracellular parasites, even with IFN-γ stimulation. As several recent studies questioned this idea, we used primary murine neuronal cultures from wild-type and transgenic mice in combination with IFN-γ stimulation and parental and transgenic parasites to reassess IFN-γ dependent neuronal clearance of intracellular parasites. We found that neurons respond to IFN-γ and that a subset of neurons clear intracellular parasites via immunity regulated GTPases. Whole neuron reconstructions from mice infected with parasites that trigger neuron GFP expression only after full invasion revealed that ~40% of these T. gondii-invaded neurons no longer harbor parasites. Finally, IFN-γ stimulated human stem cell derived neurons showed a ~ 50% decrease in parasite infection rate when compared to unstimulated cultures. This work highlights the capability of human and murine neurons to mount cytokine-dependent anti-T. gondii defense mechanisms 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.

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