39 results on '"Oren Kobiler"'
Search Results
2. Dynamic Proteomics of Herpes Simplex Virus Infection
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Nir Drayman, Omer Karin, Avi Mayo, Tamar Danon, Lev Shapira, Dor Rafael, Anat Zimmer, Anat Bren, Oren Kobiler, and Uri Alon
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cell cycle ,geminin ,RFX7 ,RPAP3 ,SLTM ,single-cell infection ,Microbiology ,QR1-502 - Abstract
ABSTRACT The cellular response to viral infection is usually studied at the level of cell populations. Currently, it remains an open question whether and to what extent cell-to-cell variability impacts the course of infection. Here we address this by dynamic proteomics—imaging and tracking 400 yellow fluorescent protein (YFP)-tagged host proteins in individual cells infected by herpes simplex virus 1. By quantifying time-lapse fluorescence imaging, we analyze how cell-to-cell variability impacts gene expression from the viral genome. We identify two proteins, RFX7 and geminin, whose levels at the time of infection correlate with successful initiation of gene expression. These proteins are cell cycle markers, and we find that the position in the cell cycle at the time of infection (along with the cell motility and local cell density) can reasonably predict in which individual cells gene expression from the viral genome will commence. We find that the onset of cell division dramatically impacts the progress of infection, with 70% of dividing cells showing no additional gene expression after mitosis. Last, we identify four host proteins that are specifically modulated in infected cells, of which only one has been previously recognized. SUMO2 and RPAP3 levels are rapidly reduced, while SLTM and YTHDC1 are redistributed to form nuclear foci. These modulations are dependent on the expression of ICP0, as shown by infection with two mutant viruses that lack ICP0. Taken together, our results provide experimental validation for the long-held notion that the success of infection is dependent on the state of the host cell at the time of infection. IMPORTANCE High-throughput assays have revolutionized many fields in biology, both by allowing a more global understanding of biological processes and by deciphering rare events in subpopulations. Here we use such an assay, dynamic proteomics, to study viral infection at the single-cell level. We follow tens of thousands of individual cells infected by herpes simplex virus using fluorescence live imaging. Our results link the state of a cell at the time of virus infection with its probability to successfully initiate gene expression from the viral genome. Further, we identified three cellular proteins that were previously unknown to respond to viral infection. We conclude that dynamic proteomics provides a powerful tool to study single-cell differences during viral infection.
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- 2017
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3. Promoting Simultaneous Onset of Viral Gene Expression Among Cells Infected with Herpes Simplex Virus-1
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Maya Ralph, Marina Bednarchik, Enosh Tomer, Dor Rafael, Sefi Zargarian, Motti Gerlic, and Oren Kobiler
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single cell ,biological noise ,timing of infection ,temperature sensitive ,herpesviruses ,Microbiology ,QR1-502 - Abstract
Synchronous viral infection facilitates the study of viral gene expression, viral host interactions, and viral replication processes. However, the protocols for achieving synchronous infections were hardly ever tested in proper temporal resolution at the single-cell level. We set up a fluorescence-based, time lapse microscopy assay to study sources of variability in the timing of gene expression during herpes simplex virus-1 (HSV-1) infection. We found that with the common protocol, the onset of gene expression within different cells can vary by more than 3 h. We showed that simultaneous viral genome entry to the nucleus can be achieved with a derivative of the previously characterized temperature sensitive mutant tsB7, however, this did not improve gene expression synchrony. We found that elevating the temperature in which the infection is done and increasing the multiplicity of infection (MOI) significantly promoted simultaneous onset of viral gene expression among infected cells. Further, elevated temperature result in a decrease in the coefficient of variation (a standardized measure of dispersion) of viral replication compartments (RCs) sizes among cells as well as a slight increment of viral late gene expression synchrony. We conclude that simultaneous viral gene expression can be improved by simple modifications to the infection process and may reduce the effect of single-cell variability on population-based assays.
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- 2017
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4. Gene Expression Correlates with the Number of Herpes Viral Genomes Initiating Infection in Single Cells.
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Efrat M Cohen and Oren Kobiler
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Immunologic diseases. Allergy ,RC581-607 ,Biology (General) ,QH301-705.5 - Abstract
Viral gene expression varies significantly among genetically identical cells. The sources of these variations are not well understood and have been suggested to involve both deterministic host differences and stochastic viral host interactions. For herpesviruses, only a limited number of incoming viral genomes initiate expression and replication in each infected cell. To elucidate the effect of this limited number of productively infecting genomes on viral gene expression in single cells, we constructed a set of fluorescence-expressing genetically tagged herpes recombinants. The number of different barcodes originating from a single cell is a good representative of the number of incoming viral genomes replicating (NOIVGR) in that cell. We identified a positive correlation between the NOIVGR and viral gene expression, as measured by the fluorescent protein expressed from the viral genome. This correlation was identified in three distinct cell-types, although the average NOIVGR per cell differed among these cell-types. Among clonal single cells, high housekeeping gene expression levels are not supportive of high viral gene expression, suggesting specific host determinants effecting viral infection. We developed a model to predict NOIVGR from cellular parameters, which supports the notion that viral gene expression is tightly linked to the NOIVGR in single-cells. Our results support the hypothesis that the stochastic nature of viral infection and host cell determinants contribute together to the variability observed among infected cells.
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- 2016
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5. Histone deacetylase inhibitors reduce the number of herpes simplex virus-1 genomes initiating expression in individual cells
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Lev Shapira, Maya Ralph, Enosh Tomer, Shai Cohen, and Oren Kobiler
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Gene Expression ,intrinsic immunity ,Herpes Simplex virus-1 ,Virus Host Interactions ,ICP0 deletion ,Histone deacetylase inhibitors. ,Microbiology ,QR1-502 - Abstract
Although many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence-expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid (SBX), Valporic Acid (VPA) and Suberoylanilide Hydoxamic Acid (SAHA). We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the gene expression from the viral genomes. Different cell types (HFF, Vero and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (PML and ATRX), which may explain the lower number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.
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- 2016
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6. A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits.
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J Patrick Card, Oren Kobiler, Ethan B Ludmir, Vedant Desai, Alan F Sved, and Lynn W Enquist
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Medicine ,Science - Abstract
Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners.
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- 2011
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7. Phage lambda CIII: a protease inhibitor regulating the lysis-lysogeny decision.
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Oren Kobiler, Assaf Rokney, and Amos B Oppenheim
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Medicine ,Science - Abstract
The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic alpha helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell.
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- 2007
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8. A Single Herpes Simplex Virus 1 Genome Reactivates from Individual Cells
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Dor Rafael, Enosh Tomer, and Oren Kobiler
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Microbiology (medical) ,General Immunology and Microbiology ,Ecology ,Physiology ,Coinfection ,Herpes Simplex ,Cell Biology ,Genome, Viral ,Herpesvirus 1, Human ,Virus Latency ,Infectious Diseases ,Genetics ,Animals ,Humans ,Virus Activation - Abstract
Latent infection is a characteristic feature of herpesviruses' life cycle. Herpes simplex virus 1 is a common human pathogen that establishes lifelong latency in peripheral neurons. Symptomatic or asymptomatic periodic reactivations from the latent state allow the virus to replicate and spread among individuals. The latent viral genomes are found as several quiescent episomes inside the infected nuclei; however, it is not clear if and how many latent genomes are able to reactivate together. To address this question, we developed a quiescent infection assay, which provides a quantitative analysis of the number of genomes reactivating per cell, in cultured immortalized fibroblasts. We found that, almost always, only one viral genome reactivates per cell. We showed that different timing of entry to quiescence did not result in a significant change in the probability of reactivating. Reactivation from this quiescent state allowed only limited intergenomic recombination between two viral strains compared to lytic infection. Following coinfection with a mutant that is unable to reactivate, only coreactivation with a reactivation-proficient recombinant can provide the opportunity for the mutant to reactivate. We speculate that each individual quiescent viral genome has a low and stochastic chance to reactivate in each cell, an assumption that can explain the limited number of genomes reactivating per cell.
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- 2022
9. Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors
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Meital Gal-Tanamy, David Hagin, Cameron J. Nowell, Dor Rafael, Ksenia Polonsky, Natalia T. Freund, Ben A. Croker, Gur Yaari, Sandra L Leibel, Evgeny Kiner, Michael Mor, Jonathan M. Gershoni, Eric R. Griffis, Moshe Dassau, Modi Safra, Hila Sharim, Noam Ben-Shalom, Elad Chomsky, Alex E. Clark, Michal Navon, Aaron F. Carlin, Oren Kobiler, Oren Zimhony, Smadar Hada-Neeman, Anna Roitburd-Berman, Michal Werbner, and Joel Alter
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education.field_of_study ,Population ,B-cell receptor ,breakpoint cluster region ,Biology ,Virology ,Epitope ,Neutralization ,Article ,Immune system ,medicine.anatomical_structure ,medicine ,biology.protein ,Antibody ,education ,B cell - Abstract
The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe and not mild infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of viral inhibition. B cell receptor (BCR) sequencing revealed two VH genes, VH3-38 and VH3-53, that were enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against live SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and RBD mutagenesis, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19.
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- 2020
10. Conserved interactions required for in vitro inhibition of the main protease of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
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Daniel Zaidman, Ori Kalid, Idit Buch, Efrat Ben-Zeev, Tali Yarnizky, Itai Bloch, Idan Segev, Elad Segev, Dvir Doron, Alina Shitrit, and Oren Kobiler
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Protease ,biology ,Chemistry ,medicine.medical_treatment ,In silico ,Rational design ,Active site ,In vitro ,Recognition sequence ,Biochemistry ,Docking (molecular) ,biology.protein ,medicine ,DrugBank - Abstract
The COVID-19 pandemic caused by the SARS-CoV-2 requires a fast development of antiviral drugs. SARS-CoV-2 viral main protease (Mpro, also called 3C-like protease, 3CLpro) is a potential target for drug design. Crystal and co-crystal structures of the SARS-CoV-2 Mpro have been solved, enabling the rational design of inhibitory compounds. In this study we analyzed the available SARS-CoV-2 and the highly similar SARS-CoV-1 crystal structures. We identified within the active site of the Mpro, in addition to the inhibitory ligands’ interaction with the catalytic C145, two key H-bond interactions with the conserved H163 and E166 residues. Both H-bond interactions are present in almost all co-crystals and are likely to occur also during the viral polypeptide cleavage process as suggested from docking of the Mpro cleavage recognition sequence. We screened in silico a library of 6,900 FDA-approved drugs (ChEMBL) and filtered using these key interactions and selected 29 non-covalent compounds predicted to bind to the protease. Additional screen, using DOCKovalent was carried out on DrugBank library (11,414 experimental and approved drugs) and resulted in 6 covalent compounds. The selected compounds from both screens were tested in vitro by a protease activity inhibition assay. Two compounds showed activity at the 50μM concentration range. Our analysis and findings can facilitate and focus the development of highly potent inhibitors against SARS-CoV-2 infection.
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- 2020
11. The Fate of Incoming HSV-1 Genomes Entering the Nucleus
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Amichay Afriat and Oren Kobiler
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0301 basic medicine ,viruses ,HSL and HSV ,Genome, Viral ,Herpesvirus 1, Human ,Biology ,Genome ,Virus ,Nucleus ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,medicine ,Animals ,Humans ,Genomes ,Incoming ,Genetics ,Cell Nucleus ,Herpes Simplex ,General Medicine ,HSV-1 ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Lytic cycle ,Naked DNA ,030220 oncology & carcinogenesis ,Entering ,DNA, Viral ,Double stranded ,DNA - Abstract
Herpesvirus genomes enter the eukaryotic nucleus as large linear double stranded DNA molecules that are free of any proteins (naked DNA). Once inside the nucleus, the HSV-1 genomes immediately associate with proteins that will be instrumental in the organization and regulation of these genomes. These initial interactions are thought to determine the fate of the infecting genomes. In general, the host cell has evolved several mechanisms to suppress viral genomes and induce latent or abortive infections. On the other hand, the virus has evolved to use viral and cellular factors to promote lytic infection. Recent findings suggest that not all viral genomes in the infected nucleus will develop progeny and that not all genetically identical cells will support successful virus propagation. Thus, the decision between different fates of infection is determined at both single-cell and single-genome levels. Here we summarize current knowledge on the conditions and interactions that lead to each outcome and discuss the unknown determinants.
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- 2020
12. Abortive herpes simplex virus infection of nonneuronal cells results in quiescent viral genomes that can reactivate
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Meir Shamay, Nir Avital, Efrat M. Cohen, and Oren Kobiler
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Herpes simplex virus infection ,Gene Expression Regulation, Viral ,viruses ,Cell ,Genome, Viral ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,HeLa ,Chlorocebus aethiops ,medicine ,Animals ,Humans ,Latency (engineering) ,Vero Cells ,Multidisciplinary ,Herpes Simplex ,Biological Sciences ,biology.organism_classification ,Virology ,Virus Latency ,medicine.anatomical_structure ,Histone ,Herpes simplex virus ,Lytic cycle ,Viral genomes ,biology.protein ,Virus Activation ,Single-Cell Analysis ,HeLa Cells - Abstract
Abortive viral infections are usually studied in populations of susceptible but nonpermissive cells. Single-cell studies of viral infections have demonstrated that even in susceptible and permissive cell populations, abortive infections can be detected in subpopulations of the infected cells. We have previously identified abortive infections in HeLa cells infected with herpes simplex virus 1 (HSV-1) at high multiplicity of infection (MOI). Here, we tested 4 additional human-derived nonneuronal cell lines (cancerous or immortalized) and found significant subpopulations that remain abortive. To characterize these abortive cells, we recovered cell populations that survived infection with HSV-1 at high MOI. The surviving cells retained proliferative potential and the ability to be reinfected. These recovered cell populations maintained the viral genomes in a quiescent state for at least 5 wk postinfection. Our results indicate that these viral genomes are maintained inside the nucleus, bound to cellular histones and occasionally reactivated to produce new progeny viruses. We conclude that abortive HSV-1 infection is a common feature during infection of nonneuronal cells and results in a latency-like state in the infected cells. Our findings question the longstanding paradigm that alphaherpesviruses can establish spontaneous latency only in neuronal cells and emphasize the stochastic nature of lytic versus latency decision of HSV-1 in nonneuronal cells.
- Published
- 2019
13. Insular cortex neurons encode and retrieve specific immune responses
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Maya Schiller, Maria Krot, Itay Zalayat, Hedva Haykin, Haitham Hajjo, Mariam Amer, Nadia Boshnak, Re’ee Yifa, Ben Korin, Asya Rolls, Tamar Koren, Oren Kobiler, Eden Avishai, Tamar L. Ben-Shaanan, Kobi Rosenblum, Hilla Azulay-Debby, Fahed Hakim, and Dorit Farfara
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Male ,Colon ,Inflammation ,Engram ,Peritonitis ,Biology ,Immunological memory ,ENCODE ,Insular cortex ,General Biochemistry, Genetics and Molecular Biology ,Mice ,Immune system ,Immunity ,medicine ,Animals ,Insular Cortex ,Dextran Sulfate Sodium ,Neurons ,Dextran Sulfate ,Zymosan ,Colitis ,Mice, Inbred C57BL ,Synapses ,Female ,Peritoneum ,medicine.symptom ,Neuroscience - Abstract
Increasing evidence indicates that the brain regulates peripheral immunity, yet whether and how the brain represents the state of the immune system remains unclear. Here, we show that the brain's insular cortex (InsCtx) stores immune-related information. Using activity-dependent cell labeling in mice (FosTRAP), we captured neuronal ensembles in the InsCtx that were active under two different inflammatory conditions (dextran sulfate sodium [DSS]-induced colitis and zymosan-induced peritonitis). Chemogenetic reactivation of these neuronal ensembles was sufficient to broadly retrieve the inflammatory state under which these neurons were captured. Thus, we show that the brain can store and retrieve specific immune responses, extending the classical concept of immunological memory to neuronal representations of inflammatory information.
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- 2021
14. Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses
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Matthew D. Weitzman, Enosh Tomer, Assaf Zaritsky, Oren Kobiler, Nir Drayman, Efrat M. Cohen, and Amichay Afriat
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0301 basic medicine ,DNA Replication ,viruses ,Genome, Viral ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Virus Replication ,Biochemistry ,Genome ,law.invention ,03 medical and health sciences ,chemistry.chemical_compound ,0302 clinical medicine ,law ,Cell Line, Tumor ,Chlorocebus aethiops ,Genetics ,medicine ,Animals ,Humans ,Molecular Biology ,Vero Cells ,In Situ Hybridization, Fluorescence ,Recombination, Genetic ,Research ,030104 developmental biology ,medicine.anatomical_structure ,Herpes simplex virus ,chemistry ,Viral replication ,Recombinant DNA ,Female ,Homologous recombination ,Nucleus ,030217 neurology & neurosurgery ,DNA ,Recombination ,Biotechnology - Abstract
Homologous recombination (HR) is considered a major driving force of evolution because it generates and expands genetic diversity. Evidence of HR between coinfecting herpesvirus DNA genomes can be found frequently both in vitro and in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when coinfecting viral genomes interact. To study the spatiotemporal requirements for intergenomic recombination, we developed an assay with dual-color FISH that enables detection of HR between different pairs of coinfecting HSV-1 genomes. Our results revealed that HR increases intermingling of RCs derived from different genomes. Furthermore, inhibition of RC movement reduces the rate of HR events among coinfecting viruses. Finally, we observed correlation between nuclear size and the number of RCs per nucleus. Our findings suggest that both viral replication and recombination are subject to nuclear spatial constraints. Other DNA viruses and cellular DNA are likely to encounter similar restrictions.—Tomer, E., Cohen, E. M., Drayman, N., Afriat, A., Weitzman, M. D., Zaritsky, A., Kobiler, O. Coalescing replication compartments provide the opportunity for recombination between coinfecting herpesviruses.
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- 2019
15. Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors
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Meital Gal-Tanamy, Smadar Hada-Neeman, Modi Safra, Anna Roitburd-Berman, Hila Sharim, Michael Mor, David Hagin, Sandra L Leibel, Ben A. Croker, Aaron F. Carlin, Ksenia Polonsky, Joel Alter, Dor Rafael, Eric R. Griffis, Gur Yaari, Michal Werbner, Oren Zimhony, Alex E. Clark, Michal Navon, Noam Ben-Shalom, Elad Chomsky, Moshe Dessau, Oren Kobiler, Natalia T. Freund, Cameron J. Nowell, Evgeny Kiner, Jamie C. Lee, and Jonathan M. Gershoni
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Male ,RNA viruses ,Viral Diseases ,B Cells ,Coronaviruses ,Physiology ,Antibody Response ,Antibodies, Viral ,Biochemistry ,Epitope ,Epitopes ,White Blood Cells ,Medical Conditions ,0302 clinical medicine ,Animal Cells ,Immune Physiology ,Chlorocebus aethiops ,Cloning, Molecular ,Enzyme-Linked Immunoassays ,Biology (General) ,Immune Response ,Pathology and laboratory medicine ,Staining ,0303 health sciences ,education.field_of_study ,Immune System Proteins ,biology ,breakpoint cluster region ,Antibodies, Monoclonal ,Cell Staining ,Middle Aged ,Medical microbiology ,Infectious Diseases ,medicine.anatomical_structure ,030220 oncology & carcinogenesis ,Spike Glycoprotein, Coronavirus ,Viruses ,Female ,SARS CoV 2 ,Pathogens ,Cellular Types ,Antibody ,Research Article ,Adult ,SARS coronavirus ,QH301-705.5 ,Immune Cells ,Immunology ,B-cell receptor ,Population ,Research and Analysis Methods ,Microbiology ,Article ,Antibodies ,03 medical and health sciences ,Immune system ,Virology ,Genetics ,medicine ,Animals ,Humans ,Antibody-Producing Cells ,Immunoassays ,Molecular Biology Techniques ,education ,Vero Cells ,Molecular Biology ,B cell ,Aged ,030304 developmental biology ,Medicine and health sciences ,Blood Cells ,Biology and life sciences ,SARS-CoV-2 ,Organisms ,Viral pathogens ,COVID-19 ,Proteins ,Convalescence ,Covid 19 ,Cell Biology ,RC581-607 ,Antibodies, Neutralizing ,Microbial pathogens ,Epitope mapping ,Specimen Preparation and Treatment ,Immunoglobulin G ,Immunologic Techniques ,biology.protein ,Parasitology ,Immunologic diseases. Allergy ,Epitope Mapping ,Cloning - Abstract
The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection. Severe, and not mild, infection correlated with high titers of IgG against Spike receptor binding domain (RBD) that were capable of ACE2:RBD inhibition. B cell receptor (BCR) sequencing revealed that VH3-53 was enriched during severe infection. Of the 22 antibodies cloned from two severe donors, six exhibited potent neutralization against authentic SARS-CoV-2, and inhibited syncytia formation. Using peptide libraries, competition ELISA and mutagenesis of RBD, we mapped the epitopes of the neutralizing antibodies (nAbs) to three different sites on the Spike. Finally, we used combinations of nAbs targeting different immune-sites to efficiently block SARS-CoV-2 infection. Analysis of 49 healthy BCR repertoires revealed that the nAbs germline VHJH precursors comprise up to 2.7% of all VHJHs. We demonstrate that severe COVID-19 is associated with unique BCR signatures and multi-clonal neutralizing responses that are relatively frequent in the population. Moreover, our data support the use of combination antibody therapy to prevent and treat COVID-19., Author summary The correlates of effective durable antibody response to SARS-CoV-2 infection are still unclear. In this study, we compared B cell receptor signatures in 8 Severe versus 10 Mild SARS-CoV-2 infected Israeli donors, at 1.5 months post infection using molecular and bioinformatic approaches. We found distinct features between the two groups with higher anti-SARS-CoV-2 receptor binding domain (RBD) plasma IgG titers and increased B cell expansion in donors with severe disease manifestations. We further isolated 22 monoclonal antibodies from these donors, 6 of which were highly potent neutralizing the live virus and inhibited the fusion of infected cells. Using mutagenesis and peptide libraries we mapped the binding sites of the neutralizing antibodies on the RBD of the SARS-CoV-2 Spike. We next demonstrated that combinations of different classes of neutralizing mAbs can completely block the live virus from spreading in culture. Lastly, we performed a bioinformatic search in 49 healthy BCR repertoires identifying precursors for these neutralizing antibodies in the top 30 most common precursors, suggesting that these antibodies can be readily produced by the majority of the uninfected population upon antigenic stimulation.
- Published
- 2021
16. Recombination between co-infecting herpesviruses occurs where replication compartments coalesce
- Author
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Matthew D. Weitzman, Assaf Zaritsky, Amichay Afriaat, Nir Drayman, Enosh Tomer, Oren Kobiler, and Efrat M. Cohen
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Genetics ,0303 health sciences ,medicine.diagnostic_test ,viruses ,030302 biochemistry & molecular biology ,Biology ,medicine.disease_cause ,Genome ,Virus ,3. Good health ,03 medical and health sciences ,chemistry.chemical_compound ,Herpes simplex virus ,medicine.anatomical_structure ,chemistry ,medicine ,Homologous recombination ,Nucleus ,Recombination ,DNA ,030304 developmental biology ,Fluorescence in situ hybridization - Abstract
Homologous recombination (HR) is considered a major driving force of evolution since it generates and expands genetic diversity. Evidence of HR between co-infecting herpesvirus DNA genomes can be found frequently, bothin vitroand in clinical isolates. Each herpes simplex virus type 1 (HSV-1) replication compartment (RC) derives from a single incoming genome and maintains a specific territory within the nucleus. This raises intriguing questions about where and when co-infecting viral genomes interact. To study the spatiotemporal requirements for inter-genomic recombination, we developed an assay with dual-color fluorescencein situhybridization which enables detection of HR between different pairs of co-infecting HSV-1 genomes. Our results revealed that when viral RCs enlarge towards each other, there is detectable overlap between territories of genomes from each virus. Infection with paired viruses that allow visualization of HR correlates with increased overlap of RCs. Further, inhibition of RC movement reduces the rate of HR events among co-infecting viruses. Taken together, these findings suggest that inter-genomic HR events take place during replication of HSV-1 DNA and are mainly confined to the periphery of RCs when they coalesce. Our observations have implications on understanding the recombination restrictions of other DNA viruses and cellular DNA.
- Published
- 2018
17. Non-homologous recombination in HSV1
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Alina Shitrit, Enosh Tomer, and Oren Kobiler
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CATS ,medicine ,Antimicrobial susceptibility ,Biology ,Antimicrobial ,medicine.disease_cause ,Oral cavity ,Applied Microbiology and Biotechnology ,Biochemistry ,Microbiology ,Staphylococcus ,Biotechnology - Published
- 2018
18. Promoting Simultaneous Onset of Viral Gene Expression Among Cells Infected with Herpes Simplex Virus-1
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Marina Bednarchik, Enosh Tomer, Dor Rafael, Oren Kobiler, Sefi Zargarian, Maya Ralph, and Motti Gerlic
- Subjects
0301 basic medicine ,Microbiology (medical) ,herpesviruses ,viruses ,Population ,lcsh:QR1-502 ,biological noise ,Biology ,medicine.disease_cause ,Microbiology ,Genome ,Time-lapse microscopy ,lcsh:Microbiology ,03 medical and health sciences ,Multiplicity of infection ,Gene expression ,medicine ,education ,Original Research ,education.field_of_study ,Temperature-sensitive mutant ,timing of infection ,Virology ,temperature sensitive ,3. Good health ,single cell ,030104 developmental biology ,Herpes simplex virus ,Viral replication - Abstract
Synchronous viral infection facilitates the study of viral gene expression, viral host interactions, and viral replication processes. However, the protocols for achieving synchronous infections were hardly ever tested in proper temporal resolution at the single-cell level. We set up a fluorescence-based, time lapse microscopy assay to study sources of variability in the timing of gene expression during herpes simplex virus-1 (HSV-1) infection. We found that with the common protocol, the onset of gene expression within different cells can vary by more than 3 h. We showed that simultaneous viral genome entry to the nucleus can be achieved with a derivative of the previously characterized temperature sensitive mutant tsB7, however, this did not improve gene expression synchrony. We found that elevating the temperature in which the infection is done and increasing the multiplicity of infection (MOI) significantly promoted simultaneous onset of viral gene expression among infected cells. Further, elevated temperature result in a decrease in the coefficient of variation (a standardized measure of dispersion) of viral replication compartments (RCs) sizes among cells as well as a slight increment of viral late gene expression synchrony. We conclude that simultaneous viral gene expression can be improved by simple modifications to the infection process and may reduce the effect of single-cell variability on population-based assays.
- Published
- 2017
19. Histone Deacetylase Inhibitors Reduce the Number of Herpes Simplex Virus-1 Genomes Initiating Expression in Individual Cells
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Maya Ralph, Lev Shapira, Oren Kobiler, Enosh Tomer, and Shai Cohen
- Subjects
0301 basic medicine ,Microbiology (medical) ,Intrinsic immunity ,viruses ,Histone deacetylase inhibitors ,lcsh:QR1-502 ,Biology ,ICP0 deletion ,histone deacetylase inhibitors ,medicine.disease_cause ,Microbiology ,lcsh:Microbiology ,Immediate early protein ,03 medical and health sciences ,Viral entry ,medicine ,Gene silencing ,virus host interactions ,Original Research ,030304 developmental biology ,0303 health sciences ,Innate immune system ,030102 biochemistry & molecular biology ,herpes simplex virus-1 ,030302 biochemistry & molecular biology ,intrinsic immunity ,Virology ,Cell biology ,3. Good health ,030104 developmental biology ,Herpes simplex virus ,Trichostatin A ,Histone ,Viral replication ,biology.protein ,gene expression ,Histone deacetylase ,medicine.drug - Abstract
Although many viral particles can enter a single cell, the number of viral genomes per cell that establish infection is limited. However, mechanisms underlying this restriction were not explored in depth. For herpesviruses, one of the possible mechanisms suggested is chromatinization and silencing of the incoming genomes. To test this hypothesis, we followed infection with three herpes simplex virus 1 (HSV-1) fluorescence-expressing recombinants in the presence or absence of histone deacetylases inhibitors (HDACi’s). Unexpectedly, a lower number of viral genomes initiated expression in the presence of these inhibitors. This phenomenon was observed using several HDACi: Trichostatin A (TSA), Suberohydroxamic Acid (SBX), Valporic Acid (VPA) and Suberoylanilide Hydoxamic Acid (SAHA). We found that HDACi presence did not change the progeny outcome from the infected cells but did alter the kinetic of the infection. Different cell types (HFF, Vero and U2OS), which vary in their capability to activate intrinsic and innate immunity, show a cell specific basal average number of viral genomes establishing infection. Importantly, in all cell types, treatment with TSA reduced the number of viral genomes. ND10 nuclear bodies are known to interact with the incoming herpes genomes and repress viral replication. The viral immediate early protein, ICP0, is known to disassemble the ND10 bodies and to induce degradation of some of the host proteins in these domains. HDACi treated cells expressed higher levels of some of the host ND10 proteins (PML and ATRX), which may down regulate the number of viral genomes initiating expression per cell. Corroborating this hypothesis, infection with three HSV-1 recombinants carrying a deletion in the gene coding for ICP0, show a reduction in the number of genomes being expressed in U2OS cells. We suggest that alterations in the levels of host proteins involved in intrinsic antiviral defense may result in differences in the number of genomes that initiate expression.
- Published
- 2016
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20. Vaccination strategies against respiratory syncytial virus
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Alison P. Galvani, Jeffrey P. Townsend, Shai Gertler, Forrest K. Jones, John P. DeVincenzo, Dan Yamin, and Oren Kobiler
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Adult ,Pediatrics ,medicine.medical_specialty ,Adolescent ,viruses ,Population ,Respiratory Syncytial Virus Infections ,Virus ,03 medical and health sciences ,Young Adult ,0302 clinical medicine ,030225 pediatrics ,medicine ,Humans ,030212 general & internal medicine ,Respiratory system ,education ,Child ,Aged ,education.field_of_study ,Multidisciplinary ,Transmission (medicine) ,business.industry ,Incidence (epidemiology) ,Vaccination ,Infant, Newborn ,Infant ,Biological Sciences ,Middle Aged ,Models, Theoretical ,United States ,Clinical trial ,Child, Preschool ,Respiratory Syncytial Virus, Human ,business ,Viral load - Abstract
Respiratory syncytial virus (RSV) is the most common cause of US infant hospitalization. Additionally, RSV is responsible for 10,000 deaths annually among the elderly across the United States, and accounts for nearly as many hospitalizations as influenza. Currently, several RSV vaccine candidates are under development to target different age groups. To evaluate the potential effectiveness of age-specific vaccination strategies in averting RSV incidence, we developed a transmission model that integrates data on daily infectious viral load and changes of behavior associated with RSV symptoms. Calibrating to RSV weekly incidence rates in Texas, California, Colorado, and Pennsylvania, we show that in all states considered, an infected child under 5 y of age is more than twice as likely as a person over 50 y of age to transmit the virus. Geographic variability in the effectiveness of a vaccination program across states arises from interplay between seasonality patterns, population demography, vaccination uptake, and vaccine mechanism of action. Regardless of these variabilities, our analysis showed that allocating vaccine to children under 5 y of age would be the most efficient strategy per dose to avert RSV in both children and adults. Furthermore, due to substantial indirect protection, the targeting of children is even predicted to reduce RSV in the elderly more than directly vaccinating the elderly themselves. Our results can help inform ongoing clinical trials and future recommendations on RSV vaccination.
- Published
- 2016
21. Dynamic proteomics of HSV1 infection reveals molecular events that govern non-stochastic infection outcomes
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Lev Shapira, Oren Kobiler, Tamar Danon, Uri Alon, Omer Karin, Nir Drayman, and Avi Mayo
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Herpes simplex virus ,medicine.anatomical_structure ,Systems biology ,Immunology ,Cell ,medicine ,Human pathogen ,Cell state ,Computational biology ,Biology ,medicine.disease_cause ,Proteomics ,Viral infection - Abstract
Viral infection is usually studied at the level of cell populations, averaging over hundreds of thousands of individual cells. Moreover, measurements are typically done by analyzing a few time points along the infection process. While informative, such measurements are limited in addressing how cell variability affects infection outcome. Here we employ dynamic proteomics to study virus-host interactions, using the human pathogen Herpes Simplex virus 1 as a model. We tracked >50,000 individual cells as they respond to HSV1 infection, allowing us to model infection kinetics and link infection outcome (productive or not) with the cell state at the time of initial infection. We find that single cells differ in their preexisting susceptibility to HSV1, and that this is partially mediated by their cell-cycle position. We also identify specific changes in protein levels and localization in infected cells, attesting to the power of the dynamic proteomics approach for studying virus-host interactions.
- Published
- 2016
- Full Text
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22. Virus strategies for passing the nuclear envelope barrier
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Ariella Oppenheim, Nir Drayman, Oren Kobiler, and Veronika Butin-Israeli
- Subjects
Nuclear Envelope ,viruses ,Active Transport, Cell Nucleus ,Review ,nuclear transport ,Genome, Viral ,Biology ,virus entry ,nuclear entry ,Viral entry ,medicine ,Humans ,RNA Viruses ,Nuclear pore ,Nuclear membrane ,Virus classification ,Parvoviridae ,DNA Viruses ,Cell Biology ,Virus Internalization ,biology.organism_classification ,disassembly ,Virology ,Lamins ,Polyomaviridae ,medicine.anatomical_structure ,Viral replication ,Nuclear Pore ,Nuclear transport ,Virus Physiological Phenomena - Abstract
Viruses that replicate in the nucleus need to pass the nuclear envelope barrier during infection. Research in recent years indicates that the nuclear envelope is a major hurdle for many viruses. This review describes strategies to overcome this obstacle developed by seven virus families: herpesviridae, adenoviridae, orthomyxoviridae, lentiviruses (which are part of retroviridae), Hepadnaviridae, parvoviridae and polyomaviridae. Most viruses use the canonical nuclear pore complex (NPC) in order to get their genome into the nucleus. Viral capsids that are larger than the nuclear pore disassemble before or during passing through the NPC, thus allowing genome nuclear entry. Surprisingly, increasing evidence suggest that parvoviruses and polyomaviruses may bypass the nuclear pore by trafficking directly through the nuclear membrane. Additional studies are required for better understanding these processes. Since nuclear entry emerges as the limiting step in infection for many viruses, it may serve as an ideal target for antiviral drug development.
- Published
- 2012
23. Microdissection of neural networks by conditional reporter expression from a Brainbow herpesvirus
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Zhiying Shan, Joshua McCambridge, Mohan K. Raizada, J. Patrick Card, Alan F. Sved, Lynn W. Enquist, Sommer Ebdlahad, and Oren Kobiler
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Technology ,Nerve net ,Pseudorabies ,Cre recombinase ,Biology ,Genome ,Virus ,Catecholamines ,Genes, Reporter ,medicine ,Biological neural network ,Brainbow ,Fluorescent Dyes ,Neurons ,Genetics ,Reporter gene ,Multidisciplinary ,Integrases ,Biological Transport ,Biological Sciences ,biology.organism_classification ,Herpesvirus 1, Suid ,Cell biology ,medicine.anatomical_structure ,Nerve Net ,Microdissection - Abstract
Transneuronal transport of neurotropic viruses is widely used to define the organization of neural circuitry in the mature and developing nervous system. However, interconnectivity within complex circuits limits the ability of viral tracing to define connections specifically linked to a subpopulation of neurons within a network. Here we demonstrate a unique viral tracing technology that highlights connections to defined populations of neurons within a larger labeled network. This technology was accomplished by constructing a replication-competent strain of pseudorabies virus (PRV-263) that changes the profile of fluorescent reporter expression in the presence of Cre recombinase (Cre). The viral genome carries a Brainbow cassette that expresses a default red reporter in infected cells. However, in the presence of Cre, the red reporter gene is excised from the genome and expression of yellow or cyan reporters is enabled. We used PRV-263 in combination with a unique lentivirus vector that produces Cre expression in catecholamine neurons. Projection-specific infection of central circuits containing these Cre-expressing catecholamine neurons with PRV-263 resulted in Cre-mediated recombination of the PRV-263 genome and conditional expression of cyan/yellow reporters. Replication and transneuronal transport of recombined virus produced conditional reporter expression in neurons synaptically linked to the Cre-expressing catecholamine neurons. This unique technology highlights connections specific to phenotypically defined neurons within larger networks infected by retrograde transneuronal transport of virus from a defined projection target. The availability of other technologies that restrict Cre expression to defined populations of neurons indicates that this approach can be widely applied across functionally defined systems.
- Published
- 2011
24. A Common Neuronal Response to Alphaherpesvirus Infection
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Lynn W. Enquist, Oren Kobiler, and Moriah L. Szpara
- Subjects
Gene Expression Regulation, Viral ,Pharmacology ,biology ,Neuroimmunomodulation ,viruses ,Immunology ,Neuroscience (miscellaneous) ,Pseudorabies ,Herpesviridae Infections ,Alphaherpesvirinae ,biology.organism_classification ,medicine.disease_cause ,Virology ,Article ,Virus ,Herpesviridae ,Herpes simplex virus ,Lytic cycle ,medicine ,Humans ,Immunology and Allergy ,Gene family ,Signal Transduction - Abstract
Alphaherpesviruses are a subfamily of the Herpesviridae that can invade the nervous system and establish either lytic or latent infections. The establishment of latent infection can occur only in neurons, indicating a unique virus-host interaction in these cells. Here, we compare results from seven microarray studies that focused on the host response of either neural tissue or isolated neurons to alphaherpesvirus infection. These studies utilized either herpes simplex virus type 1 or pseudorabies virus as the infectious agent. From these data, we have found common host responses spanning a variety of infection models in different species, with different herpesvirus strains, and during all phases of infection including lytic, latent, and reactivation. The repeated observation of transcriptional effects on these genes and gene families indicates their likely importance in host defenses or the viral infectious process. We discuss the possible role of these different genes and genes families in alphaherpesvirus infection.
- Published
- 2010
25. Host responses influence on the induction of lambda prophage
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Assaf Rokney, Amos B. Oppenheim, Oren Kobiler, Sankar Adhya, Amnon Amir, Donald L. Court, and Joel Stavans
- Subjects
Ultraviolet Rays ,Mitomycin ,Repressor ,Biology ,Microbiology ,Bacteriophage ,Viral Proteins ,SOS Response (Genetics) ,Lysogenic cycle ,Viral Regulatory and Accessory Proteins ,SOS response ,Promoter Regions, Genetic ,SOS Response, Genetics ,Lysogeny ,Molecular Biology ,Research Articles ,Prophage ,Temperature ,Lambda phage ,biology.organism_classification ,Bacteriophage lambda ,Molecular biology ,DNA-Binding Proteins ,Repressor Proteins ,Lytic cycle ,Virus Activation ,Transcription Factors - Abstract
Inactivation of bacteriophage lambda CI repressor leads almost exclusively to lytic development. Prophage induction can be initiated either by DNA damage or by heat treatment of a temperature-sensitive repressor. These two treatments also cause a concurrent activation of either the host SOS or heat-shock stress responses respectively. We studied the effects of these two methods of induction on the lytic pathway by monitoring the activation of different lambda promoters, and found that the lambda genetic network co-ordinates information from the host stress response networks. Our results show that the function of the CII transcriptional activator, which facilitates the lysogenic developmental pathway, is not observed following either method of induction. Mutations in the cro gene restore the CII function irrespective of the induction method. Deletion of the heat-shock protease gene ftsH can also restore CII function following heat induction but not following SOS induction. Our findings highlight the importance of the elimination of CII function during induction as a way to ensure an efficient lytic outcome. We also show that, despite the common inhibitory effect on CII function, there are significant differences in the heat- and SOS-induced pathways leading to the lytic cascade.
- Published
- 2008
26. Histone deacetylase inhibitors reduce the number of herpes simplex virus-1 genomes initiating expression in individual cells
- Author
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Lev, Shapira, primary, Maya, Ralph, additional, Enosh, Tomer, additional, Shai, Cohen, additional, and Oren, Kobiler, additional
- Published
- 2016
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27. Quantitative kinetic analysis of the bacteriophage λ genetic network
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Joel Stavans, Amos B. Oppenheim, Assaf Rokney, Oren Kobiler, Donald L. Court, and Nir Friedman
- Subjects
Genetics ,Regulation of gene expression ,Bacteriophage ,Multidisciplinary ,Lytic cycle ,Lysogenic cycle ,Negative feedback ,Biology ,biology.organism_classification ,DNA-binding protein ,Gene ,Function (biology) - Abstract
The lysis–lysogeny decision of bacteriophage λ has been a paradigm for a developmental genetic network, which is composed of interlocked positive and negative feedback loops. This genetic network is capable of responding to environmental signals and to the number of infecting phages. An interplay between CI and Cro functions suggested a bistable switch model for the lysis–lysogeny decision. Here, we present a real-time picture of the execution of lytic and lysogenic pathways with unprecedented temporal resolution. We monitor, in vivo , both the level and function of the CII and Q gene regulators. These activators are cotranscribed yet control opposite developmental pathways. Conditions that favor the lysogenic response show severe delay and down-regulation of Q activity, in both CII-dependent and CII-independent ways. Whereas CII activity correlates with its protein level, Q shows a pronounced threshold before its function is observed. Our quantitative analyses suggest that by regulating CII and CIII, Cro plays a key role in the ability of the λ genetic network to sense the difference between one and more than one phage particles infecting a cell. Thus, our results provide an improved framework to explain the longstanding puzzle of the decision process.
- Published
- 2005
28. The phage λ CII transcriptional activator carries a C-terminal domain signaling for rapid proteolysis
- Author
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Donald L. Court, Amos B. Oppenheim, Oren Kobiler, Dinah Teff, and Simi Koby
- Subjects
musculoskeletal diseases ,Transcription, Genetic ,Recombinant Fusion Proteins ,Proteolysis ,Molecular Sequence Data ,Mutant ,chemical and pharmacologic phenomena ,macromolecular substances ,Biology ,Virus Replication ,medicine.disease_cause ,Polymerase Chain Reaction ,Viral Proteins ,Escherichia coli ,medicine ,Amino Acid Sequence ,Cloning, Molecular ,skin and connective tissue diseases ,Lysogeny ,Gene ,Peptide sequence ,Mutation ,Multidisciplinary ,Base Sequence ,integumentary system ,medicine.diagnostic_test ,C-terminus ,RNA ,Biological Sciences ,Bacteriophage lambda ,Molecular biology ,Antisense RNA ,DNA, Viral ,RNA, Viral ,Plasmids ,Transcription Factors - Abstract
ATP-dependent proteases, like FtsH (HflB), recognize specific protein substrates. One of these is the λ CII protein, which plays a key role in the phage lysis-lysogeny decision. Here we provide evidence that the conserved C-terminal end of CII acts as a necessary and sufficient cis-acting target for rapid proteolysis. Deletions of this conserved tag, or a mutation that confers two aspartic residues at its C terminus do not affect the structure or activity of CII. However, the mutations abrogate CII degradation by FtsH. We have established an in vitro assay for the λ CIII protein and demonstrated that CIII directly inhibits proteolysis by FtsH to protect CII and CII mutants from degradation. Phage λ carrying mutations in the C terminus of CII show increased frequency of lysogenization, which indicates that this segment of CII may itself be sensitive to regulation that affects the lysis-lysogeny development. In addition, the region coding for the C-terminal end of CII overlaps with a gene that encodes a small antisense RNA called OOP. We show that deletion of the end of the cII gene can prevent OOP RNA, supplied in trans, interfering with CII activity. These findings provide an example of a gene that carries a region that modulates stability at the level of mRNA and protein.
- Published
- 2002
29. Characterization of a conserved α-helical, coiled-coil motif at the C-terminal domain of the ATP-dependent FtsH (HflB) protease of Escherichia coli 1 1Edited by J. Karn
- Author
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Amos B. Oppenheim, Oren Kobiler, Dinah Teff, Yoram Shotland, and Simi Koby
- Subjects
Coiled coil ,Leucine zipper ,Circular dichroism ,Protease ,C-terminus ,medicine.medical_treatment ,Mutant ,Biology ,medicine.disease_cause ,Biochemistry ,Structural Biology ,medicine ,Leucine ,Molecular Biology ,Escherichia coli - Abstract
FtsH (HflB) is an ATP-dependent protease found in prokaryotic cells, mitochondria and chloroplasts. Here, we have identified, in the carboxy-terminal region of FtsH (HfIB), a short α helix predicted of forming a coiled-coil, leucine zipper, structure. This region appears to be structurally conserved. The presence of the coiled-coil motif in the Escherichia coli FtsH (HflB) was demonstrated by circular dichroism and cross-linking experiments. Mutational analysis showed that three highly conserved leucine residues are essential for FtsH (HfIB) activity in vivo and in vitro. Purified proteins mutated in the conserved leucine residues, were found to be defective in the degradation of E. coli σ32 and the bacteriophage λ CII proteins. In addition, the mutant proteins were defective in the binding of CII The mutations did not interfere with the ATPase activity of FtsH (HflB). Finally, the mutant proteins were found to be more sensitive to trypsin degradation than the wild-type enzyme suggesting that the α helical region is an important structural element of FtsH (HflB).
- Published
- 2000
30. Alphaherpesvirus axon-to-cell spread involves limited virion transmission
- Author
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Oren Kobiler, Matthew P. Taylor, and Lynn W. Enquist
- Subjects
Swine ,viruses ,Population ,Cell Culture Techniques ,Pseudorabies ,Herpesvirus 1, Human ,Genome ,Virus ,Fluorescence ,Cell Line ,Chlorocebus aethiops ,medicine ,Animals ,Axon ,education ,Vero Cells ,Neurons ,education.field_of_study ,Multidisciplinary ,biology ,Virion ,Herpesviridae Infections ,Biological Sciences ,biology.organism_classification ,Virology ,Herpesvirus 1, Suid ,Axons ,medicine.anatomical_structure ,Capsid ,Cell culture ,Vero cell - Abstract
The spread of viral infection within a host can be restricted by bottlenecks that limit the size and diversity of the viral population. An essential process for alphaherpesvirus infection is spread from axons of peripheral nervous system neurons to cells in peripheral epithelia (anterograde-directed spread, ADS). ADS is necessary for the formation of vesicular lesions characteristic of reactivated herpesvirus infections; however, the number of virions transmitted is unknown. We have developed two methods to quantitate ADS events using a compartmentalized neuronal culture system. The first method uses HSV-1 and pseudorabies virus recombinants that express one of three different fluorescent proteins. The fluorescence profiles of cells infected with the virus mixtures are used to quantify the number of expressed viral genomes. Strikingly, although epithelial or neuronal cells express 3–10 viral genomes after infection by free virions, epithelial cells infected by HSV-1 or pseudorabies virus following ADS express fewer than two viral genomes. The second method uses live-cell fluorescence microscopy to track individual capsids involved in ADS. We observed that most ADS events involve a single capsid infecting a target epithelial cell. Together, these complementary analyses reveal that ADS events are restricted to small numbers of viral particles, most often a single virion, resulting in a single viral genome initiating infection.
- Published
- 2012
31. Herpesvirus Replication Compartments Originate with Single Incoming Viral Genomes
- Author
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Ethan B. Ludmir, Matthew P. Taylor, Oren Kobiler, Lynn W. Enquist, and P. Brodersen
- Subjects
Swine ,viruses ,Genome, Viral ,Biology ,Virus Replication ,Microbiology ,Genome ,Virus ,Viral Proteins ,03 medical and health sciences ,chemistry.chemical_compound ,Virology ,Animals ,Gene ,In Situ Hybridization, Fluorescence ,030304 developmental biology ,Cell Nucleus ,Genetics ,0303 health sciences ,Pseudorabies ,030306 microbiology ,Virus Assembly ,Herpesvirus 1, Suid ,Fusion protein ,QR1-502 ,3. Good health ,Viral replication ,chemistry ,Capsid ,Virion assembly ,DNA ,Research Article - Abstract
Previously we described a method to estimate the average number of virus genomes expressed in an infected cell. By analyzing the color spectrum of cells infected with a mixture of isogenic pseudorabies virus (PRV) recombinants expressing three fluorophores, we estimated that fewer than seven incoming genomes are expressed, replicated, and packaged into progeny per cell. In this report, we expand this work and describe experiments demonstrating the generality of the method, as well as providing more insight into herpesvirus replication. We used three isogenic PRV recombinants, each expressing a fluorescently tagged VP26 fusion protein (VP26 is a capsid protein) under the viral VP26 late promoter. We calculated a similar finite limit on the number of expressed viral genomes, indicating that this method is independent of the promoter used to transcribe the fluorophore genes, the time of expression of the fluorophore (early versus late), and the insertion site of the fluorophore gene in the PRV genome (UL versus US). Importantly, these VP26 fusion proteins are distributed equally in punctate virion assembly structures in each nucleus, which improves the signal-to-noise ratio when determining the color spectrum of each cell. To understand how the small number of genomes are distributed among the replication compartments, we used a two-color fluorescent in situ hybridization assay. Most viral replication compartments in the nucleus occupy unique nuclear territories, implying that they arose from single genomes. Our experiments suggest a correlation between the small number of expressed viral genomes and the limited number of replication compartments., IMPORTANCE Herpesviruses use nuclear factors and architecture to replicate their DNA genomes in the host nuclei. Viral replication compartments are distinct nuclear foci that appear during productive infection. We have recently developed a method that uses three viral recombinants (each expressing a different fluorescent protein) to quantify the number of incoming viral genomes that are expressed and replicated in each cell. We found that fewer than seven herpesvirus genomes can be expressed and replicated. Here we have expanded and improved upon our method and demonstrated that the phenomenon of limited genome expression is independent of the recombinants used. We correlated the small number of genomes expressed to the limited number of replication compartments by demonstrating that most replication compartments originate with a single genome. The distinction among replication compartments is maintained even when most of the nucleus is filled with viral DNA, implying that nuclear architecture constrains the compartments.
- Published
- 2011
32. A dual infection pseudorabies virus conditional reporter approach to identify projections to collateralized neurons in complex neural circuits
- Author
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Lynn W. Enquist, Alan F. Sved, Ethan B. Ludmir, Oren Kobiler, Vedant Desai, and J. Patrick Card
- Subjects
Male ,Histology ,Anatomy and Physiology ,Neural Networks ,viruses ,Science ,Pseudorabies ,Cre recombinase ,Genome, Viral ,Cardiovascular ,Green fluorescent protein ,Rats, Sprague-Dawley ,Open Reading Frames ,03 medical and health sciences ,0302 clinical medicine ,Genes, Reporter ,Biological neural network ,medicine ,Animals ,Brainbow ,Biology ,Gene ,030304 developmental biology ,Neurons ,0303 health sciences ,Reporter gene ,Multidisciplinary ,Base Sequence ,biology ,Systems Biology ,biology.organism_classification ,Herpesvirus 1, Suid ,Molecular biology ,Rats ,Neuroanatomy ,medicine.anatomical_structure ,Microscopy, Fluorescence ,Viral replication ,Cellular Neuroscience ,Medicine ,030217 neurology & neurosurgery ,Research Article ,Neuroscience - Abstract
Replication and transneuronal transport of pseudorabies virus (PRV) are widely used to define the organization of neural circuits in rodent brain. Here we report a dual infection approach that highlights connections to neurons that collateralize within complex networks. The method combines Cre recombinase (Cre) expression from a PRV recombinant (PRV-267) and Cre-dependent reporter gene expression from a second infecting strain of PRV (PRV-263). PRV-267 expresses both Cre and a monomeric red fluorescent protein (mRFP) fused to viral capsid protein VP26 (VP26-mRFP) that accumulates in infected cell nuclei. PRV-263 carries a Brainbow cassette and expresses a red (dTomato) reporter that fills the cytoplasm. However, in the presence of Cre, the dTomato gene is recombined from the cassette, eliminating expression of the red reporter and liberating expression of either yellow (EYFP) or cyan (mCerulean) cytoplasmic reporters. We conducted proof-of-principle experiments using a well-characterized model in which separate injection of recombinant viruses into the left and right kidneys produces infection of neurons in the renal preautonomic network. Neurons dedicated to one kidney expressed the unique reporters characteristic of PRV-263 (cytoplasmic dTomato) or PRV-267 (nuclear VP26-mRFP). Dual infected neurons expressed VP26-mRFP and the cyan or yellow cytoplasmic reporters activated by Cre-mediated recombination of the Brainbow cassette. Differential expression of cyan or yellow reporters in neurons lacking VP26-mRFP provided a unique marker of neurons synaptically connected to dual infected neurons, a synaptic relationship that cannot be distinguished using other dual infection tracing approaches. These data demonstrate Cre-enabled conditional reporter expression in polysynaptic circuits that permits the identification of collateralized neurons and their presynaptic partners.
- Published
- 2011
33. Phage Lambda CIII: A Protease Inhibitor Regulating the Lysis-Lysogeny Decision
- Author
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Assaf Rokney, Oren Kobiler, and Amos B. Oppenheim
- Subjects
Proteases ,medicine.medical_treatment ,Mutant ,Molecular Sequence Data ,lcsh:Medicine ,Biology ,Microbiology ,Bacteriophage ,Viral Proteins ,Multiplicity of infection ,Biopolymers ,Lysogenic cycle ,medicine ,Amino Acid Sequence ,lcsh:Science ,Promoter Regions, Genetic ,Molecular Biology ,Lysogeny ,Conserved Sequence ,Multidisciplinary ,Protease ,Molecular Structure ,Sequence Homology, Amino Acid ,lcsh:R ,Lambda phage ,biology.organism_classification ,Protease inhibitor (biology) ,Cell biology ,Biochemistry ,lcsh:Q ,lipids (amino acids, peptides, and proteins) ,Electrophoresis, Polyacrylamide Gel ,medicine.drug ,Research Article ,Transcription Factors - Abstract
The ATP-dependent protease FtsH (HflB) complexed with HflKC participates in post-translational control of the lysis-lysogeny decision of bacteriophage lambda by rapid degradation of lambda CII. Both phage-encoded proteins, the CII transcription activator and the CIII polypeptide, are required for efficient lysogenic response. The conserved CIII is both an inhibitor and substrate of FtsH. Here we show that the protease inhibitor CIII is present as oligomeric amphipathic alpha helical structures and functions as a competitive inhibitor of FtsH by preventing binding of the CII substrate. We identified single alanine substitutions in CIII that abolish its activity. We characterize a dominant negative effect of a CIII mutant. Thus, we suggest that CIII oligomrization is required for its function. Real-time analysis of CII activity demonstrates that the effect of CIII is not seen in the absence of either FtsH or HflKC. When CIII is provided ectopically, CII activity increases linearly as a function of the multiplicity of infection, suggesting that CIII enhances CII stability and the lysogenic response. FtsH function is essential for cellular viability as it regulates the balance in the synthesis of phospholipids and lipopolysaccharides. Genetic experiments confirmed that the CIII bacteriostatic effects are due to inhibition of FtsH. Thus, the early presence of CIII following infection stimulates the lysogenic response, while its degradation at later times ensures the reactivation of FtsH allowing the growth of the established lysogenic cell.
- Published
- 2007
34. Noise in timing and precision of gene activities in a genetic cascade
- Author
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Assaf Rokney, Oren Kobiler, Amnon Amir, Joel Stavans, and Amos B. Oppenheim
- Subjects
prophage induction ,noise ,Time Factors ,Genes, Viral ,Ultraviolet Rays ,Gene regulatory network ,Biology ,Article ,General Biochemistry, Genetics and Molecular Biology ,SOS Response (Genetics) ,Bacteriolysis ,Lysogenic cycle ,timing ,Gene Regulatory Networks ,SOS response ,SOS Response, Genetics ,News and Views ,Prophage ,Genetics ,General Immunology and Microbiology ,Bacteria ,Pulse (signal processing) ,Applied Mathematics ,Systems Biology ,bacteriophage λ ,Bacteriophage lambda ,Cell biology ,Computational Theory and Mathematics ,Lytic cycle ,Cascade ,Genes, Bacterial ,precision ,General Agricultural and Biological Sciences ,Information Systems - Abstract
The timing of events along the induction cascade of bacteriophage lambda is independent of UV dose and displays increased relative temporal precision with cascade progression. This behavior is reproduced by a model of a cascade consisting of independent steps that shows that higher temporal precision can be attained by a cascade consisting of a large number of fast steps. The observed cell-cell variability in cascade timing is not due to differences in uniform dilation of intervals between events among cells, but rather to the independent distribution of interval durations within the cascade, consistently with the modular architecture of the lambda genome. The single-cell time lapse study reveals a bistable regime at low UV doses in which some cells are induced while others are not, evidence for a commitment point beyond which lysis will occur, and an unexpected shutoff of the lambda pR promoter., Stochasticity or noise, an inherent property of all biological networks, is often manifested by different phenotypic behaviors in clonal populations of cells (Raser and O'Shea, 2005). Noise can arise, for instance, from sources such as cell–cell variations in small numbers of regulatory molecules or from the stochastic nature of molecular interactions (Paulsson, 2005). Besides affecting the number of molecules in a cell, noise may also lead to variability in timing of particular events along a given pathway. In this work, we studied temporal noise in the induction cascade of phage lambda. Infection of a bacterial cell by bacteriophage lambda can lead to two different fates (Ptashne, 2004; Dodd et al, 2005; Oppenheim et al, 2005): the phage can either multiply inside the host leading to its eventual lysis and the generation of progeny virions (the lytic pathway) or, alternatively, it can integrate its genome into the host's genome (prophage state), replicating passively with the latter (the lysogenic pathway). The prophage state is highly stable, being maintained by a phage-encoded repressor, which shuts off phage genes leading to lytic growth. However, the lytic pathway can be induced in a lysogenic cell, through the activation of the bacterial SOS response to DNA damage (Little, 1996), for example by UV irradiation. Once activated, the SOS response results in cleavage of the lambda repressor, leading to expression of the phage early and late genes, and culminating in the lysis of the host cell. The lambda induction cascade has been extensively characterized over the years. We built upon this knowledge to tap the cascade at different points and quantitatively analyze the progressive loss of temporal coherence between cells, as different stages along the cascade are executed, following synchronous induction. Using time-lapse microscopy, we monitored the time of activation of early and late genes in individual cells using lambda pR and pR′-tR′ promoter-GFP fusions, respectively, by means of reporter plasmids, and finally the time of lysis. Sample results are shown in Figure 2. At low UV levels (5 J/m2), the network exhibits bistability: only approximately 40% of the bacteria lyse, whereas the others continue to divide, following a lag period. At high UV levels (20 J/m2), almost all bacteria lyse. We found that the timing of events in cells that lyse is independent of UV dose. This is in contrast to the known behavior of the SOS network (Friedman et al, 2005), indicating that these two networks proceed independently. Following induction, a surprising shutoff in the activity of the pR promoter is observed in all cells (see Figure 2). Furthermore, the data show that whereas early genes are expressed in all cells irrespective of cell fate, late genes are expressed only in the lysing cells, indicating that similar to infection, a specific commitment checkpoint is operating. To characterize the temporal variability in a cell population, we used the coefficient of variation, defined as the non-dimensional ratio of the standard deviation and the mean time of occurrence of a particular event. We studied the changes in both standard deviation and coefficient of variation in timing of various events along the lambda induction cascade, from the expression of the early genes to the ultimate lysis of the cells. As shown in Figure 6, the absolute noise as measured by the standard deviation increases as the cascade progresses. In contrast, the coefficient of variation, which measures variability relative to the time of occurrence, decreases. Simple theoretical considerations described in the text yield a necessary and sufficient condition for a monotonic decrease in the coefficient of variation. Higher temporal precision can be achieved when the cascade is composed of a large number of fast steps. Further support for the independence of network modules is furnished by a correlation analysis of the times of occurrence of different steps along the lytic cascade. This analysis also indicates that the variability in lysis time is not due to differences in the global rate of cascade progression, but probably to random fluctuations in the execution time of the various cascade stages. Indeed, phage lambda gene expression architecture is well known to have evolved from a number of independent regulatory modules (Hendrix, 2003)., Biological developmental pathways require proper timing of gene expression. We investigated timing variations of defined steps along the lytic cascade of bacteriophage λ. Gene expression was followed in individual lysogenic cells, after induction with a pulse of UV irradiation. At low UV doses, some cells undergo partial induction and eventually divide, whereas others follow the lytic pathway. The timing of events in cells committed to lysis is independent of the level of activation of the SOS response, suggesting that the lambda network proceeds autonomously after induction. An increased loss of temporal coherence of specific events from prophage induction to lysis is observed, even though the coefficient of variation of timing fluctuations decreases. The observed temporal variations are not due to cell factors uniformly dilating the timing of execution of the cascade. This behavior is reproduced by a simple model composed of independent stages, which for a given mean duration predicts higher temporal precision, when a cascade consists of a large number of steps. Evidence for the independence of regulatory modules in the network is presented.
- Published
- 2006
35. Switches in bacteriophage lambda development
- Author
-
Oren Kobiler, Donald L. Court, Amos B. Oppenheim, Sankar Adhya, and Joel Stavans
- Subjects
Genetics ,Regulation of gene expression ,biology ,viruses ,Prophages ,Gene Expression Regulation, Bacterial ,Lambda ,biology.organism_classification ,Bacteriophage lambda ,Bacteriophage ,Bacteriolysis ,Lysogen ,Lytic cycle ,Transcription (biology) ,Lysogenic cycle ,Operon ,Lysogeny ,Prophage - Abstract
The lysis-lysogeny decision of bacteriophage lambda (λ) is a paradigm for developmental genetic networks. There are three key features, which characterize the network. First, after infection of the host bacterium, a decision between lytic or lysogenic development is made that is dependent upon environmental signals and the number of infecting phages per cell. Second, the lysogenic prophage state is very stable. Third, the prophage enters lytic development in response to DNA-damaging agents. The CI and Cro regulators define the lysogenic and lytic states, respectively, as a bistable genetic switch. Whereas CI maintains a stable lysogenic state, recent studies indicate that Cro sets the lytic course not by directly blocking CI expression but indirectly by lowering levels of CII which activates cI transcription. We discuss how a relatively simple phage like λ employs a complex genetic network in decision-making processes, providing a challenge for theoretical modeling.
- Published
- 2005
36. Recruitment of host ATP-dependent proteases by bacteriophage lambda
- Author
-
Amos B. Oppenheim, Oren Kobiler, and Christophe Herman
- Subjects
Genetics ,Gene Expression Regulation, Viral ,biology ,Phagemid ,biology.organism_classification ,Virus Replication ,Bacteriophage lambda ,Temperateness ,Bacteriophage ,Lysogen ,Lytic cycle ,ATP-Dependent Proteases ,Structural Biology ,Lysogenic cycle ,Host chromosome ,Lysogeny ,Prophage - Abstract
Upon infection of a bacterial cell, the temperate bacteriophage lambda executes a regulated temporal program with two possible outcomes: (1) Cell lysis and virion production or (2) establishment of a dormant state, lysogeny, in which the phage genome (prophage) is integrated into the host chromosome. The prophage is replicated passively as part of the host chromosome until it is induced to resume the lytic cycle. In this review, we summarize the evidence that implicates every known ATP-dependent protease in the regulation of specific steps in the phage life cycle. The proteolysis of specific regulatory proteins appears to fine-tune phage gene expression. The bacteriophage utilizes multiple proteases to irreversibly inactivate specific regulators resulting in a temporally regulated program of gene expression. Evolutionary forces may have favored the utilization of overlapping protease specificities for differential proteolysis of phage regulators according to different phage life styles.
- Published
- 2003
37. A ‘tool box’ for deciphering neuronal circuits in the developing chick spinal cord
- Author
-
Aharon Lev-Tov, Alex Etlin, Yoav Hadas, Oshri Avraham, Oren Kobiler, Amos Panet, Haya Falk, and Avihu Klar
- Subjects
Rhodopsin ,Nerve net ,Population ,Chick Embryo ,Biology ,Fluorescent imaging ,Molecular targeting ,Genes, Reporter ,Interneurons ,Connectome ,Genetics ,medicine ,Animals ,education ,Motor Neurons ,Genetic dissection ,education.field_of_study ,Integrases ,Anatomy ,Spinal cord ,Axons ,Enhancer Elements, Genetic ,medicine.anatomical_structure ,Spinal Cord ,nervous system ,Neuronal circuits ,Synapses ,Methods Online ,Calcium ,Nerve Net ,Neuroscience - Abstract
The genetic dissection of spinal circuits is an essential new means for understanding the neural basis of mammalian behavior. Molecular targeting of specific neuronal populations, a key instrument in the genetic dissection of neuronal circuits in the mouse model, is a complex and time-demanding process. Here we present a circuit-deciphering ‘tool box’ for fast, reliable and cheap genetic targeting of neuronal circuits in the developing spinal cord of the chick. We demonstrate targeting of motoneurons and spinal interneurons, mapping of axonal trajectories and synaptic targeting in both single and populations of spinal interneurons, and viral vector-mediated labeling of pre-motoneurons. We also demonstrate fluorescent imaging of the activity pattern of defined spinal neurons during rhythmic motor behavior, and assess the role of channel rhodopsin-targeted population of interneurons in rhythmic behavior using specific photoactivation.
- Published
- 2014
38. Proteolysis of bacteriophage lambda CII by Escherichia coli FtsH (HflB)
- Author
-
Tamar Ziv, Amos B. Oppenheim, Yoram Shotland, Simi Koby, Amir Shifrin, Oren Kobiler, and Dinah Teff
- Subjects
medicine.medical_treatment ,Proteolysis ,Molecular Sequence Data ,Structure and Function ,Biology ,medicine.disease_cause ,Microbiology ,Substrate Specificity ,Bacteriophage ,Gene product ,Viral Proteins ,Protein structure ,ATP-Dependent Proteases ,Bacterial Proteins ,Endopeptidases ,medicine ,Escherichia coli ,Amino Acid Sequence ,Molecular Biology ,Peptide sequence ,Protease ,medicine.diagnostic_test ,Escherichia coli Proteins ,Caseins ,Membrane Proteins ,Metalloendopeptidases ,biology.organism_classification ,Bacteriophage lambda ,Endopeptidase ,Biochemistry ,Transcription Factors - Abstract
FtsH (HflB) is a conserved, highly specific, ATP-dependent protease for which a number of substrates are known. The enzyme participates in the phage λ lysis-lysogeny decision by degrading the lambda CII transcriptional activator and by its response to inhibition by the λ CIII gene product. In order to gain further insight into the mechanism of the enzymatic activity of FtsH (HflB), we identified the peptides generated following proteolysis of the phage λ CII protein. It was found that FtsH (HflB) acts as an endopeptidase degrading CII into small peptides with limited amino acid specificity at the cleavage site. β-Casein, an unstructured substrate, is also degraded by FtsH (HflB), suggesting that protein structure may play a minor role in determining the products of proteolysis. The majority of the peptides produced were 13 to 20 residues long.
- Published
- 2000
39. TURNOVER OF StAR PROTEIN: ROLES FOR THE PROTEASOME AND MITOCHONDRIAL PROTEASES
- Author
-
Bin Lu, Carolyn K. Suzuki, Zvi Granot, Oren Kobiler, Amos B. Oppenheim, Sarah Eimerl, Joseph Orly, and Assaf Bahat
- Subjects
Proteases ,Reproductive Medicine ,Proteasome ,Biochemistry ,biology.protein ,Cell Biology ,General Medicine ,Star (graph theory) ,Biology ,Cell biology ,Deubiquitinating enzyme - Published
- 2007
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