scholarly journals Intracellular Complexes of Viral Spike and Cellular Receptor Accumulate during Cytopathic Murine Coronavirus Infections

1998 ◽  
Vol 72 (4) ◽  
pp. 3278-3288 ◽  
Author(s):  
Pasupuleti V. Rao ◽  
Thomas M. Gallagher
Keyword(s):  
Trypan Blue ◽  
Hepatitis Virus ◽  
Syncytium Formation ◽  
Threshold Level ◽  
Inducible Promoter ◽  
Critical Threshold ◽  
Murine Hepatitis Virus ◽  
Trypan Blue Exclusion ◽  
Hela Cell Lines

ABSTRACT Murine hepatitis virus (MHV) infections exhibit remarkable variability in cytopathology, ranging from acutely cytolytic to essentially asymptomatic levels. In this report, we assess the role of the MHV receptor (MHVR) in controlling this variable virus-induced cytopathology. We developed human (HeLa) cell lines in which the MHVR was produced in a regulated fashion by placing MHVR cDNA under the control of an inducible promoter. Depending on the extent of induction, MHVR levels ranged from less than ∼1,500 molecules per cell (designated Rlo) to ∼300,000 molecules per cell (designated Rhi). Throughout this range, the otherwise MHV-resistant HeLa cells were rendered susceptible to infection. However, infection in the Rlo cells occurred without any overt evidence of cytopathology, while the corresponding Rhi cells died within 14 h after infection. When the HeLa-MHVR cells were infected with vaccinia virus recombinants encoding MHV spike (S) proteins, the Rhi cells succumbed within 12 h postinfection; Rlo cells infected in parallel were intact, as judged by trypan blue exclusion. This acute cytopathology was not due solely to syncytium formation between the cells producing S and MHVR, because fusion-blocking antiviral antibodies did not prevent it. These findings raised the possibility of an intracellular interaction between S and MHVR in the acute cell death. Indeed, we identified intracellular complexes of S and MHVR via coimmunoprecipitation of endoglycosidase H-sensitive forms of the two proteins. We suggest that MHV infections can become acutely cytopathic once these intracellular complexes rise above a critical threshold level.

scholarly journals The role of incoherent feedforward circuits in regulating precision of event timing

2020 ◽  
Author(s):  
Supravat Dey ◽  
Sherin Kannoly ◽  
Pavol Bokes ◽  
John J Dennehy ◽  
Abhyudai Singh
Keyword(s):  
Single Cells ◽  
Threshold Level ◽  
Cellular Growth ◽  
Critical Threshold ◽  
E Coli ◽  
Cellular Events ◽  
Event Timing ◽  
Simple Model System ◽  
Cell Data

AbstractTriggering of cellular events often relies on the level of a key gene product crossing a critical threshold. Achieving precision in event timing in spite of noisy gene expression facilitates high-fidelity functioning of diverse processes from biomolecular clocks, apoptosis and cellular differentiation. Here we investigate the role of an incoherent feedforward circuit in regulating the time taken by a bacterial virus (bacteriophage lambda) to lyse an infected Escherichia coli cell. Lysis timing is the result of expression and accumulation of a single lambda protein (holin) in the E. coli cell membrane up to a critical threshold level, which triggers the formation of membrane lesions. This easily visualized process provides a simple model system for characterizing event-timing stochasticity in single cells. Intriguingly, lambda’s lytic pathway synthesizes two functionally opposite proteins: holin and antiholin from the same mRNA in a 2:1 ratio. Antiholin sequesters holin and inhibits the formation of lethal membrane lesions, thus creating an incoherent feedforward circuit. We develop and analyze a stochastic model for this feedforward circuit that considers correlated bursty expression of holin/antiholin, and their concentrations are diluted from cellular growth. Interestingly, our analysis shows the noise in timing is minimized when both proteins are expressed at an optimal ratio, hence revealing an important regulatory role for antiholin. These results are in agreement with single cell data, where removal of antiholin results in enhanced stochasticity in lysis timing.

scholarly journals An MHV macrodomain mutant predicted to lack ADP-ribose binding activity is severely attenuated, indicating multiple roles for the macrodomain in coronavirus replication

2021 ◽  
Author(s):  
Lynden S Voth ◽  
Joseph J O'Connor ◽  
Catherine M Kerr ◽  
Ethan Doerger ◽  
Nancy Schwarting ◽  
...  
Keyword(s):  
Hepatitis Virus ◽  
Single Point ◽  
Binding Activity ◽  
Multiple Roles ◽  
Structural Protein ◽  
Transcript Accumulation ◽  
Murine Hepatitis Virus ◽  
Point Mutant

All coronaviruses (CoVs) contain a macrodomain, also termed Mac1, in non-structural protein 3 (nsp3) which binds and hydrolyzes ADP-ribose covalently attached to proteins. Despite several reports demonstrating that Mac1 is a prominent virulence factor, there is still a limited understanding of its cellular roles during infection. Currently, most of the information regarding the role of CoV Mac1 during infection is based on a single point mutant of a highly conserved asparagine-to-alanine mutation, which is known to largely eliminate Mac1 ADP-ribosylhydrolase activity. To determine if Mac1 ADP-ribose binding separately contributes to CoV replication, we compared the replication of a murine hepatitis virus (MHV) Mac1 mutant predicted to dramatically reduce ADP-ribose binding, D1329A, to the previously mentioned asparagine mutant, N1347A. D1329A and N1347A both replicated poorly in bone-marrow derived macrophages (BMDMs), were inhibited by PARP enzymes, and were highly attenuated in vivo. However, D1329A was significantly more attenuated than N1347A in all cell lines tested that were susceptible to MHV infection. In addition, D1329A retained some ability to block IFN-β transcript accumulation compared to N1347A, indicating that these two mutants impacted distinct Mac1 functions. Mac1 mutants predicted to eliminate both binding and hydrolysis activities were unrecoverable, suggesting that the combined activities of Mac1 may be essential for MHV replication. We conclude that Mac1 has multiple roles in promoting the replication of MHV, and that these results provide further evidence that Mac1 could be a prominent target for anti-CoV therapeutics.

scholarly journals Murine Hepatitis Virus Replicase Protein nsp10 Is a Critical Regulator of Viral RNA Synthesis

2007 ◽  
Vol 81 (12) ◽  
pp. 6356-6368 ◽  
Author(s):  
Eric F. Donaldson ◽  
Amy C. Sims ◽  
Rachel L. Graham ◽  
Mark R. Denison ◽  
Ralph S. Baric
Keyword(s):  
Rna Synthesis ◽  
Hepatitis Virus ◽  
Infectious Clone ◽  
Proteolytic Processing ◽  
Central Core ◽  
Viral Rna ◽  
Respiratory Syndrome Virus ◽  
Murine Hepatitis Virus ◽  
Mutant Phenotypes

ABSTRACT Coronavirus replication requires proteolytic processing of the large polyprotein encoded by ORF1a/ab into putative functional intermediates and eventually ∼15 mature proteins. The C-terminal ORF1a protein nsp10 colocalizes with viral replication complexes, but its role in transcription/replication is not well defined. To investigate the role of nsp10 in coronavirus transcription/replication, alanine replacements were engineered into a murine hepatitis virus (MHV) infectious clone in place of conserved residues in predicted functional domains or charged amino acid pairs/triplets, and rescued viruses were analyzed for mutant phenotypes. Of the 16 engineered clones, 5 viable viruses were rescued, 3 mutant viruses generated no cytopathic effect but were competent to synthesize viral subgenomic RNAs, and 8 were not viable. All viable mutants showed reductions in growth kinetics and overall viral RNA synthesis, implicating nsp10 as being a cofactor in positive- or negative-strand synthesis. Viable mutant nsp10-E2 was compromised in its ability to process the nascent polyprotein, as processing intermediates were detected in cells infected with this virus that were not detectable in wild-type infections. Mapping the mutations onto the crystal structure of severe acute respiratory syndrome virus nsp10 identified a central core resistant to mutation. Mutations targeting residues in or near either zinc-binding finger generated nonviable phenotypes, demonstrating that both domains are essential to nsp10 function and MHV replication. All mutations resulting in viable phenotypes mapped to loops outside the central core and were characterized by a global decrease in RNA synthesis. These results demonstrate that nsp10 is a critical regulator of coronavirus RNA synthesis and may play an important role in polyprotein processing.

scholarly journals E2f3 is critical for normal cellular proliferation

2000 ◽  
Vol 14 (6) ◽  
pp. 690-703 ◽  
Author(s):  
Patrick O. Humbert ◽  
Raluca Verona ◽  
Jeffrey M. Trimarchi ◽  
Catherine Rogers ◽  
Savita Dandapani ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Cellular Proliferation ◽  
Threshold Level ◽  
Critical Threshold ◽  
Cellular Immortalization ◽  
Proliferation And Differentiation ◽  
Rate Limiting ◽  
Deficient Mice

E2F is a family of transcription factors that regulate both cellular proliferation and differentiation. To establish the role of E2F3 in vivo, we generated an E2f3 mutant mouse strain. E2F3-deficient mice arise at one-quarter of the expected frequency, demonstrating that E2F3 is important for normal development. To determine the molecular consequences of E2F3 deficiency, we analyzed the properties of embryonic fibroblasts derived from E2f3 mutant mice. Mutation of E2f3 dramatically impairs the mitogen-induced, transcriptional activation of numerous E2F-responsive genes. We have been able to identify a number of genes, including B-myb,cyclin A, cdc2, cdc6, and DHFR, whose expression is dependent on the presence of E2F3 but not E2F1. We further show that a critical threshold level of one or more of the E2F3-regulated genes determines the timing of the G1/S transition, the rate of DNA synthesis, and thereby the rate of cellular proliferation. Finally, we show that E2F3 is not required for cellular immortalization but is rate limiting for the proliferation of the resulting tumor cell lines. We conclude that E2F3 is critical for the transcriptional activation of genes that control the rate of proliferation of both primary and tumor cells.

scholarly journals Severe Acute Respiratory Syndrome Coronavirus Replication Is Severely Impaired by MG132 due to Proteasome-Independent Inhibition of M-Calpain

2012 ◽  
Vol 86 (18) ◽  
pp. 10112-10122 ◽  
Author(s):  
Martha Schneider ◽  
Kerstin Ackermann ◽  
Melissa Stuart ◽  
Claudia Wex ◽  
Ulrike Protzer ◽  
...  
Keyword(s):  
Life Cycle ◽  
Severe Acute Respiratory Syndrome ◽  
Cysteine Protease ◽  
Hepatitis Virus ◽  
Ubiquitin Proteasome System ◽  
Murine Hepatitis Virus ◽  
Angiotensin Converting Enzyme 2 ◽  
Ubiquitin Proteasome ◽  
Calpain Inhibition

The ubiquitin-proteasome system (UPS) is involved in the replication of a broad range of viruses. Since replication of the murine hepatitis virus (MHV) is impaired upon proteasomal inhibition, the relevance of the UPS for the replication of the severe acute respiratory syndrome coronavirus (SARS-CoV) was investigated in this study. We demonstrate that the proteasomal inhibitor MG132 strongly inhibits SARS-CoV replication by interfering with early steps of the viral life cycle. Surprisingly, other proteasomal inhibitors (e.g., lactacystin and bortezomib) only marginally affected viral replication, indicating that the effect of MG132 is independent of proteasomal impairment. Induction of autophagy by MG132 treatment was excluded from playing a role, and no changes in SARS-CoV titers were observed during infection of wild-type or autophagy-deficient ATG5−/−mouse embryonic fibroblasts overexpressing the human SARS-CoV receptor, angiotensin-converting enzyme 2 (ACE2). Since MG132 also inhibits the cysteine protease m-calpain, we addressed the role of calpains in the early SARS-CoV life cycle using calpain inhibitors III (MDL28170) and VI (SJA6017). In fact, m-calpain inhibition with MDL28170 resulted in an even more pronounced inhibition of SARS-CoV replication (>7 orders of magnitude) than did MG132. Additional m-calpain knockdown experiments confirmed the dependence of SARS-CoV replication on the activity of the cysteine protease m-calpain. Taken together, we provide strong experimental evidence that SARS-CoV has unique replication requirements which are independent of functional UPS or autophagy pathways compared to other coronaviruses. Additionally, this work highlights an important role for m-calpain during early steps of the SARS-CoV life cycle.

scholarly journals A Sensitivity Analysis on the Consequences Assessment in Cases of Toxic Substance Released by Pipelines

2008 ◽  
Vol 4 (1) ◽  
pp. 39-56
Author(s):  
Gagliardi Valentina ◽  
Citro Lucia
Keyword(s):  
Sensitivity Analysis ◽  
Toxic Substance ◽  
Threshold Level ◽  
Critical Threshold ◽  
Toxic Substances ◽  
Threshold Values ◽  
Hazard Area ◽  
The Impact ◽  
Harmful Effects

A Sensitivity Analysis on the Consequences Assessment in Cases of Toxic Substance Released by Pipelines This paper presents preliminary results of a study aimed at evaluating the impact on man and environment of major accident hazards related to transmission pipelines carrying toxic substances. In particular, it describes the assessment of the consequences of potential toxic release from pipelines, expressed in terms of hazard area, that is the zone in which the concentration of a toxic substance exceed a critical threshold level and induce harmful effects on people and the environment. Moreover, a sensitivity analysis has been undertaken, emphasizing the role of threshold values on the results obtained.

Role of intracellular calcium in hydrogen peroxide-induced renal tubular cell injury

1992 ◽  
Vol 263 (2) ◽  
pp. F214-F221 ◽  
Author(s):  
N. Ueda ◽  
S. V. Shah
Keyword(s):  
Trypan Blue ◽  
Cell Injury ◽  
Tubular Cell ◽  
Renal Tubular Cell ◽  
Reactive Oxygen Metabolites ◽  
Acetoxymethyl Ester ◽  
Trypan Blue Exclusion ◽  
Renal Tubular Cells ◽  
Renal Tubular

Both reactive oxygen metabolites and calcium have been implicated in ischemic and toxic renal tubular cell injury. However, the role of calcium in oxidant injury to renal tubular cells has not been previously examined. In the present study we examined the role of intracellular free Ca2+ ([Ca2+]i) in H2O2-mediated injury to LLC-PK1 cells, a renal tubular epithelial cell line. H2O2 induced a significant rise in [Ca2+]i within 1 min after exposure of cells to 5 mM H2O2, with a sustained rise in [Ca2+]i during the course of experiments, reaching a value of 1.3 microM at 60 min (n = 10). The rise in [Ca2+]i preceded sublethal cell injury as measured by [3H]adenine release or irreversible cell injury as determined by trypan blue exclusion. Buffering [Ca2+]i with quin-2 (50 microM) and 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 50 microM) was highly protective against the H2O2-induced cell injury. For example, at 120 min after exposure of cells to 5 mM H2O2, irreversible cell injury was reduced from 45 +/- 8 to 9 +/- 1% (n = 3) by quin-2. The acetoxymethyl ester of quin-2 (quin-2/AM) and BAPTA/AM did not interfere with the trypan blue exclusion assay or scavenge H2O2. Preventing mobilization of Ca2+ from intracellular storage sites using 8-(N,N-dimethylamino)octyl 3,4,5-trimethoxybenzoate hydrochloride (TMB-8, 10(-4) M) significantly reduced the rise in [Ca2+]i and thus prevented H2O2-mediated cytotoxicity to LLC-PK1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

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