scholarly journals Specificity of resistance and tolerance to cucumber vein yellowing virus in melon accessions and evidence for resistance breaking associated with a single mutation in VPg

2021 ◽  
Author(s):  
Cecile Desbiez ◽  
Maria Luisa Domingo Calap ◽  
Michel Pitrat ◽  
Catherine Wipf-Scheibel ◽  
Gregory Girardot ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Cultural Practices ◽  
Durable Resistance ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Coding Region ◽  
Resistance Breaking ◽  
Single Amino Acid Change ◽  
Emerging Virus ◽  
Yellowing Virus

Cucumber vein yellowing virus (CVYV) is an emerging virus on cucurbits in the Mediterranean Basin, against which few resistance sources are available, particularly in melon. The melon accession PI 164323 displays complete resistance to isolate CVYV-Esp, and accession HSD 2458 presents a tolerance, i.e. very mild symptoms in spite of virus accumulation in inoculated plants. The resistance is controlled by a dominant allele Cvy-11, while the tolerance is controlled by a recessive allele cvy-2, independent from Cvy-11. Before introducing the resistance or tolerance in commercial cultivars through a long breeding process, it is important to estimate their specificity and durability. Upon inoculation with eight molecularly diverse CVYV isolates, the resistance was found to be isolate-specific since many CVYV isolates induced necrosis on PI 164323, whereas the tolerance presented a broader range. A resistance-breaking isolate inducing severe mosaic on PI 164323 was obtained. This isolate differed from the parental strain by a single amino acid change in the VPg coding region. An infectious CVYV cDNA clone was obtained, and the effect of the mutation in the VPg cistron on resistance to PI 164323 was confirmed by reverse genetics. This represents the first determinant for resistance-breaking in an ipomovirus. Our results indicate that the use of the Cvy-11 allele alone will not provide durable resistance to CVYV and that, if used in the field, it should be combined with other control methods such as cultural practices and pyramiding of resistance genes to achieve long-lasting resistance against CVYV.

scholarly journals Emergence of a Large-Plaque Variant in Mice Infected with Coxsackievirus B3

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Yao Wang ◽  
Julie K. Pfeiffer
Keyword(s):  
Cell Culture ◽  
Viral Replication ◽  
Coxsackievirus B3 ◽  
Plaque Formation ◽  
Single Amino Acid ◽  
Growth Defect ◽  
Single Mutation ◽  
Wild Type ◽  
Large Plaque ◽  
Single Amino Acid Change

ABSTRACTCoxsackieviruses are enteric viruses that frequently infect humans. To examine coxsackievirus pathogenesis, we orally inoculated mice with the coxsackievirus B3 (CVB3) Nancy strain. Using HeLa cell plaque assays with agar overlays, we noticed that some fecal viruses generated plaques >100 times as large as inoculum viruses. These large-plaque variants emerged following viral replication in several different tissues. We identified a single amino acid change, N63Y, in the VP3 capsid protein that was sufficient to confer the large-plaque phenotype. Wild-type CVB3 and N63Y mutant CVB3 had similar plaque sizes when agarose was used in the overlay instead of agar. We determined that sulfated glycans in agar inhibited plaque formation by wild-type CVB3 but not by N63Y mutant CVB3. Furthermore, N63Y mutant CVB3 bound heparin, a sulfated glycan, less efficiently than wild-type CVB3 did. While N63Y mutant CVB3 had a growth defect in cultured cells and reduced attachment, it had enhanced replication and pathogenesis in mice. Infection with N63Y mutant CVB3 induced more severe hepatic damage than infection with wild-type CVB3, likely because N63Y mutant CVB3 disseminates more efficiently to the liver. Our data reinforce the idea that culture-adapted laboratory virus strains can have reduced fitnessin vivo. N63Y mutant CVB3 may be useful as a platform to understand viral adaptation and pathogenesis in animal studies.IMPORTANCECoxsackieviruses frequently infect humans, and although many infections are mild or asymptomatic, there can be severe outcomes, including heart inflammation. Most studies with coxsackieviruses and other viruses use laboratory-adapted viral strains because of their efficient replication in cell culture. We used a cell culture-adapted strain of CVB3, Nancy, to examine viral replication and pathogenesis in orally inoculated mice. We found that mice shed viruses distinct from input viruses because they formed extremely large plaques in cell culture. We identified a single mutation, VP3 N63Y, that was sufficient for large-plaque formation. N63Y mutant viruses have reduced glycan binding and replication in cell culture; however, they have enhanced replication and virulence in mice. We are now using N63Y mutant CVB3 as an improved system for viral pathogenesis studies.

scholarly journals Structural Determinants for Antitoxin Identity and Insulation of Cross Talk between Homologous Toxin-Antitoxin Systems

2016 ◽  
Vol 198 (24) ◽  
pp. 3287-3295 ◽  
Author(s):  
Lauren R. Walling ◽  
J. Scott Butler
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Antibiotic Treatment ◽  
Bacterial Growth ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Bacterial Persistence ◽  
Content Type ◽  
Single Amino Acid Change ◽  
Specificity Determinants

ABSTRACT Toxin-antitoxin (TA) systems are ubiquitous in bacteria and archaea, where they play a pivotal role in the establishment and maintenance of dormancy. Under normal growth conditions, the antitoxin neutralizes the toxin. However, under conditions of stress, such as nutrient starvation or antibiotic treatment, cellular proteases degrade the antitoxin, and the toxin functions to arrest bacterial growth. We characterized the specificity determinants of the interactions between VapB antitoxins and VapC toxins from nontypeable Haemophilus influenzae (NTHi) in an effort to gain a better understanding of how antitoxins control toxin activity and bacterial persistence. We studied truncated and full-length antitoxins with single amino acid mutations in the toxin-binding domain. Coexpressing the toxin and antitoxin in Escherichia coli and measuring bacterial growth by dilution plating assayed the ability of the mutant antitoxins to neutralize the toxin. Our results identified two single amino acid residues (W48 and F52) in the C-terminal region of the VapB2 antitoxin necessary for its ability to neutralize its cognate VapC2 toxin. Additionally, we attempted to alter the specificity of VapB1 by making a mutation that would allow it to neutralize its noncognate toxin. A mutation in VapB1 to contain the tryptophan residue identified herein as important in the VapB2-VapC2 interaction resulted in a VapB1 mutant (the T47W mutant) that binds to and neutralizes both its cognate VapC1 and noncognate VapC2 toxins. This represents the first example of a single mutation causing relaxed specificity in a type II antitoxin. IMPORTANCE Toxin-antitoxin systems are of particular concern in pathogenic organisms, such as nontypeable Haemophilus influenzae , as they can elicit dormancy and persistence, leading to chronic infections and failure of antibiotic treatment. Despite the importance of the TA interaction, the specificity determinants for VapB-VapC complex formation remain uncharacterized. Thus, our understanding of how antitoxins control toxin-induced dormancy and bacterial persistence requires thorough investigation of antitoxin specificity for its cognate toxin. This study characterizes the crucial residues of the VapB2 antitoxin from NTHi necessary for its interaction with VapC2 and provides the first example of a single amino acid change altering the toxin specificity of an antitoxin.

scholarly journals A Point Mutation in the Rhesus Rotavirus VP4 Protein Generated through a Rotavirus Reverse Genetics System Attenuates Biliary Atresia in the Murine Model

2017 ◽  
Vol 91 (15) ◽  
Author(s):  
Sujit K. Mohanty ◽  
Bryan Donnelly ◽  
Phylicia Dupree ◽  
Inna Lobeck ◽  
Sarah Mowery ◽  
...  
Keyword(s):  
Amino Acid ◽  
Biliary Atresia ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Wild Type ◽  
Neonatal Mice ◽  
Single Amino Acid Change

ABSTRACT Rotavirus infection is one of the most common causes of diarrheal illness in humans. In neonatal mice, rhesus rotavirus (RRV) can induce biliary atresia (BA), a disease resulting in inflammatory obstruction of the extrahepatic biliary tract and intrahepatic bile ducts. We previously showed that the amino acid arginine (R) within the sequence SRL (amino acids 445 to 447) in the RRV VP4 protein is required for viral binding and entry into biliary epithelial cells. To determine if this single amino acid (R) influences the pathogenicity of the virus, we generated a recombinant virus with a single amino acid mutation at this site through a reverse genetics system. We demonstrated that the RRV mutant (RRVVP4-R446G) produced less symptomatology and replicated to lower titers both in vivo and in vitro than those seen with wild-type RRV, with reduced binding in cholangiocytes. Our results demonstrate that a single amino acid change in the RRV VP4 gene influences cholangiocyte tropism and reduces pathogenicity in mice. IMPORTANCE Rotavirus is the leading cause of diarrhea in humans. Rhesus rotavirus (RRV) can also lead to biliary atresia (a neonatal human disease) in mice. We developed a reverse genetics system to create a mutant of RRV (RRVVP4-R446G) with a single amino acid change in the VP4 protein compared to that of wild-type RRV. In vitro, the mutant virus had reduced binding and infectivity in cholangiocytes. In vivo, it produced fewer symptoms and lower mortality in neonatal mice, resulting in an attenuated form of biliary atresia.

scholarly journals A Single Amino Acid Change in Viral Genome-Associated Protein of Potato Virus Y Correlates with Resistance Breaking in ‘Virgin A Mutant’ Tobacco

1999 ◽  
Vol 89 (2) ◽  
pp. 118-123 ◽  
Author(s):  
Chikara Masuta ◽  
Mitsuyo Nishimura ◽  
Hiroshi Morishita ◽  
Tatsuji Hataya
Keyword(s):  
Amino Acid ◽  
Potato Virus ◽  
Viral Genome ◽  
Potato Virus Y ◽  
Cell Movement ◽  
Amino Acid Sequences ◽  
Single Amino Acid ◽  
Viral Gene ◽  
Resistance Breaking ◽  
Single Amino Acid Change

Tobacco cultivar Virgin A Mutant (VAM) is reported to have the recessive potyvirus resistance gene va. Varied levels of resistance were observed in VAM plants inoculated with Japanese potato virus Y (PVY) isolates. VAM was highly resistant to most of the PVY isolates tested and tolerant to three necrotic strain isolates of PVY-T. Based on data obtained from tissue printing and press blotting, the resistance appeared to be mainly at the level of cell-to-cell movement. PVY replicated in VAM proto-plasts, but the replication was 30% lower than in susceptible tobacco, suggesting that impairment of replication also contributes to resistance. To identify the viral gene product or products involved in VAM resistance, we isolated spontaneous resistance-breaking mutants by passing vein-banding (O strain) isolates several times through VAM plants. By comparing the amino acid sequences of the mutants with their original isolates, we identified a single amino acid substitution in the viral genome-associated protein (VPg) domain that is correlated with VAM resistance breaking. Together, these results suggest that, in addition to its role in replication, VPg plays an important role in the cell-to-cell movement of PVY.

scholarly journals The host-range tdCE phenotype of Chandipura virus is determined by mutations in the polymerase gene

2014 ◽  
Vol 95 (1) ◽  
pp. 38-43
Author(s):  
Emily L. Stock ◽  
Anthony C. Marriott ◽  
Andrew J. Easton
Keyword(s):  
Host Range ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Single Amino Acid ◽  
Permissive Temperature ◽  
Dependent Manner ◽  
Polymerase Gene ◽  
Temperature Dependent ◽  
Chandipura Virus ◽  
Single Amino Acid Change

The emerging arbovirus Chandipura virus (CV) has been implicated in epidemics of acute encephalitis in India with high mortality rates. The isolation of temperature-dependent host-range (tdCE) mutants, which are impaired in growth at 39 °C in chick embryo (CE) cells but not in monkey cells, highlights a dependence on undetermined host factors. We have characterized three tdCE mutants, each containing one or more coding mutations in the RNA polymerase gene and two containing additional mutations in the attachment protein gene. Using reverse genetics, we showed that a single amino acid change in the virus polymerase of each mutant was responsible for the host-range specificity. In CE cells at the non-permissive temperature, the discrete cytoplasmic replication complexes seen in mammalian cells or at the permissive temperature in CE cells were absent with the tdCE mutants, consistent with the tdCE lesions causing disruption of the replication complexes in a host-dependent manner.

scholarly journals A single mutation increases heavy-chain heterodimer assembly of bispecific antibodies by inducing structural disorder in one homodimer species

2020 ◽  
Vol 295 (28) ◽  
pp. 9392-9408
Author(s):  
Cian Stutz ◽  
Stanislas Blein
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Heavy Chain ◽  
Cell Receptor ◽  
Bispecific Antibodies ◽  
Single Amino Acid ◽  
Minimal Risk ◽  
Single Mutation ◽  
Protein Interface ◽  
Single Amino Acid Change

We previously reported efficient heavy-chain assembly of heterodimeric bispecific antibodies by exchanging the interdomain protein interface of the human IgG1 CH3 dimer with the protein interface of the constant α and β domains of the human T-cell receptor, a technology known as bispecific engagement by antibodies based on the T-cell receptor (BEAT). Efficient heterodimerization in mammalian cell transient transfections was observed, but levels were influenced by the nature of the binding arms, particularly in the Fab-scFv-Fc format. In this study, we report a single amino acid change that significantly and consistently improved the heterodimerization rate of this format (≥95%) by inducing partial disorder in one homodimer species without affecting the heterodimer. Correct folding and assembly of the heterodimer were confirmed by the high-resolution (1.88–1.98 Å) crystal structure presented here. Thermal stability and 1-anilinonaphthalene-8-sulfonic acid–binding experiments, comparing original BEAT, mutated BEAT, and “knobs-into-holes” interfaces, suggested a cooperative assembly process of heavy chains in heterodimers. The observed gain in stability of the interfaces could be classified in the following rank order: mutated BEAT > original BEAT > knobs-into-holes. We therefore propose that the superior cooperativity found in BEAT interfaces is the key driver of their greater performance. Furthermore, we show how the mutated BEAT interface can be exploited for the routine preparation of drug candidates, with minimal risk of homodimer contamination using a single Protein A chromatography step.

scholarly journals A Single Coxsackievirus B2 Capsid Residue Controls Cytolysis and Apoptosis in Rhabdomyosarcoma Cells

2010 ◽  
Vol 84 (12) ◽  
pp. 5868-5879 ◽  
Author(s):  
Maria Gullberg ◽  
Conny Tolf ◽  
Nina Jonsson ◽  
Charlotta Polacek ◽  
Jana Precechtelova ◽  
...  
Keyword(s):  
Reverse Genetics ◽  
Wide Spectrum ◽  
Prototype Strain ◽  
Single Amino Acid ◽  
Human Pathogens ◽  
Adaptive Mutations ◽  
Single Amino Acid Change ◽  
Rd Cells ◽  
Group B ◽  
Induced Apoptosis

ABSTRACT Coxsackievirus B2 (CVB2), one of six human pathogens of the group B coxsackieviruses within the enterovirus genus of Picornaviridae, causes a wide spectrum of human diseases ranging from mild upper respiratory illnesses to myocarditis and meningitis. The CVB2 prototype strain Ohio-1 (CVB2O) was originally isolated from a patient with summer grippe in the 1950s. Later on, CVB2O was adapted to cytolytic replication in rhabdomyosarcoma (RD) cells. Here, we present analyses of the correlation between the adaptive mutations of this RD variant and the cytolytic infection in RD cells. Using reverse genetics, we identified a single amino acid change within the exposed region of the VP1 protein (glutamine to lysine at position 164) as the determinant for the acquired cytolytic trait. Moreover, this cytolytic virus induced apoptosis, including caspase activation and DNA degradation, in RD cells. These findings contribute to our understanding of the host cell adaptation process of CVB2O and provide a valuable tool for further studies of virus-host interactions.

scholarly journals Inhibition of IRF-3 Activation by VP35 Is Critical for the High Level of Virulence of Ebola Virus

2008 ◽  
Vol 82 (6) ◽  
pp. 2699-2704 ◽  
Author(s):  
Amy L. Hartman ◽  
Brian H. Bird ◽  
Jonathan S. Towner ◽  
Zoi-Anna Antoniadou ◽  
Sherif R. Zaki ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Change ◽  
Reverse Genetics ◽  
Ebola Virus ◽  
Single Amino Acid ◽  
Wild Type Virus ◽  
Hemorrhagic Disease ◽  
Viral Virulence ◽  
Single Amino Acid Change ◽  
High Level

ABSTRACT Zaire ebolavirus causes a rapidly progressing hemorrhagic disease with high mortality. Identification of the viral virulence factors that contribute to the severity of disease induced by Ebola virus is critical for the design of therapeutics and vaccines against the disease. Given the rapidity of disease progression, virus interaction with the innate immune system early in the course of infection likely plays an important role in determining the outcome of the disease. The Ebola virus VP35 protein inhibits the activation of IRF-3, a critical transcription factor for the induction of early antiviral immunity. Previous studies revealed that a single amino acid change (R312A) in VP35 renders the protein unable to inhibit IRF-3 activation. A reverse-genetics-generated, mouse-adapted, recombinant Ebola virus that encodes the R312A mutation in VP35 was produced. We found that relative to the case for wild-type virus containing the authentic VP35 sequence, this single amino acid change in VP35 renders the virus completely attenuated in mice. Given that these viruses differ by only a single amino acid in the IRF-3 inhibitory domain of VP35, the level of alteration of virulence is remarkable and highlights the importance of VP35 for the pathogenesis of Ebola virus.

scholarly journals A Single Amino Acid Change in the Marburg Virus Glycoprotein Arises during Serial Cell Culture Passages and Attenuates the Virus in a Macaque Model of Disease

mSphere ◽  
2018 ◽  
Vol 3 (1) ◽  
Author(s):  
Kendra J. Alfson ◽  
Laura E. Avena ◽  
Jenny Delgado ◽  
Michael W. Beadles ◽  
Jean L. Patterson ◽  
...  
Keyword(s):  
Cell Culture ◽  
Cynomolgus Macaque ◽  
Case Fatality ◽  
Marburg Virus ◽  
Single Amino Acid ◽  
Single Mutation ◽  
Macaque Model ◽  
Culture Passage ◽  
Single Amino Acid Change ◽  
Cell Culture Passage

Marburg virus (MARV) causes disease with a high case fatality rate, and there are no approved vaccines or therapies. Serial amplification of viruses in cell culture often results in accumulation of mutations, but the effect of such cell culture passage on MARV is unclear. Serial passages of MARV resulted in a single mutation in the region encoding the glycoprotein (GP). This is a region where mutations can have important consequences on outbreaks and human disease [S. Mahanty and M. Bray, Lancet Infect Dis 4:487–498, 2004,https://doi.org/10.1016/S1473-3099(04)01103-X]. We thus investigated whether this mutation impacted disease by using a cynomolgus macaque model of MARV infection. Monkeys exposed to virus containing the mutation had better clinical outcomes than monkeys exposed to virus without the mutation. We also observed that a remarkably low number of MARV particles was sufficient to cause death. Our results could have a significant impact on how future studies are designed to model MARV disease and test vaccines and therapeutics.

scholarly journals Neuraminidase Inhibitor-Resistant Influenza Viruses May Differ Substantially in Fitness and Transmissibility

2005 ◽  
Vol 49 (10) ◽  
pp. 4075-4084 ◽  
Author(s):  
Hui-Ling Yen ◽  
Louise M. Herlocher ◽  
Erich Hoffmann ◽  
Mikhail N. Matrosovich ◽  
Arnold S. Monto ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Reverse Genetics ◽  
Neuraminidase Inhibitor ◽  
Influenza Viruses ◽  
Viral Fitness ◽  
Single Amino Acid ◽  
Cross Resistance ◽  
Oseltamivir Carboxylate ◽  
Sialidase Activity ◽  
Single Amino Acid Change

ABSTRACT Mutations of the conserved residues of influenza virus neuraminidase (NA) that are associated with NA inhibitor (NAI) resistance decrease the sialidase activity and/or stability of the NA, thus compromising viral fitness. In fact, clinically derived NAI-resistant variants with different NA mutations have shown different transmissibilities in ferrets (M. L. Herlocher, R. Truscon, S. Elias, H. Yen, N. A. Roberts, S. E. Ohmit, and A. S. Monto, J. Infect. Dis. 190:1627-1630, 2004). Molecular characterization of mutant viruses that have a homogeneous genetic background is required to determine the effect of single mutations at conserved NA residues. We generated recombinant viruses containing either the wild-type NA (RG WT virus) or a single amino acid change at NA residue 119 (RG E119V-NA virus) or 292 (RG R292K-NA virus) in the A/Wuhan/359/95 (H3N2) influenza virus background by reverse genetics. Both mutants showed decreased sensitivity to oseltamivir carboxylate, and the RG R292K-NA virus showed cross-resistance to zanamivir. We also observed differences between the two mutants in NA enzymatic activity and thermostability. The R292K mutation caused greater reduction of sialidase activity and thermostability than the E119V mutation. The NA defect caused by the R292K mutation was associated with compromised growth and transmissibility, whereas the growth and transmissibility of the RG E119V-NA virus were comparable to those of RG WT virus. Our results suggest that NAI-resistant influenza virus variants may differ substantially in fitness and transmissibility, depending on different levels of NA functional loss.

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