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.

Auxin and Cell Wall Crosstalk as Revealed by the Arabidopsis thaliana Cellulose Synthase Mutant Radially Swollen 1

2019 ◽  
Vol 60 (7) ◽  
pp. 1487-1503 ◽  
Author(s):  
Thiel A. Lehman ◽  
Karen A Sanguinet
Keyword(s):  
Arabidopsis Thaliana ◽  
Cell Wall ◽  
Cellulose Synthase ◽  
Cellulose Microfibrils ◽  
Single Amino Acid ◽  
Cell Wall Biosynthesis ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Anisotropic Expansion ◽  
Single Amino Acid Change

AbstractPlant cells sheath themselves in a complex lattice of polysaccharides, proteins and enzymes forming an integral matrix known as the cell wall. Cellulose microfibrils, the primary component of cell walls, are synthesized at the plasma membrane by CELLULOSE SYNTHASE A (CESA) proteins throughout cellular growth and are responsible for turgor-driven anisotropic expansion. Associations between hormone signaling and cell wall biosynthesis have long been suggested, but recently direct links have been found revealing hormones play key regulatory roles in cellulose biosynthesis. The radially swollen 1 (rsw1) allele of Arabidopsis thaliana CESA1 harbors a single amino acid change that renders the protein unstable at high temperatures. We used the conditional nature of rsw1 to investigate how auxin contributes to isotropic growth. We found that exogenous auxin treatment reduces isotropic swelling in rsw1 roots at the restrictive temperature of 30�C. We also discovered decreases in auxin influx between rsw1 and wild-type roots via confocal imaging of AUX1-YFP, even at the permissive temperature of 19�C. Moreover, rsw1 displayed mis-expression of auxin-responsive and CESA genes. Additionally, we found altered auxin maxima in rsw1 mutant roots at the onset of swelling using DII-VENUS and DR5:vYFP auxin reporters. Overall, we conclude disrupted cell wall biosynthesis perturbs auxin transport leading to altered auxin homeostasis impacting both anisotropic and isotropic growth that affects overall root morphology.

scholarly journals A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3

2009 ◽  
Vol 206 (12) ◽  
pp. 2747-2760 ◽  
Author(s):  
Masahiro Yamamoto ◽  
Daron M. Standley ◽  
Seiji Takashima ◽  
Hiroyuki Saiga ◽  
Megumi Okuyama ◽  
...  
Keyword(s):  
Amino Acid ◽  
Toxoplasma Gondii ◽  
Immune Responses ◽  
Mammalian Cells ◽  
Single Amino Acid ◽  
Type I ◽  
Dependent Manner ◽  
Type Ii ◽  
Enhanced Production ◽  
Stat3 Phosphorylation

Infection by Toxoplasma gondii down-regulates the host innate immune responses, such as proinflammatory cytokine production, in a Stat3-dependent manner. A forward genetic approach recently demonstrated that the type II strain fails to suppress immune responses because of a potential defect in a highly polymorphic parasite-derived kinase, ROP16. We generated ROP16-deficient type I parasites by reverse genetics and found a severe defect in parasite-induced Stat3 activation, culminating in enhanced production of interleukin (IL) 6 and IL-12 p40 in the infected macrophages. Furthermore, overexpression of ROP16 but not ROP18 in mammalian cells resulted in Stat3 phosphorylation and strong activation of Stat3-dependent promoters. In addition, kinase-inactive ROP16 failed to activate Stat3. Comparison of type I and type II ROP16 revealed that a single amino acid substitution in the kinase domain determined the strain difference in terms of Stat3 activation. Moreover, ROP16 bound Stat3 and directly induced phosphorylation of this transcription factor. These results formally establish an essential and direct requirement of ROP16 in parasite-induced Stat3 activation and the significance of a single amino acid replacement in the function of type II ROP16.

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 Molecular Basis of Replication of Duck H5N1 Influenza Viruses in a Mammalian Mouse Model

2005 ◽  
Vol 79 (18) ◽  
pp. 12058-12064 ◽  
Author(s):  
Zejun Li ◽  
Hualan Chen ◽  
Peirong Jiao ◽  
Guohua Deng ◽  
Guobin Tian ◽  
...  
Keyword(s):  
Amino Acid ◽  
Host Range ◽  
Reverse Genetics ◽  
Southern China ◽  
Single Gene ◽  
Influenza Viruses ◽  
Single Amino Acid ◽  
Species Barrier ◽  
H5n1 Influenza ◽  
The One

ABSTRACT We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice. A/duck/Guangxi/22/2001 (DKGX/22) is nonpathogenic in mice, whereas A/duck/Guangxi/35/2001 (DKGX/35) is highly pathogenic. We used reverse genetics to create a series of single-gene recombinants that contained one gene from DKGX/22 and the remaining seven gene segments from DKGX/35. We find that the PA, NA, and NS genes of DKGX/22 could attenuate DKGX/35 virus to some extent, but PB2 of DKGX/22 virus attenuated the DKGX/35 virus dramatically, and an Asn-to-Asp substitution at position 701 of PB2 plays a key role in this function. Conversely, of the recombinant viruses in the DKGX/22 background, only the one that contains the PB2 gene of DKGX/35 was able to replicate in mice. A single amino acid substitution (Asp to Asn) at position 701 of PB2 enabled DKGX/22 to infect and become lethal for mice. These results demonstrate that amino acid Asn 701 of PB2 is one of the important determinants for this avian influenza virus to cross the host species barrier and infect mice, though the replication and lethality of H5N1 influenza viruses involve multiple genes and may result from a constellation of genes. Our findings may help to explain the expansion of the host range and lethality of the H5N1 influenza viruses to humans.

scholarly journals Utilizing fowlpox virus recombinants to generate defective RNAs of the coronavirus infectious bronchitis virus

2000 ◽  
Vol 81 (12) ◽  
pp. 2855-2865 ◽  
Author(s):  
Sharon Evans ◽  
David Cavanagh ◽  
Paul Britton
Keyword(s):  
Infectious Bronchitis Virus ◽  
Mammalian Cells ◽  
Reverse Genetics ◽  
Leader Sequence ◽  
Dependent Manner ◽  
T7 Promoter ◽  
Fowlpox Virus ◽  
Infectious Bronchitis ◽  
Defective Rnas

Coronavirus defective RNAs (D-RNAs) have been used as RNA vectors for the expression of heterologous genes and as vehicles for reverse genetics by modifying coronavirus genomes by targetted recombination. D-RNAs based on the avian coronavirus infectious bronchitis virus (IBV) D-RNA CD-61 have been rescued (replicated and packaged into virions) in a helper virus-dependent manner following electroporation of in vitro-generated T7 transcripts into IBV-infected cells. In order to increase the efficiency of rescue of IBV D-RNAs, cDNAs based on CD-61, under the control of a T7 promoter, were integrated into the fowlpox virus (FPV) genome. The 3′-UTR of the D-RNAs was flanked by a hepatitis delta antigenomic ribozyme and T7 terminator sequence to generate suitable 3′ ends for rescue by helper IBV. Cells were co-infected simultaneously with IBV, the recombinant FPV (rFPV) containing the D-RNA sequence and a second rFPV expressing T7 RNA polymerase for the initial expression of the D-RNA transcript, subsequently rescued by helper IBV. Rescue of rFPV-derived CD-61 occurred earlier and with higher efficiency than demonstrated previously for electroporation of in vitro T7-generated RNA transcripts in avian cells. Rescue of CD-61 was also demonstrated for the first time in mammalian cells. The rescue of rFPV-derived CD-61 by M41 helper IBV resulted in leader switching, in which the Beaudette-type leader sequence on CD-61 was replaced with the M41 leader sequence, confirming that helper IBV virus replicated the rFPV-derived D-RNA. An rFPV-derived D-RNA containing the luciferase gene under the control of an IBV transcription-associated sequence was also rescued and expressed luciferase on serial passage.

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 Influenza A Viruses with Mutations in the M1 Helix Six Domain Display a Wide Variety of Morphological Phenotypes

2005 ◽  
Vol 79 (2) ◽  
pp. 1262-1270 ◽  
Author(s):  
Laura M. Burleigh ◽  
Lesley J. Calder ◽  
John J. Skehel ◽  
David A. Steinhauer
Keyword(s):  
Nuclear Export ◽  
Influenza A ◽  
Matrix Protein ◽  
Reverse Genetics ◽  
Virus Assembly ◽  
Single Amino Acid ◽  
Dependent Manner ◽  
Influenza A Viruses ◽  
Binding Function ◽  
Wide Range

ABSTRACT Several functions required for the replication of influenza A viruses have been attributed to the viral matrix protein (M1), and a number of studies have focused on a region of the M1 protein designated “helix six.” This region contains an exposed positively charged stretch of amino acids, including the motif 101-RKLKR-105, which has been identified as a nuclear localization signal, but several studies suggest that this domain is also involved in functions such as binding to the ribonucleoprotein genome segments (RNPs), membrane association, interaction with the viral nuclear export protein, and virus assembly. In order to define M1 functions in more detail, a series of mutants containing alanine substitutions in the helix six region were generated in A/WSN/33 virus. These were analyzed for RNP-binding function, their capacity to incorporate into infectious viruses by using reverse genetics, the replication properties of rescued viruses, and the morphological phenotypes of the mutant virus particles. The most notable effect that was identified concerned single amino acid substitution mutants that caused significant alterations to the morphology of budded viruses. Whereas A/WSN/33 virus generally forms particles that are predominantly spherical, observations made by negative stain electron microscopy showed that several of the mutant virions, such as K95A, K98A, R101A, and K102A, display a wide range of shapes and sizes that varied in a temperature-dependent manner. The K102A mutant is particularly interesting in that it can form extended filamentous particles. These results support the proposition that the helix six domain is involved in the process of virus assembly.

scholarly journals Filovirus receptor NPC1 contributes to species-specific patterns of ebolavirus susceptibility in bats

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Melinda Ng ◽  
Esther Ndungo ◽  
Maria E Kaczmarek ◽  
Andrew S Herbert ◽  
Tabea Binger ◽  
...  
Keyword(s):  
Host Range ◽  
Ebola Virus ◽  
Viral Evolution ◽  
Single Amino Acid ◽  
Mammalian Cell Lines ◽  
Single Amino Acid Change ◽  
Ebov Infection ◽  
Species Specific ◽  
Receptor Interface ◽  
Eidolon Helvum

Biological factors that influence the host range and spillover of Ebola virus (EBOV) and other filoviruses remain enigmatic. While filoviruses infect diverse mammalian cell lines, we report that cells from African straw-colored fruit bats (Eidolon helvum) are refractory to EBOV infection. This could be explained by a single amino acid change in the filovirus receptor, NPC1, which greatly reduces the affinity of EBOV-NPC1 interaction. We found signatures of positive selection in bat NPC1 concentrated at the virus-receptor interface, with the strongest signal at the same residue that controls EBOV infection in Eidolon helvum cells. Our work identifies NPC1 as a genetic determinant of filovirus susceptibility in bats, and suggests that some NPC1 variations reflect host adaptations to reduce filovirus replication and virulence. A single viral mutation afforded escape from receptor control, revealing a pathway for compensatory viral evolution and a potential avenue for expansion of filovirus host range in nature.

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.

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