scholarly journals A Single Amino Acid Substitution in the Murine Norovirus Capsid Protein Is Sufficient for Attenuation In Vivo

2008 ◽  
Vol 82 (15) ◽  
pp. 7725-7728 ◽  
Author(s):  
D. Bailey ◽  
L. B. Thackray ◽  
I. G. Goodfellow
Keyword(s):  
Amino Acid ◽  
Capsid Protein ◽  
Single Amino Acid ◽  
Murine Norovirus ◽  
Molecular Determinant ◽  
Capsid Protein Vp1 ◽  
Attenuated Viruses ◽  
Lysine Substitution ◽  
First Time

ABSTRACT Murine norovirus (MNV), a prevalent pathogen of laboratory mice, shares many characteristics with human noroviruses. Previous results indicated that passage of MNV1 in the macrophage cell line RAW 264.7 results in attenuation in STAT1-deficient mice (C. E. Wobus, S. M. Karst, L. B. Thackray, K. O. Chang, S. V. Sosnovtsev, G. Belliot, A. Krug, J. M. Mackenzie, K. Y. Green, and H. W. Virgin, PLoS. Biol. 2:e432, 2004). Sequence analysis revealed two amino acid differences between the virulent and attenuated viruses. Using an infectious cDNA clone of the attenuated virus, we demonstrated that a glutamate-to-lysine substitution at position 296 in the capsid protein (VP1) is sufficient to restore virulence in vivo, identifying, for the first time, a virus-encoded molecular determinant of norovirus virulence.

scholarly journals A Single-Amino-Acid Substitution in the P2 Domain of VP1 of Murine Norovirus Is Sufficient for Escape from Antibody Neutralization

2007 ◽  
Vol 81 (22) ◽  
pp. 12316-12322 ◽  
Author(s):  
Vance P. Lochridge ◽  
Michele E. Hardy
Keyword(s):  
Amino Acid ◽  
Host Cells ◽  
Single Amino Acid ◽  
Carbohydrate Binding ◽  
Viral Gastroenteritis ◽  
Murine Norovirus ◽  
Norwalk Virus ◽  
Human Norovirus ◽  
Disulfide Linkage ◽  
Capsid Protein Vp1

ABSTRACT Noroviruses cause epidemic outbreaks of acute viral gastroenteritis worldwide, and the number of reported outbreaks is increasing. Human norovirus strains do not grow in cell culture. However, murine norovirus (MNV) replicates in the RAW 264.7 macrophage cell line and thus provides a tractable model to investigate norovirus interactions with host cells. Epitopes recognized by monoclonal antibodies (MAbs) against the human norovirus strains Norwalk virus and Snow Mountain virus (SMV) identified regions in the P domain of major capsid protein VP1 important for interactions with putative cellular receptors. To determine if there was a relationship between domains of MNV VP1 and VP1 of human norovirus strains involved in cell binding, epitope mapping by phage display was performed with an MNV-1-neutralizing MAb, A6.2.1. A consensus peptide, GWWEDHGQL, was derived from 20 third-round phage clones. A synthetic peptide containing this sequence and constrained through a disulfide linkage reacted strongly with the A6.2.1 MAb, whereas the linear sequence did not. Four residues in the A6.2.1-selected peptide, G327, G333, Q334, and L335, aligned with amino acid residues in the P2 domain of MNV-1 VP1. This sequence is immediately adjacent to the epitope recognized by anti-SMV MAb 61.21. Neutralization escape mutants selected with MAb A6.2.1 contained a leucine-to-phenylalanine substitution at position 386 in the P2 domain. The predicted location of these residues on VP1 suggests that the phage peptide and the mutation in the neutralization-resistant viruses may be in close proximity to each other and to residues reported to be important for carbohydrate binding to VP1 of human norovirus strains.

scholarly journals Site-saturation mutagenesis of the PALTAVETG motif in coxsackievirus A9 capsid protein VP1 reveals evidence of conservation of a periodic hydrophobicity profile

1999 ◽  
Vol 80 (8) ◽  
pp. 1919-1927 ◽  
Author(s):  
Antero Airaksinen ◽  
Merja Roivainen ◽  
Glyn Stanway ◽  
Tapani Hovi
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Capsid Protein ◽  
Amino Acid Substitution ◽  
Saturation Mutagenesis ◽  
Single Amino Acid ◽  
Site Saturation ◽  
Coxsackievirus A9 ◽  
Capsid Protein Vp1 ◽  
Hydrophobicity Profile

Enteroviruses possess a highly conserved 9 amino acid stretch of mainly hydrophobic character in the capsid protein VP1. A novel strategy, combining site-saturation mutagenesis and a single-tube cloning and transfection procedure, has been developed for the analysis of this motif in coxsackievirus A9 (CAV-9). Four individual amino acids were separately mutated. Mutagenesis of three of the four positions in CAV-9 resulted in a number of viable but impaired mutant strains, each containing a single amino acid substitution. In contrast, no mutants with amino acid substitutions at leucine 31 were isolated, although three different leucine codons were found among the viruses recovered. Small plaque size was regularly associated with reduced yields of infectious virus and an amino acid substitution at the target site in the viruses isolated from the site-saturated virus pools. From the range of amino acids observed in viable mutants, it was possible to estimate the characteristics that are required at individual amino acid positions. It seems that in the motif studied here, a periodic hydrophobicity profile needs to be conserved. The constraints observed on the ranges of acceptable amino acids presumably reflect the structural–functional requirements that have resulted in the conservation of the motif.

Single Amino Acid Based Self-Assemblies of Cysteine and Methionine

2018 ◽  
Author(s):  
Nidhi Gour ◽  
Bharti Koshti ◽  
Chandra Kanth P. ◽  
Dhruvi Shah ◽  
Vivek Shinh Kshatriya ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Self Assembly ◽  
Aromatic Amino Acids ◽  
Neutral Condition ◽  
Single Amino Acid ◽  
Binding Assays ◽  
Human Neuroblastoma ◽  
Cytotoxicity Assays ◽  
First Time

We report for the very first time self-assembly of Cysteine and Methionine to discrenible strucutres under neutral condition. To get insights into the structure formation, thioflavin T and Congo red binding assays were done which revealed that aggregates may not have amyloid like characteristics. The nature of interactions which lead to such self-assemblies was purported by coincubating assemblies in urea and mercaptoethanol. Further interaction of aggregates with short amyloidogenic dipeptide diphenylalanine (FF) was assessed. While cysteine aggregates completely disrupted FF fibres, methionine albeit triggered fibrillation. The cytotoxicity assays of cysteine and methionine structures were performed on Human Neuroblastoma IMR-32 cells which suggested that aggregates are not cytotoxic in nature and thus, may not have amyloid like etiology. The results presented in the manuscript are striking, since to the best of our knowledge,this is the first report which demonstrates that even non-aromatic amino acids (cysteine and methionine) can undergo spontaneous self-assembly to form ordered aggregates.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

Amino acid changes in the capsid protein of a reassortant betanodavirus strain: Effect on viral replication in vitro and in vivo

2018 ◽  
Vol 42 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Sandra Souto ◽  
José G. Olveira ◽  
Esther García-Rosado ◽  
Carlos P. Dopazo ◽  
Isabel Bandín
Keyword(s):  
Amino Acid ◽  
Viral Replication ◽  
Capsid Protein ◽  
Strain Effect

scholarly journals Single amino acid mutations effect Zika virus replication in vitro and virulence in vivo

2020 ◽  
Author(s):  
Nicole M. Collette ◽  
Victoria H.I. Lao ◽  
Dina R. Weilhammer ◽  
Barbara Zingg ◽  
Shoshana D. Cohen ◽  
...  
Keyword(s):  
Amino Acid ◽  
Zika Virus ◽  
Model Systems ◽  
Single Amino Acid ◽  
Replication Rate ◽  
Adult Mice ◽  
Naturally Occurring ◽  
Viral Virulence

AbstractThe 2014-2016 Zika virus (ZIKV) epidemic in the Americas resulted in large deposits of next-generation sequencing data from clinical samples. This resource was mined to identify emerging mutations and trends in mutations as the outbreak progressed over time. Information on transmission dynamics, prevalence and persistence of intra-host mutants, and the position of a mutation on a protein were then used to prioritize 544 reported mutations based on their ability to impact ZIKV phenotype. Using this criteria, six mutants (representing naturally occurring mutations) were generated as synthetic infectious clones using a 2015 Puerto Rican epidemic strain PRVABC59 as the parental backbone. The phenotypes of these naturally occurring variants were examined using both cell culture and murine model systems. Mutants had distinct phenotypes, including changes in replication rate, embryo death, and decreased head size. In particular, a NS2B mutant previously detected during in vivo studies in rhesus macaques was found to cause lethal infections in adult mice, abortions in pregnant females, and increased viral genome copies in both brain tissue and blood of female mice. Additionally, mutants with changes in the region of NS3 that interfaces with NS5 during replication displayed reduced replication in the blood of adult mice. This analytical pathway, integrating both bioinformatic and wet lab experiments, provides a foundation for understanding how naturally occurring single mutations affect disease outcome and can be used to predict the of severity of future ZIKV outbreaks.Author summaryTo determine if naturally occurring individual mutations in the Zika virus epidemic genotype effect viral virulence or replication rate in vitro or in vivo, we generated an infectious clone representing the epidemic genotype of stain Puerto Rico, 2015. Using this clone, six mutants were created by changing nucleotides in the genome to cause one to two amino acid substitutions in the encoded proteins. The six mutants we generated represent mutations that differentiated the early epidemic genotype from genotypes that were either ancestral or that occurred later in the epidemic. We assayed each mutant for changes in growth rate, and for virulence in adult mice and pregnant mice. Three of the mutants caused catastrophic embryo effects including increased embryonic death or significant decrease in head diameter. Three other mutants that had mutations in a genome region associated with replication resulted in changes in in vitro and in vivo replication rates. These results illustrate the potential impact of individual mutations in viral phenotype.

scholarly journals Dynamics of intravital concentration of amino acid metabolites in human brain in post-traumatic period

2019 ◽  
Vol 484 (2) ◽  
pp. 238-242
Author(s):  
N. A. Semenova ◽  
P. E. Menshchikov ◽  
A. V. Manzhurtsev ◽  
M. V. Ublinskiy ◽  
T. A. Akhadov ◽  
...  
Keyword(s):  
Traumatic Brain Injury ◽  
Amino Acid ◽  
Human Brain ◽  
Leading Role ◽  
Acid Metabolites ◽  
Post Traumatic ◽  
First Time

Intracellular concentrations of N acetyaspartate (NAA), aspartate (Asp) and glutamate (Glu) were determined for the first time in human brain in vivo, and the effect of severe traumatic brain injury on NAA synthesis in acute and late post-traumatic period was investigated. In MRI‑negative frontal lobes one day after injury Asp and Glu levels were found to decrease by 45 and 35%, respectively, while NAA level decreased by only 16%. A negative correlation between NAA concentration and the ratio of Asp/Glu concentrations was found. In the long-term period, Glu level returned to normal, Asp level remained below normal by 60%, NAA level was reduced by 65% relative to normal, and Asp/Glu ratio significantly decreased. The obtained results revealed leading role of the neuronal aspartate-malate shuttle in violation of NAA synthesis.

scholarly journals Antibody escape by polyomavirus capsid mutation facilitates neurovirulence

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Matthew D Lauver ◽  
Daniel J Goetschius ◽  
Colleen S Netherby-Winslow ◽  
Katelyn N Ayers ◽  
Ge Jin ◽  
...  
Keyword(s):  
Progressive Multifocal Leukoencephalopathy ◽  
Capsid Protein ◽  
Neutralizing Antibody ◽  
Viral Capsid ◽  
Cell Tropism ◽  
Capsid Protein Vp1 ◽  
Particle Refinement ◽  
Immunomodulatory Therapies ◽  
Major Viral Capsid Protein

JCPyV polyomavirus, a member of the human virome, causes progressive multifocal leukoencephalopathy (PML), an oft-fatal demyelinating brain disease in individuals receiving immunomodulatory therapies. Mutations in the major viral capsid protein, VP1, are common in JCPyV from PML patients (JCPyV-PML) but whether they confer neurovirulence or escape from virus-neutralizing antibody (nAb) in vivo is unknown. A mouse polyomavirus (MuPyV) with a sequence-equivalent JCPyV-PML VP1 mutation replicated poorly in the kidney, a major reservoir for JCPyV persistence, but retained the CNS infectivity, cell tropism, and neuropathology of the parental virus. This mutation rendered MuPyV resistant to a monoclonal Ab (mAb), whose specificity overlapped the endogenous anti-VP1 response. Using cryo-EM and a custom sub-particle refinement approach, we resolved an MuPyV:Fab complex map to 3.2 Å resolution. The structure revealed the mechanism of mAb evasion. Our findings demonstrate convergence between nAb evasion and CNS neurovirulence in vivo by a frequent JCPyV-PML VP1 mutation.

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