A Single Amino Acid Substitution in the Core Protein of West Nile Virus Increases Resistance to Acidotropic Compounds

West Nile virus (WNV) is a worldwide distributed mosquito-borne flavivirus that naturally cycles between birds and mosquitoes, although it can infect multiple vertebrate hosts including horses and humans. This virus is responsible for recurrent epidemics of febrile illness and encephalitis, and has recently become a global concern. WNV requires to transit through intracellular acidic compartments at two different steps to complete its infectious cycle. These include fusion between the viral envelope and the membrane of endosomes during viral entry, and virus maturation in the trans-Golgi network. In this study, we followed a genetic approach to study the connections between viral components and acidic pH. A WNV mutant with increased resistance to the acidotropic compound NH₄Cl, which blocks organelle acidification and inhibits WNV infection, was selected. Nucleotide sequencing revealed that this mutant displayed a single amino acid substitution (Lys 3 to Glu) on the highly basic internal capsid or core (C) protein. The functional role of this replacement was confirmed by its introduction into a WNV infectious clone. This single amino acid substitution also increased resistance to other acidification inhibitor (concanamycin A) and induced a reduction of the neurovirulence in mice. Interestingly, a naturally occurring accompanying mutation found on prM protein abolished the resistant phenotype, supporting the idea of a genetic crosstalk between the internal C protein and the external glycoproteins of the virion. The findings here reported unveil a non-previously assessed connection between the C viral protein and the acidic pH necessary for entry and proper exit of flaviviruses.     

A Single Amino Acid Substitution in the NS2A Protein of Japanese Encephalitis Virus Affects Virus Propagation In Vitro but Not In Vivo

We identified a unique amino acid of NS2A₁₁₃, phenylalanine, that affects the efficient propagation of two Japanese encephalitis virus strains, JaTH160 and JaOArS982, in neuroblastoma Neuro-2a cells but not in cell lines of extraneural origin. This amino acid did not affect viral loads in the brain or survival curves in mice. These findings suggest that virus propagation in vitro may not reflect the level of virus neuroinvasiveness in vivo.     

A Single Amino Acid Substitution in the Novel H7N9 Influenza A Virus NS1 Protein Increases CPSF30 Binding and Virulence

Although an effective interferon antagonist in human and avian cells, the novel H7N9 influenza virus NS1 protein is defective at inhibiting CPSF30. An I106M substitution in H7N9 NS1 can restore CPSF30 binding together with the ability to block host gene expression. Furthermore, a recombinant virus expressing H7N9 NS1-I106M replicates to higher titers in vivo, and is subtly more virulent, than the parental virus. Natural polymorphisms in H7N9 NS1 that enhance CPSF30 binding may be cause for concern.     

A Single Amino Acid Change in Turnip Crinkle Virus Movement Protein p8 Affects RNA Binding and Virulence on Arabidopsis thaliana

Comparison of the symptoms caused by turnip crinkle virus strain M (TCV-M) and TCV-B infection of a resistant Arabidopsis thaliana line termed Di-17 demonstrates that TCV-B has a greater ability to spread in planta. This ability is due to a single amino acid change in the viral movement protein p8 and inversely correlates with p8 RNA binding affinity.     

A Single Amino Acid Substitution in the Group 1 Trypanosoma brucei gambiense Haptoglobin-Hemoglobin Receptor Abolishes TLF-1 Binding

Critical to human innate immunity against African trypanosomes is a minor subclass of human high-density lipoproteins, termed Trypanosome Lytic Factor-1 (TLF-1). This primate-specific molecule binds to a haptoglobin-hemoglobin receptor (HpHbR) on the surface of susceptible trypanosomes, initiating a lytic pathway. Group 1 Trypanosoma brucei gambiense causes human African Trypanosomiasis (HAT), escaping TLF-1 killing due to reduced uptake. Previously, we found that group 1 T. b. gambiense HpHbR (TbgHpHbR) mRNA levels were greatly reduced and the gene contained substitutions within the open reading frame. Here we show that a single, highly conserved amino acid in the TbgHpHbR ablates high affinity TLF-1 binding and subsequent endocytosis, thus evading TLF-1 killing. In addition, we show that over-expression of TbgHpHbR failed to rescue TLF-1 susceptibility. These findings suggest that the single substitution present in the TbgHpHbR directly contributes to the reduced uptake and resistance to TLF-1 seen in these important human pathogens.     

Lineage-Specific Differences in the Amino Acid Substitution Process

In Darwinian evolution, mutations occur approximately at random in a gene, turned into amino acid mutations by the genetic code. Some mutations are fixed to become substitutions and some are eliminated from the population. Partitioning pairs of closely related species with complete genome sequences by average population size of each pair, we looked at the substitution matrices generated for these partitions and compared the substitution patterns between species. We estimated a population genetic model that relates the relative fixation probabilities of different types of mutations to the selective pressure and population size. Parameterizations of the average and distribution of selective pressures for different amino acid substitution types in different population size comparisons were generated with a Bayesian framework. We found that partitions in population size as well as in substitution type are required to explain the substitution data. Selection coefficients were found to decrease with increasingly radical amino acid substitution and with increasing effective population size.To further explore the role of underlying processes in amino acid substitution, we analyzed embryophyte (plant) gene families from TAED (The Adaptive Evolution Database), where solved structures for at least one member exist in the Protein Data Bank. Using PAML, we assigned branches to three categories: strong negative selection, moderate negative selection/neutrality, and positive diversifying selection. Focusing on the first and third categories, we identified sites changing along gene family lineages and observed the spatial patterns of substitution. Selective sweeps were expected to create primary sequence clustering under positive diversifying selection. Co-evolution through direct physical interaction was expected to cause tertiary structural clustering. Under both positive and negative selection, the substitution patterns were found to be nonrandom. Under positive diversifying selection, significant independent signals were found for primary and tertiary sequence clustering, suggesting roles for both selective sweeps and direct physical interaction. Under strong negative selection, the signals were not found to be independent. All together, a complex interplay of population genetic and protein thermodynamics forces is suggested.     

Enhancement of Thermostability of Firefly Luciferase from Luciola lateralis by a Single Amino Acid Substitution

We constructed firefly luciferase mutants from Luciola lateralis in which Ala at position 217 was replaced by each of three hydrophobic amino acid residues (lie, Leu, and Val). These mutants were superior to the wild-type in thermostability. Especially, the purified Ala217Leu mutant still maintained over 70% of the initial activity after 60 min at 50°C. This mutant is the most thermostable firefly luciferase obtained.     

A Single Amino Acid Substitution in the v-Eyk Intracellular Domain Results in Activation of Stat3 and Enhances Cellular Transformation

The receptor tyrosine kinase Eyk, a member of the Axl/Tyro3 subfamily, activates the STAT pathway and transforms cells when constitutively activated. Here, we compared the potentials of the intracellular domains of Eyk molecules derived from c-Eyk and v-Eyk to transform rat 3Y1 fibroblasts. The v-Eyk molecule induced higher numbers of transformants in soft agar and stronger activation of Stat3; levels of Stat1 activation by the two Eyk molecules were similar. A mutation in the sequence Y⁹³³VPL, present in c-Eyk, to the v-Eyk sequence Y⁹³³VPQ led to increased activation of Stat3 and increased transformation efficiency. However, altering another sequence, Y⁸⁶²VNT, present in both Eyk molecules to F⁸⁶²VNT markedly decreased transformation without impairing Stat3 activation. These results indicate that activation of Stat3 enhances transformation efficiency and cooperates with another pathway to induce transformation.     

CodonTest: Modeling Amino Acid Substitution Preferences in Coding Sequences

Codon models of evolution have facilitated the interpretation of selective forces operating on genomes. These models, however, assume a single rate of non-synonymous substitution irrespective of the nature of amino acids being exchanged. Recent developments have shown that models which allow for amino acid pairs to have independent rates of substitution offer improved fit over single rate models. However, these approaches have been limited by the necessity for large alignments in their estimation. An alternative approach is to assume that substitution rates between amino acid pairs can be subdivided into rate classes, dependent on the information content of the alignment. However, given the combinatorially large number of such models, an efficient model search strategy is needed. Here we develop a Genetic Algorithm (GA) method for the estimation of such models. A GA is used to assign amino acid substitution pairs to a series of rate classes, where is estimated from the alignment. Other parameters of the phylogenetic Markov model, including substitution rates, character frequencies and branch lengths are estimated using standard maximum likelihood optimization procedures. We apply the GA to empirical alignments and show improved model fit over existing models of codon evolution. Our results suggest that current models are poor approximations of protein evolution and thus gene and organism specific multi-rate models that incorporate amino acid substitution biases are preferred. We further anticipate that the clustering of amino acid substitution rates into classes will be biologically informative, such that genes with similar functions exhibit similar clustering, and hence this clustering will be useful for the evolutionary fingerprinting of genes.     

Identification of Single Amino Acid Substitutions in Proteogenomics

An important aim of proteogenomics, which combines data of high throughput nucleic acid and protein analysis, is to reliably identify single amino acid substitutions representing a main type of coding genome variants. Exact knowledge of deviations from the consensus genome can be utilized in several biomedical fields, such as studies of expression of mutated proteins in cancer, deciphering heterozygosity mechanisms, identification of neoantigens in anticancer vaccine production, search for RNA editing sites at the level of the proteome, etc. Generation of this new knowledge requires processing of large data arrays from high–resolution mass spectrometry, where information on single–point protein variation is often difficult to extract. Accordingly, a significant problem in proteogenomic analysis is the presence of high levels of false positive results for variant–containing peptides in the produced results. Here we review recently suggested approaches of high quality proteomics data processing that may provide more reliable identification of single amino acid substitutions, especially contrary to residue modifications occurring in vitro and in vivo. Optimized methods for assessment of false discovery rate save instrumental and computational time spent for validation of interesting findings of amino acid polymorphism by orthogonal methods.     

A Single Amino Acid Substitution Converts γ-Glutamyltranspeptidase to a Class IV Cephalosporin Acylase (Glutaryl-7-Aminocephalosporanic Acid Acylase)

The aspartyl residue at position 433 of γ-glutamyltranspeptidase of Escherichia coli K-12 was replaced by an asparaginyl residue. This substitution enabled γ-glutamyltranspeptidase to deacylate glutaryl-7-aminocephalosporanic acid, producing 7-aminocephalosporanic acid, which is a starting material for the synthesis of semisynthetic cephalosporins.     

氨基酸在蛋白质空间结构中的深度倾向性因子

用统计和几何方法给出了氨基酸在蛋白质空间结构中的深度计算,并利用PDB数据库得到了不同氨基酸在蛋白质中的深度倾向性因子,并得到了这些倾向性因子与氨基酸的物理、化学综合特性的相关性质.     

Amino Acid Metabolic Origin as an Evolutionary Influence on Protein Sequence in Yeast

The metabolic cycle of Saccharomyces cerevisiae consists of alternating oxidative (respiration) and reductive (glycolysis) energy-yielding reactions. The intracellular concentrations of amino acid precursors generated by these reactions oscillate accordingly, attaining maximal concentration during the middle of their respective yeast metabolic cycle phases. Typically, the amino acids themselves are most abundant at the end of their precursor’s phase. We show that this metabolic cycling has likely biased the amino acid composition of proteins across the S. cerevisiae genome. In particular, we observed that the metabolic source of amino acids is the single most important source of variation in the amino acid compositions of functionally related proteins and that this signal appears only in (facultative) organisms using both oxidative and reductive metabolism. Periodically expressed proteins are enriched for amino acids generated in the preceding phase of the metabolic cycle. Proteins expressed during the oxidative phase contain more glycolysis-derived amino acids, whereas proteins expressed during the reductive phase contain more respiration-derived amino acids. Rare amino acids (e.g., tryptophan) are greatly overrepresented or underrepresented, relative to the proteomic average, in periodically expressed proteins, whereas common amino acids vary by a few percent. Genome-wide, we infer that 20,000 to 60,000 residues have been modified by this previously unappreciated pressure. This trend is strongest in ancient proteins, suggesting that oscillating endogenous amino acid availability exerted genome-wide selective pressure on protein sequences across evolutionary time. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Benjamin L. de Bivort and Ethan O. Perlstein have contributed equally to this work.     

Amino Acid Biosynthetic Cost and Protein Conservation

Protein products of highly expressed genes tend to favor amino acids that have lower average biosynthetic costs (i.e., they exhibit metabolic efficiency). While this trend has been observed in several studies, the specific sites where cost-reducing substitutions accumulate have not been well characterized. Toward that end, weighted costs in conserved and variable positions were evaluated across a total of 9,119 homologous proteins in four mammalian orders (primate, carnivore, rodent, and artiodactyls), which together contain a total of 20,457,072 amino acids. Degree of conservation at homologous positions in these mammalian proteins and average-weighted cost across all positions within a single protein are significantly correlated. Dividing human genes into two classes (those with and those without CpG islands in their promoters) suggests that humans also preferentially utilize less costly amino acids in highly expressed genes. In contrast to the intuitive expectation that the relatively weak selective force associated with metabolic efficiency would be a selection pressure in complex multicellular organisms, the overall level of selective constraint within the variable regions of mammalian proteins allows the metabolic efficiency to derive a reduction of overall biosynthetic cost, particularly in genes with the highest levels of expression.