Conservation Of Motifs Research Articles

Gamma Carbonic Anhydrases from Hydrothermal Vent Bacteria: Cases of Alternating Active Site Due to a Long Loop with Proton Shuttle Residue

Accelerated CO2 sequestration uses carbonic anhydrases (CAs) as catalysts; thus, there is much research on these enzymes. The γ-CA from Escherichia coli (EcoCA-γ) was the first γ-CA to display an active site that switches between “open” and “closed” states through Zn2+ coordination by the proton-shuttling His residue. Here, we explored this occurrence in γ-CAs from hydrothermal vent bacteria and also the γ-CA from Methanosarcina thermophila (Cam) using molecular dynamics. Ten sequences were analyzed through multiple sequence alignment and motif analysis, along with three others from a previous study. Conservation of residues and motifs was high, and phylogeny indicated a close relationship amongst the sequences. All structures, like EcoCA-γ, had a long loop harboring the proton-shuttling residue. Trimeric structures were modeled and simulated for 100 ns at 423 K, with all the structures displaying thermostability. A shift between “open” and “closed” active sites was observed in the 10 models simulated through monitoring the behavior of the His proton-shuttling residue. Cam, which has two Glu proton shuttling residues on long loops (Glu62 and Glu84), also showed an active site switch affected by the first Glu proton shuttle, Glu62. This switch was thus concluded to be common amongst γ-CAs and not an isolated occurrence.

An l-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) possesses agglutination activity and regulates inflammation, phagocytosis and respiratory burst of monocytes/macrophages

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are widely present in vertebrates and invertebrates, which involve in the innate immune response. However, the functional characterization and related regulation mechanisms of RBLs remain unclear in teleost fish. In this study, an l-rhamnose-binding lectin-like (OnRBL-L) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-L is 678 bp encoding 225 aa. The sequence of OnRBL-L has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-L was abundantly distributed in intestine tissue, and widely existed in all detected tissues. Meanwhile, the expression of OnRBL-L increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (monocytes/macrophages) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. In addition, the recombinant OnRBL-L was found to bind and agglutinate S. agalactiae and A. hydrophila. Furthermore, OnRBL-L could participate in non-specific cellular immune defense, including reducing the expression of pro-inflammatory factors (IL-6、IL-8 and TNF-α), and enhancement of the phagocytosis and respiratory burst of MO/MФ. Overall, our results provide new insights into the understanding of RBL as an important pattern recognition molecule and regulator in non-specific cell immunity in an early vertebrate.

Genome-Wide Identification and Expression Analysis of AP2/EREBP Transcription Factors in Litchi (Litchi chinensis Sonn.)

APETALA2/ethylene response element binding proteins (AP2/EREBP) are a vital type of TF involved in plant organ development and embryogenesis. In this study we identified 202 Litchi AP2/EREBP TFs from the litchi genome. They were classified into four subfamilies by phylogenetic clustering, including AP2s (20), ERFs (112), DREBs (64), and RAVs (6). Analysis of conserved domains, motifs, gene structure, and genome localization were carried out to investigate the evolutionary features of litchi AP2/EREBPs. Over 35% of DREBs and ERFs involved in the expansion of litchi AP2/EREBPs resulted from tandem duplication. The majority of genomic organizations were conservative, except those of the AP2 subfamily, which had no intron and contained less conservative motif numbers. The expression profiles of litchi AP2/EREBPs in ten tissues were investigated using RNA-Seq data and fifty-nine showed tissue-specific expressions. Their expression patterns were confirmed by qRT-PCR with eight tissues-specificity genes. Six genes related to embryogenesis were identified using the map of orthologous gene interaction between Arabidopsis and litchi. This paper is a comprehensive report on the characteristics of the litchi AP2/EREBP gene superfamily. It will serve to further explore the regulatory mechanisms of AP2/EREBP TFs in the litchi somatic embryogenesis and provide information for litchi molecular breeding.

Reference Gene Selection for Analyzing the Transcription Patterns of Two Fatty Acyl-CoA Reductase Genes From Paracoccus marginatus (Hemiptera: Pseudococcidae).

Paracoccus marginatus (Hemiptera: Pseudococcidae), known as the papaya mealybug, could cause considerable yield loss of several plants. To date, there is no molecular-based study of P. marginatus. Fatty acyl-CoA reductases (FARs) are key enzymes involved in wax synthesis. In the present study, we cloned and characterized coding sequences (CDS) of two FAR genes from P. marginatus. The results showed that PmFAR1 and PmFAR2 CDS were 1,590 and 1,497 bp in length, respectively, and sequence analysis indicated that these two genes both had the conservative motifs belonging to FAR_C superfamily. Furthermore, seven candidate reference genes were analyzed for their expression stability by using common algorithms including comparative ΔCq method, geNorm, NormFinder, BestKeeper, and RefFinder. Eventually, β-actin and GAPDH were the best reference genes in evaluating the expression of those two FAR genes. We found that PmFAR1 and PmFAR2 showed distinct expression patterns in different life stages. Moreover, the transcription of PmFAR1 and PmFAR2 in P. marginatus fed on resistant cassava cultivars was significantly lower compared with those fed on susceptible ones, indicating the potential function of FAR genes in cassava resistance to P. marginatus. The present study might help in better understanding the molecular mechanism of cassava resistance to mealybug.

Genome-wide detection and classification of terpene synthase genes in Aquilaria agallochum.

The online version contains supplementary material available at 10.1007/s12298-021-01040-z.

Expression and functional characterization of an L-rhamnose-binding lectin from Nile tilapia (Oreochromis niloticus) in host defense against bacterial infection

Rhamnose-binding lectins (RBLs), a Ca2+-independent lectin family, are able to recognize varieties of pathogens and involve in the innate immune response. In this study, an L-rhamnose-binding lectin-like (OnRBL-1) was identified and functionally characterized from Nile tilapia (Oreochromis niloticus). The open reading frame of OnRBL-1 is 702 bp encoding 233 amino acids. The sequence of OnRBL-1 has relatively conservative characteristic peptide motifs, including YGR, DPC, and KYL-motif. Expression analysis showed that OnRBL-1 was abundantly distributed in liver tissue, and widely existed in all detected tissues. Meanwhile, the expressions of OnRBL-1 increased significantly in vivo (liver, spleen, head kidney, intestine, gills and peripheral blood) and in vitro (hepatocytes) following challenges with two important tilapia pathogenic bacteria Streptococcus agalactiae and Aeromonas hydrophila. Moreover, the recombinant OnRBL-1 was expressed and purified, which was found to bind and agglutinate S. agalactiae and A. hydrophila. Overall, This study indicated that OnRBL-1 may function as an important pattern recognition molecule, possessing recognition and agglutination activity to bacterial pathogens, thus involving in the innate immune defense of O. niloticus.

Shotgun proteomics of Brassica rapa seed proteins identifies vicilin as a major seed storage protein in the mature seed.

Proteins make up a large percentage of the Brassica seed and are second only to the oil in economic importance with uses for both animal and human nutrition. The most abundant proteins reported in the seeds of Brassica are the seed storage proteins cruciferin and napin, belonging to the 12S globulin and 2S albumin families of proteins, respectively. To gain insight into the Brassica rapa seed proteome and to confirm the presence and relative quantity of proteins encoded by candidate seed storage genes in the mature seed, shotgun proteomics was carried out on protein extracts from seeds of B. rapa inbred line R-o-18. Following liquid chromatography tandem mass spectrometry, a total of 34016 spectra were mapped to 323 proteins, where 233 proteins were identified in 3 out of 4 biological replicates by at least 2 unique peptides. 2S albumin like napin seed storage proteins (SSPs), 11/12S globulin like cruciferin SSPs and 7S globulin like vicilin SSPs were identified in the samples, along with other notable proteins including oil body proteins, namely ten oleosins and two oil body-associated proteins. The identification of vicilin like proteins in the mature B. rapa seed represents the first account of these proteins in the Brassicaceae and analysis indicates high conservation of sequence motifs to other 7S vicilin-like allergenic proteins as well as conservation of major allergenic epitopes in the proteins. This study enriches our existing knowledge on rapeseed seed proteins and provides a robust foundation and rational basis for plant bioengineering of seed storage proteins.

Evolution of plant telomerase RNAs: farther to the past, deeper to the roots.

The enormous sequence heterogeneity of telomerase RNA (TR) subunits has thus far complicated their characterization in a wider phylogenetic range. Our recent finding that land plant TRs are, similarly to known ciliate TRs, transcribed by RNA polymerase III and under the control of the type-3 promoter, allowed us to design a novel strategy to characterize TRs in early diverging Viridiplantae taxa, as well as in ciliates and other Diaphoretickes lineages. Starting with the characterization of the upstream sequence element of the type 3 promoter that is conserved in a number of small nuclear RNAs, and the expected minimum TR template region as search features, we identified candidate TRs in selected Diaphoretickes genomes. Homologous TRs were then used to build covariance models to identify TRs in more distant species. Transcripts of the identified TRs were confirmed by transcriptomic data, RT-PCR and Northern hybridization. A templating role for one of our candidates was validated in Physcomitrium patens. Analysis of secondary structure demonstrated a deep conservation of motifs (pseudoknot and template boundary element) observed in all published TRs. These results elucidate the evolution of the earliest eukaryotic TRs, linking the common origin of TRs across Diaphoretickes, and underlying evolutionary transitions in telomere repeats.

Plant RNA Binding Proteins as Critical Modulators in Drought, High Salinity, Heat, and Cold Stress Responses: An Updated Overview.

Plant abiotic stress responses are tightly regulated by different players at multiple levels. At transcriptional or post-transcriptional levels, several RNA binding proteins (RBPs) regulate stress response genes through RNA metabolism. They are increasingly recognized as critical modulators of a myriad of biological processes, including stress responses. Plant RBPs are heterogeneous with one or more conservative RNA motifs that constitute canonical/novel RNA binding domains (RBDs), which can bind to target RNAs to determine their regulation as per the plant requirements at given environmental conditions. Given its biological significance and possible consideration as a potential tool in genetic manipulation programs to improve key agronomic traits amidst frequent episodes of climate anomalies, studies concerning the identification and functional characterization of RBP candidate genes are steadily mounting. This paper presents a comprehensive overview of canonical and novel RBPs and their functions in major abiotic stresses including drought, heat, salt, and cold stress conditions. To some extent, we also briefly describe the basic motif structure of RBPs that would be useful in forthcoming studies. Additionally, we also collected RBP genes that were modulated by stress, but that lacked functional characterization, providing an impetus to conduct further research.

Replacement of Arg in the conserved N-terminal RLFDQxFG motif affects physico-chemical properties and chaperone-like activity of human small heat shock protein HspB8 (Hsp22).

The small heat shock protein (sHsp) called HspB8 (formerly, Hsp22) is one of the least typical sHsp members, whose oligomerization status remains debatable. Here we analyze the effect of mutations in a highly conservative sequence located in the N-terminal domain of human HspB8 on its physico-chemical properties and chaperone-like activity. According to size-exclusion chromatography coupled to multi-angle light scattering, the wild type (WT) HspB8 is present as dominating monomeric species (~24 kDa) and a small fraction of oligomers (~60 kDa). The R29A amino acid substitution leads to the predominant formation of 60-kDa oligomers, leaving only a small fraction of monomers. Deletion of the 28–32 pentapeptide (Δ mutant) results in the formation of minor quantities of dimers (~49 kDa) and large quantities of the 24-kDa monomers. Both the WT protein and its Δ mutant efficiently bind a hydrophobic probe bis-ANS and are relatively rapidly hydrolyzed by chymotrypsin, whereas the R29A mutant weakly binds bis-ANS and resists chymotrypsinolysis. In contrast to HspB8 WT and its Δ mutant, which are well phosphorylated by cAMP-dependent and ERK1 protein kinases, the R29A mutant is poorly phosphorylated. R29A mutation affects the chaperone-like activity of HspB8 measured in vitro. It is concluded that the irreplaceable Arg residue located in the only highly conservative motif in the N-terminal domain of all sHsp proteins affects the oligomeric structure and key properties of HspB8.

Identification of the Group III WRKY Subfamily and the Functional Analysis of GhWRKY53 in Gossypium hirsutum L.

WRKY transcription factors had multiple functions in plant secondary metabolism, leaf senescence, fruit ripening, adaptation to biotic and abiotic stress, and plant growth and development. However, knowledge of the group III WRKY subfamily in fiber development in upland cotton (Gossypium hirsutum L.) is largely absent. Previous studies have shown that there were 21 putative group III WRKY members in G. hirsutum L. These putative amino acid sequences from the III WRKY group were phylogenetically clustered into three clades. Multiple alignment, conservative motif analysis, and gene structure analysis showed that the members clustered together in the phylogenetic tree had similar motifs and gene structures. Expression pattern analysis revealed that variation in the expression levels of these genes in different tissues and fiber development stages. To better understand the functions of putative group III WRKY genes in G. hirsutum L., we selected the cotton fiber initiation-related gene GhWRKY53 for cloning and functional identification. The subcellular localization experiment of GhWRKY53 in Nicotiana tabacum leaves showed that it was located in the nucleus. The heterologous expression of GhWRKY53 in Arabidopsis thaliana could significantly increase the density of trichomes. Twelve proteins that interacted with GhWRKY53 were screened from the cotton fiber cDNA library by yeast two-hybrid experiment. This study findings lay a foundation for further research on the role of the GhWRKY53 during cotton fiber development and provide a new insight for further studying putative group III WRKY genes in G. hirsutum L. Our research results also provide vital information for the genetic mechanism of high-quality cotton fiber formation and essential genetic resources for cotton fiber quality improvement.

Genome‑wide identification and analysis of the trihelix transcription factors in sunflower

The trihelix genes encode plant-specific transcription factors, which play a vital role in plant morphological and developmental processes. However, information about the presence of trihelix genes in sunflower (Helianthus annuus L.) is scarce. Sunflower belongs to composite family and possesses strong drought and salt-alkali tolerance. In this study based on H. annuus genome data, we have identified and analyzed the trihelix genes with a complete description of their physical and chemical properties, phylogenetic relationships, motif composition, chromosome distribution, exon-intron structure, cis-acting elements, and chromosome collinearity. In H. annuus, 31 full-length trihelix genes were identified and categorized into six subgroups (SIP, GT1, SH4, Gδ, GT-γ, and GT2). Multiple Em for motif elicitation (MEME), used for conservative motif analysis, identified 10 distinct motifs unevenly distributed on 31 trihelix genes. In addition to that, chromosome localization analysis showed the number and distribution of these trihelix genes on 17 chromosomes of H. annuus. Transcriptional structure analysis revealed the structure of introns and exons of different gene members. Furthermore, cis-element analysis identified 19 different types of cis-elements mainly related to abiotic stress, hormones, and growth and development of plant. Results of this study manifested novel insights into phylogenetic relationships and possible functions of H. annuus trihelix genes. Moreover, these findings can assist in future studies regarding specific physiological effects of H. annuus trihelix transcription factors.

Identification and expression analysis of the AHL gene family in grape (Vitis vinifera)

AT-Hook Motif Nuclear Localized (AHL) proteins is a small DNA binding protein motif. It was first found in the high-mobility-group (HMG-I/Y) of mammalian non-histone chromosomal protein, which is different from other known DNA binding protein motifs. In order to reveal the basic characteristics of AHL gene family in grape, this study used biological information technology to identify the members of grape AHL genes, and analyzed its physical and chemical properties, cis-acting element, codon usage bias, gene structure, conservative motif and phylogenetic relationship. The study identified 14 members in the AHL gene family of grape. Phylogenetic analysis showed that AHL proteins from grape can be classified into two branches, class I and class II. And nearly all VvAHLs members in the same branch displayed similar motif distributions and gene structures, which might be related to their same executive functions. Subcellular localization showed that most of the VvAHL genes are located in Extracellular matrix, mitochondrion, nucleus, cytoplasm and other organelles related to photosynthesis and respiration. All of the 14 VvAHLs possess a number of light response-, stress response-, and hormone response-related cis-elements in the promoter sequences. The real-time fluorescence quantification technique was used to analyze the response of grape AHL genes under various hormone induction and adversity stress. The results showed that the expression of VvAHL gene was significantly regulated under the hormone induction and the adversity stress. It is suggested that VvAHLs play an important role in growth and development of grape, organ formation and defense response to adversity. This study will provide the basis for further analysis of AHL genes function, and lay the foundation for in-depth study of AHL genes in grape.

Evolutionary conservation of the DRACH signatures of potential N6-methyladenosine (m6A) sites among influenza A viruses

The addition of a methyl group to the N6-position of adenosine (m6A) is considered one of the most prevalent internal post-transcriptional modifications and is attributed to virus replication and cell biology. Viral epitranscriptome sequencing analysis has revealed that hemagglutinin (HA) mRNA of H1N1 carry eight m6A sites which are primarily enriched in 5′-DRACH-3′ sequence motif. Herein, a large-scale comparative m6A analysis was conducted to investigate the conservation patterns of the DRACH motifs that corresponding to the reference m6A sites among influenza A viruses. A total of 70,030 complete HA sequences that comprise all known HA subtypes (H1–18) collected over several years, countries, and affected host species were analysed on both mRNA and vRNA strands. The bioinformatic analysis revealed the highest degree of DRACHs conservation among all H1 sequences that clustered largely in the middle and in the vicinity to 3′ end with at least four DRACH motifs were conserved in all mRNA sequences. The major HA-containing subtypes displayed a modest DRACH motif conservation located either in the middle region of HA transcript (H3) or at the 3′ end (H5) or were distributed across the length of HA sequence (H9). The lowest conservation was demonstrated in HA subtypes that infect mostly the wild type avian species and bats. Interestingly, the total number and the conserved DRACH motifs in the vRNA were found to be much lower than those observed in the mRNA. Collectively, the identification of putative m6A topology provides a foundation for the future intervention of influenza infection, replication, and pathobiology in susceptible hosts.

Aestivation motifs explain hypertension and muscle mass loss in mice with psoriatic skin barrier defect.

Recent evidence suggests that arterial hypertension could be alternatively explained as a physiological adaptation response to water shortage, termed aestivation, which relies on complex multi-organ metabolic adjustments to prevent dehydration. Here, we tested the hypothesis that chronic water loss across diseased skin leads to similar adaptive water conservation responses as observed in experimental renal failure or high salt diet. We studied mice with keratinocyte-specific overexpression of IL-17A which develop severe psoriasis-like skin disease. We measured transepidermal water loss and solute and water excretion in the urine. We quantified glomerular filtration rate (GFR) by intravital microscopy, and energy and nitrogen pathways by metabolomics. We measured skin blood flow and transepidermal water loss (TEWL) in conjunction with renal resistive indices and arterial blood pressure. Psoriatic animals lost large amounts of water across their defective cutaneous epithelial barrier. Metabolic adaptive water conservation included mobilization of nitrogen and energy from muscle to increase organic osmolyte production, solute-driven maximal anti-diuresis at normal GFR, increased metanephrine and angiotensin 2 levels, and cutaneous vasoconstriction to limit TEWL. Heat exposure led to cutaneous vasodilation and blood pressure normalization without parallel changes in renal resistive index, albeit at the expense of further increased TEWL. Severe cutaneous water loss predisposes psoriatic mice to lethal dehydration. In response to this dehydration stress, the mice activate aestivation-like water conservation motifs to maintain their body hydration status. The circulatory water conservation response explains their arterial hypertension. The nitrogen-dependency of the metabolic water conservation response explains their catabolic muscle wasting.

Genome-wide identification of the SOD gene family and expression analysis under drought and salt stress in barley

Superoxide dismutase (SOD) plays a vital role in the removal of active oxygen in plant. In this study, bioinformatics research and expression analysis of SOD gene family under drought and salt stress has been done in barley (Hordeum vulgare L., Hv). Seven SOD genes were identified, including four Cu/Zn-SODs, two Fe-SODs and one Mn-SOD. Those three kinds of SOD genes were distributed in 2, 4 and 7 chromosomes, respectively. Phylogenetic analysis revealed that SODs of barley, rice and Arabidopsis thaliana can be divided into three subfamilies (A–C). Motif analysis founded six conservative motifs. Most SOD genes had relatively conservative intron/exon and motif composition, but the number of intron/exons varied greatly among different genes. Based on RNA-seq data and qRT-PCR analysis of expression changes of barley SOD family gene under stress, HvCSD1, HvCSD4 and HvFSD1 were more valuable for in-depth research due to their significant changes in their expression after stress treatment. These outcomes help to lay the foundation for studying the gene function of barley SOD family and provide the improvement of drought and salt tolerance barley genetic breeding.

G-quadruplex motifs are functionally conserved in cis-regulatory regions of pathogenic bacteria: An in-silico evaluation

The role of G-quadruplexes in the cellular physiology of human pathogenesis is an intriguing area of research. Nonetheless, their functional roles and evolutionary conservation have not been compared comprehensively in pathogenic forms of various bacterial genera and species. In the current in silico study, we addressed the role of G-quadruplex-forming sequences (G4 motifs) in the context of cis-regulation, expression variation, regulatory networks, gene orthology and ontology. Genome-wide screening across seven pathogenic genomes using the G4Hunter tool revealed the significant prevalence of G4 motifs in cis-regulatory regions compared to the intragenic regions. Significant conservation of G4 motifs was observed in the regulatory region of 300 orthologous genes. Further analysis of published ChIP-Seq data (Minch et al., 2015) of 91 DNA-binding proteins of the M. tuberculosis genome revealed significant links between G4 motifs and target sites of transcriptional regulators. Interestingly, the transcription factors entangled with virulence, in specific, CsoR, Rv0081, DevR/DosR, and TetR family are found to have G4 motifs in their target regulatory regions. Overall the current study applies positional-functional relationship computation to delve into the cis-regulation of G-quadruplex structures in the context of gene orthology in pathogenic bacteria.

Molecular organization of 5S rDNA intergenic spacer in Gentiana pneumonanthe L. and G. punctata L.

Aim. The study was aimed at cloning and analysis of molecular organization of 5S rDNA intergenic spacer (IGS) in two Gentiana species of Ukrainian flora, G. pneumonanthe L. and G. punctata L. Methods. 5S rDNA IGS sequence was amplified using polymerase chain reaction (PCR) with a pair of primers specific for the gene coding region. The produced PCR products were fractionated by gel-electrophoresis, isolated, ligated into plasmid pUC18, cloned into E. coli, and then sequenced. Nucleotide sequences were aligned using the Muscle algorithm and analyzed in the Unipro UGENE software. Results. The intergenic spacer region of the 5S rRNA genes was cloned and sequenced for two Gentiana species of Ukrainian flora, G. pneumonanthe and G. punctata. Based on the analysis of the alignment of the IGS sequences of five Gentiana species from three sections, some features of molecular organization of IGS of 5S rRNA genes in the studied species were established. In particular, motifs typical for other angiosperm families were identified, such as conservative oligo-dT motif at the IGS 3'-end that served as a transcription termination site and AT-rich region preceding the coding region of 5S rRNA gene. However, in the region of transcription initiation, conservative GC-element in position -13 is changed to AC. Conclusions. The interspecific variation of molecular organization of 5S rDNA IGS was identified among Gentiana species that can be used to clarify the phylogenetic relationships between members of this genus.Keywords: Gentiana species, 5S rDNA intergenic spacer, molecular organization, phylogeny.

Purinergic GPCR transmembrane residues involved in ligand recognition and dimerization.

We compare the GPCR-ligand interactions and highlight important residues for recognition in purinergic receptors-from both X-ray crystallographic and cryo-EM structures. These include A1 and A2A adenosine receptors, and P2Y1 and P2Y12 receptors that respond to ADP and other nucleotides. These receptors are important drug discovery targets for immune, metabolic and nervous system disorders. In most cases, orthosteric ligands are represented, except for one allosteric P2Y1 antagonist. This review catalogs the residues and regions that engage in contacts with ligands or with other GPCR protomers in dimeric forms. Residues that are in proximity to bound ligands within purinergic GPCR families are correlated. There is extensive conservation of recognition motifs between adenosine receptors, but the P2Y1 and P2Y12 receptors are each structurally distinct in their ligand recognition. Identifying common interaction features for ligand recognition within a receptor class that has multiple structures available can aid in the drug discovery process.

Genetic Variability of Tick-Borne Encephalitis Virus Genome 5'-UTR from Northern Eurasia

This paper reports the analysis of the nucleotide sequences of the 5'-untranslated region (5'-UTR) of tick-borne encephalitis virus (TBEV) genomic RNA isolated from 39 individual taiga ticks collected in several regions of Northern Eurasia. The sequences of 5'-UTRs of the Siberian and Far East TBEV genotypes were 89% and 95% identical to the prototype strains (Zausaev and 205), respectively. The detected nucleotide substitutions were typical for these two TBEV genotypes, which made possible unambiguous identification. Both conservative and variable motifs were detected in the 5'-UTR RNA. The B2, C1, and C2 elements of the Y-shaped 5'-UTR structure and the presumable viral RNA-dependent RNA-polymerase binding site were the most variable. The A2, CS A, CS В elements as well as the start codon were conservative. Interestingly, five substitutions in the 5'-UTR C1 variable element of the TBEVs isolated in different geographical regions were strictly conservative, while 11 different substitutions were detected in this element among the laboratory TBEV variants. A little less that a third of all nucleotide substitutions were mapped outside the main elements of the Y-shaped structure. In general, nucleotide substitutions were localized to stem structures, not being found in the hairpin regions of the TBEV 5'-UTR. The results indicated significant variability of the genomic RNA 5'-UTR in the TBEV laboratory strains and field isolates obtained from different geographical regions. It has been suggested that genetic variability of 5'-UTR is characteristic of the TBEV genome 5'-UTR organization and may serve as a structural basis for virus efficient replication in various avian, mammalian, and ixodic tick cells.