A novel nanobody broadly neutralizes SARS-CoV-2 via induction of spike trimer dimers conformation.

The ongoing mutations of the SARS-CoV-2 pose serious challenges to the efficacy of the available antiviral drugs, and new drugs with fantastic efficacy are always deserved investigation. Here, a nanobody called IBT-CoV144 is reported, which exhibits broad neutralizing activity against SARS-CoV-2 by inducing the conformation of spike trimer dimers. IBT-CoV144 was isolated from an immunized alpaca using the RBD of wild-type SARS-CoV-2, and it showed strong cross-reactive binding and neutralizing potency against diverse SARS-CoV-2 variants, including Omicron subvariants. Moreover, the prophylactically and therapeutically intranasal administration of IBT-CoV144 confers fantastic protective efficacy against the challenge of Omicron BA.1 variant in BALB/c mice model. The structure analysis of the complex between spike (S) protein, conducted using Cryo-EM, revealed a special conformation known as the trimer dimers. This conformation is formed by two trimers, with six RBDs in the "up" state and bound by six VHHs. IBT-CoV144 binds to the lateral region of the RBD on the S protein, facilitating the aggregation of S proteins. This aggregation results in steric hindrance, which disrupts the recognition of the virus by ACE2 on host cells. The discovery of IBT-CoV144 will provide valuable insights for the development of advanced therapeutics and the design of next-generation vaccines.

Consequences of asymmetric mutational pressure for the dynamic of linear B-cell epitopes repertoire of influenza a virus neuraminidase rearrangement.

Full-length nucleotide sequences of avian influenza A virus neuraminidase coding region (20,631 sequences) were analyzed and compared with those isolated from viruses infecting human and swine (63,750 sequences). If in fourfold degenerate sites there is asymmetric A-bias that may be more or less asymmetric depending on the type of neuraminidase and the host, than in twofold degenerate sites from third codon positions there is a strong asymmetric U-bias in coding regions of N4, N5, and N8 isolated from viruses infecting birds, as well as in those of N1 and N2 isolated from viruses infecting human, swine, and birds, while in coding regions of N9 isolated from birds, there is surprisingly strong C-bias, and in sequences of N3, N6, and N7 the usage of C is quite close to the level of U. Revealed stabilization of both U and C in twofold degenerate sites is the evidence of frequent changes in mutational pressure direction. Asymmetric mutational pressure was one of the sources of amino acid replacements that resulted in an equal percentage of sites with appeared and disappeared linear B-cell epitopes in N1, N2, N4, and N5 (33.62-35.33% vs. 32.41-36.45%, respectively), and controlled by the immune pressure it resulted in a stronger tendency to disappear for B-cell epitopes of N3, N6, N7, N8, and N9 of avian viruses (8.74-28.77% vs. 28.96-38.89%). The lack of correlation between nucleotide usages in fourfold and twofold degenerate sites for three nucleotides, except U, is a strong evidence of mutational pressure theory.

Peptide Models of the Cytoplasmic Tail of Influenza A/H1N1 Virus Hemagglutinin Expand Understanding its pH-Dependent Modes of Interaction with Matrix Protein M1.

Influenza A virus hemagglutinin (HA) is a major virus antigen. No cryo-electron microscopy or X-ray data can be obtained for the HA intraviral (cytoplasmic) domain (CT) post-translationally modified with long fatty acid residues bound to three highly conserved cysteines. We recently proposed a model of HA CT of Influenza A/H1N1 virus possessing an antiparallel beta structure based on the experimental secondary structure analysis of four 14-15 amino acid long synthetic peptides, corresponding to the HA CT sequence, with free or acetaminomethylated cysteines. To dispel doubts about possible non-specific "amyloid-like" aggregation of those synthetic peptides in phosphate buffer solution, we have determined the order of oligomers based on blue native gel electrophoresis, membrane filtration, fluorescence spectroscopy and molecular modeling approaches. We have found that unmodified peptides form only low molecular weight oligomers, while modified peptides form both oligomers of low order similar to those found for unmodified peptides and high order conglomerates, which however are not of beta-amyloid-like fold. This study confirms that the beta structure previously detected by circular dichroism spectroscopy analysis is more likely the result of intrinsic propensity of the HA CT amino acid sequence than the consequence of aggregation. The structures of low order oligomers of the synthetic peptides were used for in silico experiments on modeling of HA CT interactions with matrix protein M1 at physiological and acidic pH levels and revealed two different areas of binding. Finally, tripeptides capable of blocking interactions between HA CT and M1 were proposed.

Germline mutations directions are different between introns of the same gene: case study of the gene coding for amyloid-beta precursor protein.

Amyloid-beta precursor protein (APP) is highly conserved in mammals. This feature allowed us to compare nucleotide usage biases in fourfold degenerated sites along the length of its coding region for 146 species of mammals and birds in search of fragments with significant deviations. Even though cytosine usage has the highest value in fourfold degenerated sites in APP coding region from all tested placental mammals, in contrast to marsupial mammals with the bias toward thymine usage, the most frequent germline and somatic mutations in human APP coding region are C to T and G to A transitions. The same mutational AT-pressure is characteristic for germline mutations in introns of human APP gene. However, surprisingly, there are several exceptional introns with deviations in germline mutations rates. The most of those introns surround exons with exceptional biases in nucleotide usage in fourfold degenerated sites. Existence of such fragments in exons 4 and 5, as well as in exon 14, can be connected with the presence of lncRNA genes in complementary strand of DNA. Exceptional nucleotide usage bias in exons 16 and 17 that contain a sequence encoding amyloid-beta peptides can be explained either by the presence of yet unmapped lncRNA(s), or by the autonomous expression of a short mRNA that encodes just C-terminal part of the APP providing an alternative source of amyloid-beta peptides. This hypothesis is supported by the increased rate of T to C transitions in introns 16-17 and 17-18 of Human APP gene relatively to other introns.

Equilibrium Between Dimeric and Monomeric Forms of Human Epidermal Growth Factor is Shifted Towards Dimers in a Solution.

An interplay between monomeric and dimeric forms of human epidermal growth factor (EGF) affecting its interaction with EGF receptor (EGFR) is poorly understood. While EGF dimeric structure was resolved at pH 8.1, the possibility of EGF dimerization under physiological conditions is still unclear. This study aimed to describe the oligomeric state of EGF in a solution at physiological pH value. With centrifugal ultrafiltration followed by blue native gel electrophoresis, we showed that synthetic human EGF in a solution at a concentration of 0.1 mg/ml exists mainly in the dimeric form at pH 7.4 and temperature of 37 °C, although a small fraction of its monomers was also observed. Based on bioinformatics predictions, we introduced the D46G substitution to examine if EGF C-terminal part is directly involved in the intermolecular interface formation of the observed dimers. We found a reduced ability of the resulting EGF D46G dimers to dissociate at temperatures up to 50 °C. The D46G substitution also increased the intermolecular antiparallel ß-structure content within the EGF peptide in a solution according to the CD spectra analysis that was confirmed by HATR-FTIR results. Additionally, the energy transfer between Tyr and Trp residues was detected by fluorescence spectroscopy for the EGF D46G mutant, but not for the native EGF. This allowed us to suggest the elongation and rearrangement of the intermolecular ß-structure that leads to the observed stabilization of EGF D46G dimers. The results imply EGF dimerization under physiological pH value and temperature and the involvement of EGF C-terminal part in this process.

The PentUnFOLD algorithm as a tool to distinguish the dark and the light sides of the structural instability of proteins.

Intrinsically disordered proteins are frequently involved in important regulatory processes in the cell thanks to their ability to bind several different targets performing sometimes even opposite functions. The PentUnFOLD algorithm is a physicochemical method that is based on new propensity scales for disordered, nonstable and stable elements of secondary structure and on the counting of stabilizing and destabilizing intraprotein contacts. Unlike other methods, it works with a PDB file, and it can determine not only those fragments of alpha helices, beta strands, and random coils that can turn into disordered state (the "dark" side of the disorder), but also nonstable regions of alpha helices and beta strands which are able to turn into random coils (the "light" side), and vice versa (H ↔ C, E ↔ C). The scales have been obtained from structural data on disordered regions from the middle parts of amino acid sequences only, and not on their expectedly disordered N- and C-termini. Among other tendencies we have found that regions of both alpha helices and beta strands that can turn into the disordered state are relatively enriched in residues of Ala, Met, Asp, and Lys, while regions of both alpha helices and beta strands that can turn into random coil are relatively enriched in hydrophilic residues, and Cys, Pro, and Gly. Moreover, PentUnFOLD has the option to determine the effect of secondary structure transitions on the stability of a given region of a protein. The PentUnFOLD algorithm is freely available at http://3.17.12.213/pent-un-fold and http://chemres.bsmu.by/PentUnFOLD.htm .

Translation-Associated Mutational U-Pressure in the First ORF of SARS-CoV-2 and Other Coronaviruses.

Within 4 months of the ongoing COVID-19 pandemic caused by SARS-CoV-2, more than 250 nucleotide mutations have been detected in ORF1ab of the virus isolated from infected persons from different parts of the globe. These observations open up an obvious question about the rate and direction of mutational pressure for further vaccine and therapeutics designing. In this study, we did a comparative analysis of ORF1a and ORF1b by using the first isolate (Wuhan strain) as the parent sequence. We observed that most of the nucleotide mutations are C to U transitions. The rate of synonymous C to U transitions is significantly higher than the rate of non-synonymous ones, indicating negative selection on amino acid substitutions. Further, trends in nucleotide usage bias have been investigated in 49 coronaviruses species. A strong bias in nucleotide usage in fourfold degenerate sites toward uracil residues is seen in ORF1ab of all the studied coronaviruses: both in the ORF1a and in the ORF1b translated thanks to the programmed ribosomal frameshifting that has an efficiency of 14 - 45% in different species. A more substantial mutational U-pressure is observed in ORF1a than in ORF1b perhaps because ORF1a is translated more frequently than ORF1b. Mutational U-pressure is there even in ORFs that are not translated from genomic RNA plus strands, but the bias is weaker than in ORF1ab. Unlike other nucleotide mutations, mutational U-pressure caused by cytosine deamination, mostly occurring during the RNA plus strand replication and also translation, cannot be corrected by the proof-reading machinery of coronaviruses. The knowledge generated on the mutational U-pressure that becomes stronger during translation of viral RNA plus strands has implications for vaccine and nucleoside analog development for treating COVID-19 and other coronavirus infections.

The history of mutational pressure changes during the evolution of adeno-associated viruses: A message to gene therapy and DNA-vaccine vectors designers.

The use of virus-associated vectors for gene therapy and vaccination have emerged as safe and effective delivery system. Like all other genetic materials, these vehicles are also prone to spontaneous mutations. To understand what types of nucleotide mutations are expected in the vector, one needs to know distinct characteristics of mutational process in the corresponding virus. In this study we analyzed mutational pressure directions along the length of the genomes of all types of primate adeno-associated viruses (AAV) that are frequently used in gene therapy or DNA-vaccines. We observed clear evidences of transcription-associated mutational pressure in AAV: nucleotide usage biases are changing drastically after each of the three promoters: the higher the rate of transcription, the stronger the bias towards GC to AT mutations. Moreover, the usage of G decreased at the lower transcription rate (after P19 promoter) than the usage of C (after P40 promoter). Since nucleotide usage biases are retrospective indices, we created a scenario of changes in transcriptional map during the AAV evolution. Current mutational pressure directions are different for AAV types, while all of them demonstrate high rates of T to C transitions in the second long ORF. Since transcription rate and cell tropism are the main factors determining the preferable direction of nucleotide mutations in AAV, mutational pressure should be checked experimentally in DNA vectors before their final design with the aim to make the transferred gene more stable against those mutations.

Cobalt(ii) cation binding by proteins.

Herein, a set of non-homologous proteins (238) that could bind the cobalt(ii) cations was selected from all the available Protein Data Bank structures with Co2+ cations. The secondary structure motifs around the amino acid residues that most frequently bind the Co2+ cations (His, Asp, and Glu) as well as the amino acid contents of the inner and outer spheres of complexes were studied. The residues forming coordination bonds to Co2+ (from the inner spheres of the complexes) are overrepresented in the regions of random coil between two ß strands, between a ß strand and α helix, and in all types of ß strands, except that situated between an α helix and ß strand. The residues situated at a distance of less than 5 Å from the Co2+ cations, but unable to form coordination bond to them (from the outer spheres of the complexes), are overrepresented in the regions of coil between the ß strand and α helix and between two ß strands. The data obtained for the Co2+ binding sites was compared with the data obtained for the Mg2+ and Mn2+ binding sites. Although the preferable motifs of the secondary structure for Co2+ binding (beta strand-loop-beta strand and beta strand-loop-alpha helix) are the same as those for Mg2+ and Mn2+, there are some differences in the amino acid contents of the inner and outer spheres of these complexes. The Co2+ cations are preferably coordinated by a combination of His and Glu residues, whereas the Mn2+ and Mg2+ cations prefer a combination of His and Asp and just Asp residues, respectively. As a result, two computer algorithms were developed that could evaluate the possibility of Mg2+ and Mn2+ replacement by the Co2+ cations (chemres.bsmu.by). These algorithms should help to investigate the pathogenesis of cobalt intoxication occurring in patients with cobalt-containing artificial joints.

Microenvironment of tryptophan residues in proteins of four structural classes: applications for fluorescence and circular dichroism spectroscopy.

In this study we examined microenvironment of Trp residues in "dry" sets of nonhomologous proteins that belong to four structural classes, as well as in a "wet" set. In silico experiments showed that residues of Trp demonstrate higher surface accessibility in proteins of "alpha/beta" class where they are rarely included in beta strands. However, this feature has not caused "red" shift in fluorescence spectra in "alpha/beta" proteins in vitro, since there are several factors that should be combined together to cause it: high surface accessibility and high hydrophilicity of the microenvironment, the presence of destabilizing contacts with Asp, Asn, Leu, and multiple Tyr residues, as well as the lack of stabilizing interactions with Arg, Thr, and Pro. The occurrence of Trp residues has the highest value in beta-structural proteins, while they are not involved in aromatic-aromatic interactions with each other as frequently, as they do in proteins of "alpha + beta" class in which Trp residues are overrepresented near each other in the primary sequence. That is why the deformation of circular dichroism spectra because of Trp-Trp interactions is expected to be more frequent in proteins of "alpha + beta" class. In all four classes of proteins Trp residues are involved in long-range interactions with some hydrophobic (Leu, Val, Ile) and aromatic residues (Trp, Phe, and Tyr) more frequently than it is expected. They are involved in long-range interactions with some hydrophilic residues (Asp, Glu, Ser, and Lys) rarely than it is expected. Short-range interactions between Arg and Trp are overrepresented just in alpha-helical proteins.

Mutational pressure and natural selection in epidermal growth factor receptor gene during germline and somatic mutagenesis in cancer cells.

In this study we investigated nucleotide usage biases along the length of a gene encoding human epidermal growth factor receptor (EGFR) and found out that there had been mutational GC-pressure with stronger asymmetric C-pressure in that gene before the preferable direction of nucleotide mutations changed. Current preferable direction of germline mutations in EGFR gene has been estimated with the help of Ensembl data base of gene variations. Preferable direction of somatic mutations in EGFR gene from cancer cells has been estimated with the help of COSMIC data base. Both germline and somatic mutations in cancer cells have the same GC to AT preferable direction in EGFR gene. These data have been used with the aim to find fragments of EGFR gene that have lower probability of missense C to T and G to A transitions to occur. So, the less mutable parts of extracellular EGFR domain are: C-terminal part of the first beta barrel and the central part of the second beta barrel. The less mutable parts of tyrosine kinase EGFR domain are: ATP-binding site (partially), regulatory alpha helix, and fragments that change their secondary structure during the activation process. These parts of EGFR should be considered as the best targets for new types of therapy development. Such criterion as low mutability is especially important for the selection of targets for anti-tumor therapy, since we have detected positive selection of amino acid replacements during somatic mutagenesis of EGFR gene in cancer cells.

Selection and structural analysis of the NY25 peptide - A vaccine candidate from hemagglutinin of swine-origin Influenza H1N1.

The aim of this study was to construct a vaccine peptide candidate against pandemic Influenza H1N1 hemagglutinin and to test its structure. With the help of bioinformatic algorithms we showed that the sequence encoding the second polypeptide of pandemic Influenza H1N1 hemagglutinin (HA2) is protected from nonsynonymous mutations better than the sequence encoding its first polypeptide (HA1). With the help of secondary and ternary structure predicting algorithms we found the fragment of HA2 with the most reproducible secondary structure and synthesized the NY25 peptide corresponding to the residues Asn117 - Tyr141 of HA2. According to the circular dichroism spectra analysis, the peptide has short helix and beta hairpin. According to the analysis of differential fluorescence quenching results, two tyrosine residues are situated on a long distance from each other. These facts taken together with the positive results of affine chromatography with the serum of a person immunized by full-length hemagglutinin confirm that the structure of the fragment of viral full-length protein has been reproduced in the synthetic NY25 peptide. Amino acid sequence of the NY25 peptide (NLYEKVRSQLKNNAKEIGNGCFEFY) is relatively conserved in 18 subtypes of Influenza A virus hemagglutinin.

Amino acid content of beta strands and alpha helices depends on their flanking secondary structure elements.

The influence of flanking structures (alpha helices and beta strands in the primary sequence) on amino acid content of the elements of secondary structure has been analyzed in seven sets of nonhomologous proteins. Elevated usage of beta structural amino acid residues and pentapeptides in beta strands between two alpha helices can be explained by the stabilization of secondary structure of those beta strands by natural selection. High usage of alpha helical amino acids and pentapeptides in beta strands situated between two other beta strands is an evidence of the relaxation of natural selection: "passive" beta strands in these fragments of polypeptide chains are frequently formed due to the influence of flanking "active" beta strands. Alpha helices situated between alpha helix and beta strand are enriched by alpha helical pentapeptides and have lower usage of beta structural pentapeptides than those situated between beta strand and alpha helix, their N-termini are more frequently protected from helix to beta transitions by Glu residues. Alpha helices between two beta strands are stabilized by Ala residues and pentapeptides specific to alpha helices. Dipeptide content of the most stable alpha helices and beta strands was used for the creation of the PentaFOLD 2.0 algorithm (http://chemres.bsmu.by) that finds stable fragments of these elements of secondary structure in PDB files.

The alpha helix 1 from the first conserved region of HIV1 gp120 is reconstructed in the short NQ21 peptide.

Investigations of short peptides that can be used in the next phase of synthetic HIV1 vaccine development are an urgent goal, as well as investigations of peptides that can be used in immunological tests with the aim to check the titer of antibodies against the alpha helix 1 from the first conserved region of HIV1 gp120 that are known to cause antibody-dependent cellular cytotoxicity (ADCC). The aim of this work was to study the structure of the NQ21 peptide corresponding to the less mutable part of the first conserved region of HIV1 gp120 (residues 94-114). The NQ21 peptide and its conjugate with biotin (biotin-NQ21) are absolutely alpha-helical in phosphate buffer solutions at pH = 6.8, 7.4 and 8.0, as well as in the dried form, according to the results of surface-enhanced Raman scattering (SERS) spectroscopy. Results of the native gel electrophoresis and thermal analysis under the control of spectrofluorometer and near UV circular dichroism (CD) showed that the peptide exists in form of octamers and tetramers at pH = 7.4, that is important information for further vaccine development. Strong signal of interacting Trp residues in oligomers in the far UV CD obscures the signal from secondary structure, but becomes less intensive during the heating.

Ethanol binding sites on proteins.

This study is on the analysis of ethanol binding sites on 3D structures of nonredundant proteins from the Protein Data Bank. The only one amino acid residue that is significantly overrepresented around ethanol molecules is Tyr. There are usually two or more Tyr residues in the same ethanol binding site, while residues of Thr, Asp and Gln are underrepresented around them. Residues of Ala and Pro are significantly underrepresented in ethanol binding surfaces. Several residues (Phe, Val, Pro, Ala, Arg, His, Ser, Asp) bind ethanol significantly more frequent if they are not included in beta strands. Residues of Ala, Ile and Arg preferably bind ethanol when they are included in an alpha helix. Ethanol molecules often make hydrogen bonds with oxygen and nitrogen atoms from the main chain of a protein. Because of this reason, the binding of ethanol may be associated with the decrease of the length of alpha helices and the disappearance of 3/10 helices. Obtained data should be useful for studies on new targets of the direct action of ethanol on enzymes, receptors, and transcription factors.

Transcription-associated mutational pressure in the Parvovirus B19 genome: Reactivated genomes contribute to the variability of viral populations.

In this study we used non-overlapping parts of the two long open reading frames coding for nonstructural (NS) and capsid (VP) proteins of all available sequences of the Parvovirus B19 subgenotype 1a genome and found out that the rates of A to G, C to T and A to T mutations are higher in the first long reading frame (NS) of the virus than in the second one (VP). This difference in mutational pressure directions for two parts of the same viral genome can be explained by the fact of transcription of just the first long reading frame during the lifelong latency in nonerythroid cells. Adenine deamination (producing A to G and A to T mutations) and cytosine deamination (producing C to T mutations) occur more frequently in transcriptional bubbles formed by DNA "plus" strand of the first open reading frame. These mutations can be inherited only in case of reactivation of the infectious virus due to the help of Adenovirus that allows latent Parvovirus B19 to start transcription of the second reading frame and then to replicate its genome by the rolling circle mechanism using the specific origin. Results of this study provide evidence that the genomes reactivated from latency make significant contributions to the variability of Parvovirus B19.

The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein.

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C-half of the second helix and N-half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179-Cys214) failed because of the beta hairpin formation in the region 206-MERVVEQMC-214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn(2+) cations, which causes small temperature-associated structural shifts at concentrations of 2 - 10·10(-6) M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184-IKQHTVT-190 and 197-TETDVKM-205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462-1479. © 2016 Wiley Periodicals, Inc.

Magnesium and manganese binding sites on proteins have the same predominant motif of secondary structure.

Manganese ion (Mn(2+)) can substitute magnesium ion (Mg(2+)) in active sites of numerous enzymes. Binding sites for these two ions have been studied in two sets of protein 3D structures from the Protein Data Bank with the homology level lower than 25%. The structural motif "beta strand - binder - random coil" is predominant in both Mn(2+) and Mg(2+) coordination spheres, especially in functionally relevant ones. That predominant motif works as an active binder of those divalent cations which can then attract additional ligands, such as different phosphate-containing compounds. In contrast, such Mg(2+) and Mn(2+) binding motif as "GK(T/S)T" being the N-terminal part of alpha helices works as an active binder of phosphates which can then attract divalent cations. There are few differences between Mg(2+) and Mn(2+) coordination spheres responsible of the cation specificity. His residues are underrepresented in certain positions around Asp and Glu residues involved in Mg(2+) coordination, while they are overrepresented in certain positions around Asp and Glu residues coordinating Mn(2+). The random coil region in the "beta strand - random coil - alpha helix" motif for Mg(2+) binding is usually shorter than that in the same motif for Mn(2+) coordination. This feature is associated with the lower number of binding amino acids (and lower levels of usage of such "major" binders as Asp and Glu) for Mg(2+) (which is a hard Lewis acid) in comparison with those for Mn(2+) (an intermediate Lewis acid).

Opposite nucleotide usage biases in different parts of the Corynebacterium diphtheriae spaC gene.

In this work we described a bacterial open reading frame with two different directions of nucleotide usage biases in its two parts. The level of GC-content in third codon positions (3GC) is equal to 40.17 ± 0.22% during the most of the length of Corynebacterium diphtheriae spaC gene. However, in the 3'-end of the same gene (from codon #1600 to codon #1873) 3GC level is equal to 64.61 ± 0.91%. Using original methodology ('VVTAK Sliding window' and 'VVTAK VarInvar') we approved that there is an ongoing mutational AT-pressure during the most of the length of spaC gene (up to codon #1599), and there is an ongoing mutational G-pressure in the 3′-end of spaC. Intragenic promoters predicted by three different methods may be the cause of the differences in preferable types of nucleotide mutations in spaC parts because of their autonomous transcription.

Local Mutational Pressures in Genomes of Zaire Ebolavirus and Marburg Virus.

Heterogeneities in nucleotide content distribution along the length of Zaire ebolavirus and Marburg virus genomes have been analyzed. Results showed that there is asymmetric mutational A-pressure in the majority of Zaire ebolavirus genes; there is mutational AC-pressure in the coding region of the matrix protein VP40, probably, caused by its high expression at the end of the infection process; there is also AC-pressure in the 3'-part of the nucleoprotein (NP) coding gene associated with low amount of secondary structure formed by the 3'-part of its mRNA; in the middle of the glycoprotein (GP) coding gene that kind of mutational bias is linked with the high amount of secondary structure formed by the corresponding fragment of RNA negative (-) strand; there is relatively symmetric mutational AU-pressure in the polymerase (Pol) coding gene caused by its low expression level. In Marburg virus all genes, including C-rich fragment of GP coding region, demonstrate asymmetric mutational A-bias, while the last gene (Pol) demonstrates more symmetric mutational AU-pressure. The hypothesis of a newly synthesized RNA negative (-) strand shielding by complementary fragments of mRNAs has been described in this work: shielded fragments of RNA negative (-) strand should be better protected from oxidative damage and prone to ADAR-editing.