Magdalena Alejska

Genetic variability: the key problem in the prevention and therapy of RNA-based virus infections.

Despite extraordinary progress that has recently been made in biomedical sciences, viral infectious diseases still remain one of the most serious world health problems. Among the different types of viruses, those using RNA as their genetic material (RNA viruses and retroviruses) are especially dangerous. At present there is no medicine allowing an effective treatment of RNA‐based virus infections. Many RNA viruses and retroviruses need only a few weeks to escape immune response or to produce drug‐resistant mutants. This seems to be the obvious consequence of the unusual genetic variability of RNA‐based viruses. An individual virus does not form a homogenous population but rather a set of similar but not identical variants. In consequence, RNA‐based viruses can easily adapt to environmental changes, also those resulting from immune system response or therapy. The modifications identified within viral genes can be divided into two groups: point mutations and complex genome rearrangements. The former arises mainly during error‐prone replication, whereas RNA recombination and generic reassortment are responsible for the latter. This article shortly describes major strategies used to control virus infections. Then, it presents the various mechanisms generating the genetic diversity of RNA‐based viruses, which are most probably the main cause of clinical problems.

Homologous Crossovers among Molecules of Brome Mosaic Bromovirus RNA1 or RNA2 Segments In Vivo

ABSTRACT Previously we demonstrated frequent homologous crossovers among molecules of the RNA3 segment in the tripartite brome mosaic bromovirus (BMV) RNA genome (A. Bruyere, M. Wantroba, S. Flasinski, A. Dzianott, and J. J. Bujarski, J. Virol. 74:4214-4219, 2000). To further our knowledge about mechanisms of viral RNA genome variability, in this paper we have studied homologous recombination in BMV RNA1 and RNA2 components during infection. We have found that basal RNA-RNA crossovers could occur within coding regions of both RNAs, although recombination frequencies slightly varied at different RNA sections. In all cases, the frequencies were much lower than the rate observed for the intercistronic recombination hot spot in BMV RNA3. Probability calculations accounted for at least one homologous crossover per RNA molecule per replication cycle. In addition, we have demonstrated an efficient repair of mutations within the conserved 3′ and 5′ noncoding regions, most likely due to error-prone BMV RNA replication. Overall, our data verify that homologous crossovers are common events a during virus life cycle, and we discuss their importance for viral RNA genetics.

Hepatitis C virus quasispecies in chronically infected children subjected to interferon-ribavirin therapy.

Accumulating evidence suggests that certain features of hepatitis C virus (HCV), especially its high genetic variability, might be responsible for the low efficiency of anti-HCV treatment. Here, we present a bioinformatic analysis of HCV-1a populations isolated from 23 children with chronic hepatitis C (CHC) subjected to interferon–ribavirin therapy. The structures of the viral quasispecies were established based on a 132-amino-acid sequence derived from E1/E2 protein, including hypervariable region 1 (HVR1). Two types of HCV populations were identified. The first type, found in non-responders, contained a small number of closely related variants. The second type, characteristic for sustained responders, was composed of a large number of distantly associated equal-rank variants. Comparison of 445 HVR1 sequences showed that a significant number of variants present in non-responding patients are closely related, suggesting that certain, still unidentified properties of the pathogen may be key factors determining the result of CHC treatment.

The brome mosaic virus-based recombination vector triggers a limited gene silencing response depending on the orientation of the inserted sequence.

In some RNA viruses (e.g. in brome mosaic virus, BMV), the same factor (intra- or intermolecular hybridization between viral RNA molecules) is capable of inducing two different processes: RNA silencing and RNA recombination. To determine whether there is some interplay between these two phenomena, we have examined if the BMV-based recombination vector containing a plant-genome-derived sequence can function as a gene-silencing vector. Surprisingly, we found that neither dsRNA forming during the replication of the BMV-based vector nor highly structured regions of its genome were effective RNAi triggers. Only mutants carrying a sequence complementary to the target mRNA functioned as gene silencing vectors and were steadily maintained in the infected plant. The constructs containing a sense sequence or inverted repeats did not induce gene silencing but instead were eliminated from the plant cells.

Crystal Structure of 2′-Deoxycytidine Hemidihydrogenphosphate Reveals C+·C Base Pairs and Tight, Hydrogen-Bonded (H2PO4 −)∞ Columns (1)

Abstract 2′-Deoxycytidine hemidihydrogenphosphate has been crystallized in the hexagonal space group P62 with α=25.839(3), c = 12.529(1) A. The structure has been solved using the Patterson search method. The asymmetric unit contains two protonated, base-paired 2′-deoxycytidine dimers and two H2PO4 − anions. The C+·C base pairs are composed of a protonated and a neutral species each and are triple H-bonded, the central N(3)…N(3) bonds being 2.850(7) and 2.884(5) A. The conformations of the four nucleosides fall in the same category (sugar puckers 2·-endo, glycosidic links anti) but in one of them the glycosidic torsion angle is quite low with consequences in other geometrical parameters. The H2PO4 − anions are located on twofold axes and form two types of tight columns with P…P separations about 4.18 A The neighboring units along a column are linked via two very short O…H…O hydrogen bonds (O…O about 2.49 A) leading to effective equalization of the P-O bonds. The base pairs of the two dC+·dC cations are co...

Evolution of hepatitis C virus hypervariable region 1 in chronically infected children.

Hepatitis C virus (HCV) quasispecies diversification plays an essential role in the establishment of chronic infections. Our earlier analysis of HCV population structure in children subjected to interferon-ribavirin treatment demonstrated that viral quasispecies is homogenous in patients who failed to respond to the therapy and heterogeneous in sustained responders. We also showed that certain variants of HCV hypervariable region 1 (HVR1) are conserved in non-responders. To better elucidate the pathways of HCV evolution, here we examined the changes of HVR1 in viral populations isolated from sera of eight treatment-naive pediatric patients. We found that HCV evolution in untreated chronically infected children occurs according to two pathways and results in the formation of either genetically homogenous or variable quasispecies. Variable populations are prone to quasispecies shifts. In contrast, homogenous populations are composed of closely related variants that undergo only minor changes. In addition, we observed that a phenomenon of inter-quasispecies conservation of HVR1 is associated with some of the homogenous HCV populations. The collected data suggest that there exist HVR1 variants with superior fitness, capable of persisting in different hosts.

The crystal engineering of cytidine (Cyd), 2′-deoxycytidine (dCyd) and their phosphate salts Part 3. The main stages of the transcrystallization processes of the solid-state mixture in which dCyd mono(dihydrogenphosphate) salt is transformed into dCyd hemi(dihydrogenphosphate) salt and vice versa☆

Abstract Two binary crystalline mixtures are transformed in the solid state into homogeneous dCyd dihydrogenphosphate salts. Mixture 1 is transformed into the dCyd hemi(dihydrogenphosphate) salt and mixture 2 into the dCyd mono(dihydrogenphosphate) salt. The crystal structures of both salts are drastically different. For both transformational processes we propose two different molecular mechanisms on the basis of FT-IR-PAS spectra, thermochemical data (DSC) and a simple computerographic approach. For mixture 1, we postulate a two-stage process. In the first stage, the formation of the active collision complex takes place at the border of the crystalline components of the mixture. The crucial transfer of protons from non-bonded pairs of cations (dCydH+)2 to hydrogen-bonded pairs of neutral dCyd molecules (dCyd ⋯ dCyd)n is accomplished. For mixture 2, we postulate a one-stage mechanism in which external H3PO4 molecules easily penetrate into the crystal lattice of the dCyd hemi(dihydrogenphosphate) salt and act simultaneously as a degrading agent of the crystal lattice of the hemiphosphate salt, and as a rebuilding agent of the monophosphate salt. In mixture 2 the crucial factor is the very fascinating array of polyassociation motifs forming the network of hydrogen bonds around the perpendicular polyanionic columns which create the “empty space” ready for dislocation of the external H3PO4 molecules in an ordered manner.

Crystal engineering of cytidine and deoxycytidine sulphates. I. Preparation, and unusual properties.

Abstract From three possible simple salts of Cyd (dCyd)1 with H2SO4, we succeeded in preparation of only one type of a crystalline salt in which two monoprotonated cations: 2CydH+ (2dCydH+) pair with one sulphate dianion (SO2−4) (Fig.3)4. Attempts to obtain crystalline sulphates with dimeric hemi-cations and/or hydrogen sulphate anions completely failed. The presence or absence of 2′OH group drastically changes the thermochemical properties of Cyd and dCyd sulphates (Fig.4). Cyd sulphate undergoes rapid one-step thermal decomposition within the range 205–260°C with subsequent smoth further mass loss up to 340°C, whereas dCyd sulphate (Form A and B) undergoes a three stage thermal decomposition: I. 25–170°C, II. 170–230°C, III. 230–340°C. Clear differences in the DSC and TGA curves of both types of salt may reflect dissimilar mechanisms of their crystal structure decomposition. An extremely easy transformation of dCyd sulphates into the hydrochloride salt in the crystal phase was discovered. The reaction proceeds both in finely ground stoichiometric mixtures of 2dCydH+·SO2−4 + 2NaCl or KCl, and also in a nujol suspension of pure dCyd sulphate placed between NaCl plates. Similar transformation occurs also with KBr and leads to the crystalline dCyd hydrobromide which is isostructural to dCyd hydrochloride. Form A of dCyd sulphate, which contains methanol trapped in its crystal lattice, is particularly susceptible to the above transformation. Cyd sulphate undergoes transformation to hydrochloride and hydrobromide salts by a few orders of magnitude slower. These unidirectional ion exchange reactions proceed quantitatively in crystalline phase only, showing the significance of crystal structures of nucleosides and their salts. Unusual properties of sulphate counter anions may be exploited for a controlled modulation of reactivity of nucleosides. The unidirectional ion exchange reactions take place in 100 % in crystalline phase only , revealing the significance of information coded in crystal structures of nucleosides.

A new outlook on the nature of short intramolecular non-bonded contacts between 6CH⋯O5′ in the crystals of pyrimidine nucleosides and their salts☆

Abstract Short intramolecular contacts between the 6CH group and the O5′ oxygen have been propounded for a long time in the description of structures of some native pyrimidine nucleosides and their derivatives. The character of these interactions was not, however, fully resolved. On the basis of X-ray investigation, it is assumed that these contacts have the character of hydrogen bonds. For some time, however, doubts have been growing as to the validity of this opinion. Consequently we have started detailed investigations to elucidate this problem. Studies carried out for cytidine (Cyd), ethenocytidine (ϵCyd) and their hydrochloride, nitrate, and dihydrogenphosphate salts, were based upon X-ray analysis, IR spectra, and calculations of electron density by the CNDO/2 method. A detailed comparative examination of IR spectra of the eight chosen nucleoside, four of which showed evidence for the presence of such a short contact, proved that the relevant contact could not have the character of an intramolecular hydrogen bond, but instead has a repulsive nature. This conclusion was supported by hypsochromic shifts of the absorption bands of 6CH stretching vibrations participating in short intramolecular contacts, as well as by the positive value of electron density on the carbon atom 6C. On the basis of computer diagrams of the same projectiobs for the crystal structures of all studied compounds, we also tried to find the factors responsible for the existence or absence of the short intramolecular repulsive contact, and to predict the effect of such a contact on the conformational and functional dynamics of nucleic acid fragments.

Crystal and molecular structure of putrescinium hydrogen phosphate dihydrate

AbstractThe X-ray structure of putrescinium hydrogen phosphate dihydrate,