[Structure of Potato Virus A Coat Protein Particles and Their Dissociation].

This paper reports on a complex structural analysis of the potato virus A coat protein using a set of complementary physico-chemical methods. We have demonstrated previously that this protein does not exist as individual subunits in solution and undergoes association into oligomers with subsequent transition to ß-conformation. The purpose of the present work was to study the possible mechanisms of this transformation and to search for methods that dissociate protein oligomers. To analyze the low resolution protein structure in solution, small-angle X-ray scattering was used. Stable particles representing clusters of 30 coat protein subunits were present even in an aqueous salt solution with a high ionic strength and pH (pH 10.5; 0.5 M NaCl). The particles did not dissociate in the presence of 10 mM dextran sulfates (15 and 100 kDa). Dissociation in the presence of 5.2 mM sodium dodecyl sulfate results in the formation of the subunit-detergent complexes consisting of 10-12 small particles joined together like "beads on a string". Similar effects of sodium dodecyl sulfate were shown for serum albumins (bovine and human). Denaturation of the potato virus A coat protein molecules occurs in the presence of detergent concentrations that are seven times lower than that in albumins (5.2 and 35 mM), which confirms low stability of the potato virus A coat protein. Using spectral methods, preservation of the secondary structure and loss of the tertiary structure of the protein in its complex with sodium dodecyl sulfate have been demonstrated. Possible mechanism for protein particle formation through the interaction between unordered terminal domains and their transformation into ß-structures has been suggested.

Identification of Anaplasma phagocytophilum in tick populations in Estonia, the European part of Russia and Belarus.

Anaplasma phagocytophilum is associated with diseases of goats, sheep, cattle, dogs and horses. In the beginning of the 1990s it was identified as a human pathogen, causing human granulocytic anaplasmosis (HGA) in the USA, Europe and the far east of Russia. A. phagocytophilum is maintained in nature in an enzootic cycle including ticks as the main vector and a wide range of mammalian species as reservoirs. Ixodes ricinus and I. persulcatus ticks were collected in Estonia, Belarus and the European part of Russia and screened for the presence of A. phagocytophilum by real-time PCR. Positive samples were found only among I. ricinus, in 13.4% in the European part of Russia, 4.2% in Belarus, 1.7% in mainland Estonia and 2.6% on Saaremaa Island. Positive samples were sequenced for partial 16S rRNA, groESL and ankA genes and phylogenetic analyses were performed. The results showed that A. phagocytophilum circulating in Eastern Europe belongs to different groESL lineages and 16S rRNA gene variants and also consists of variable numbers of repetitive elements within the ankA gene.

In situ spatial organization of Potato virus A coat protein subunits as assessed by tritium bombardment.

Potato virus A (PVA) particles were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the label in the amino acids of the coat protein was determined to assess their in situ steric accessibility. This method revealed that the N-terminal 15 amino acids of the PVA coat protein and a region comprising amino acids 27 to 50 are the most accessible at the particle surface to labeling with tritium atoms. A model of the spatial arrangement of the PVA coat protein polypeptide chain within the virus particle was derived from the experimental data obtained by tritium bombardment combined with predictions of secondary-structure elements and the principles of packing alpha-helices and beta-structures in proteins. The model predicts three regions of tertiary structure: (i) the surface-exposed N-terminal region, comprising an unstructured N terminus of 8 amino acids and two beta-strands, (ii) a C-terminal region including two alpha-helices, as well as three beta-strands that form a two-layer structure called an abCd unit, and (iii) a central region comprising a bundle of four alpha-helices in a fold similar to that found in tobacco mosaic virus coat protein. This is the first model of the three-dimensional structure of a potyvirus coat protein.

Biochemical and genetic evidence for interactions between potato A potyvirus-encoded proteins P1 and P3 and proteins of the putative replication complex.

Interactions of the first and third proteins (P1 and P3) of the potato A potyvirus (PVA) with the other six main proteins of PVA were studied using Escherichia coli-expressed recombinant proteins in two in vitro interaction assays and a genetic assay yeast two-hybrid system (YTHS). In overlay blotting and binding assays in liquid, P1 and P3 interacted with each other and with proteins of the putative replication complex of potyvirus: RNA-helicase (CI), viral protein genome-linked (VPg), NIa proteinase part (NIaPro), and RNA-dependent-RNA-polymerase (NIb). In addition, P1 self-interaction and interaction with helper-component proteinase (HC-Pro) also were detected. Neither P1 nor P3 interact with coat protein (CP) or with various control proteins. In the YTHS, P1 interacted only with CI and P3 with NIb. The different results obtained using the two test systems may reflect changes in interactions at different stages of potyvirus infection: in the virus genome replication and the virion accumulation stages when nonstructural proteins form inclusions. Our data are consistent with previous functional data, indicating that P1 and P3 proteins are involved in potyvirus genome amplification and provide the first direct evidence that these proteins interact with the proteins that have been shown to be part of the replication complex.

Comparison of the nucleotide sequences of the 3'-terminal regions of one aphid and two non-aphid transmissible isolates of potato A potyvirus.

The sequences of the 3'-terminal 1145 nucleotides of two non-aphid transmissible (NAT) isolates (Ali and Juliniere) and one aphid transmissible (AT) isolate (Rouge) of potato virus A (PVA) RNA were determined. Those sequences contained the complete coding region of the coat protein (CP) followed by a 3'-nontranslated region (3'-NTR) of 225 (Ali and Juliniere) and 227 (Rouge) nucleotides. The obtained sequences were compared to those of the 3'-regions of four published PVA isolates and a virus described as tamarillo mosaic virus (TamMV) which on the basis of sequence data is a strain of PVA. The analysis of the 3'-terminal region of PVA isolates indicated that the CP N-terminal variable domain (32 residues) divides PVA isolates into two subgroups, where only tripeptide DAG correlates with aphid transmissibility. In addition to DAG/DAS sequence we found four other amino acids at the N-terminus of PVA CP, which are conserved in two subgroups. The central region (core part and C-termini) of CP is highly conserved among all PVA isolates (96.6 to 99.6%). 3'-NTR, separates PVA-isolates into two subgroups on the basis of its length and homology.

The organization of potato virus X coat proteins in virus particles studied by tritium planigraphy and model building.

Potato virus X particles containing the intact, undegraded Ps form of the coat protein and particles containing the in situ degraded Pf form of the coat protein, which is devoid of 19-21 amino acids from the N-terminus, were bombarded with thermally activated tritium atoms, and the intramolecular distribution of the tritium label was studied. The tritium planigraphy revealed that the N-terminal region of the coat protein is the most accessible region for both type of PVX particles. The C-terminal region of the coat protein in the intact virus particles is almost inaccessible to the hot tritium atoms, whereas in Pf particles this region becomes available for the tritium label. A model of PVX coat protein tertiary structure was built, taking into account the predicted secondary structure of the protein, the principles of packing alpha-helices and beta-structure in globular proteins, and known biochemical, immunological, and tritium bombardment data. In the model one beta-sheet consisting of beta-strands at regions 1-12, 14-22, and 24-33 flanks the molecule and forms the outside surface of the PVX particles.

The topography of the surface of potato virus X: tritium planigraphy and immunological analysis.

Thermally activated tritium atoms were used to probe the surface topography of the coat protein of potato virus X (PVX) potexvirus. The accessibility profile of amino acid residues in the polypeptide chain was determined from data on the intramolecular distribution of tritium label in the PVX coat protein. Tryptic peptides T1 and T2, as well as parts of peptides T3 and T5, from the PVX particles were all located in the N-terminal region of the PVX coat protein and were accessible to tritium labelling, whereas the C-terminal region of the coat protein was practically inaccessible to it. Indirect ELISA and immunoblotting with two PVX-specific monoclonal antibodies confirmed that the N terminus of the coat protein (residues 1 to 56) was exposed on the virus surface, and furthermore that this region forms a highly immunogenic virus-specific antigenic region. The data obtained support the spatial model of PVX, in which the N-terminal amino acids of the coat protein are exposed at the particle surface, and the C-terminal region is buried in the particle. The spatial organization of the PVX coat proteins differs from the model proposed for other filamentous plant viruses such as potyviruses and tobamoviruses where both the N and C termini of the coat protein are located at the particles' surface.

Investigations of virus-protozoa relationships in the model of the free-living ciliate Tetrahymena pyriformis and adenovirus type 3.

Cultures of the free-living ciliate, Tetrahymena pyriformis, strain GL were experimentally infected with adenovirus type 3 (Ad3). The course of interaction was followed over a five-day period and in four successively passaged subcultures in virus-free medium. Possible presence of viral genomic DNA in the ciliates was assayed by DNA-DNA hybridization, allowing the detection of 100 pg of homologous DNA. The presence of viral protein was tested by time-resolved fluoroimmunoassay (TR FIA) enabling to detect less than 5 ng of Ad3 hexon antigen per ml. In ciliate culture supernatants the presence of virus was tested by TR FIA and by estimation of viral titers (PFU tests). Viral genome was detected in the ciliates over the first two days post-infection and not later. Antigen concentration in the supernatant was negative by the fourth day of the experiment and on the tenth day no virus was detected by PFU tests. During passages in virus-free medium, viral genome was detected only in the ciliates of the first subculture, the antigen concentration was negative after the second passage. In addition, no viral titer could be detected after this time. Our data do not support the hypothesis that Ad3-replication takes place in T. pyriformis.

Simultaneous quantification of two plant viruses by double-label time-resolved immunofluorometric assay.

For simultaneous and sensitive detection of two antigens in one sample, monoclonal antibody (MAb) to potato virus M (PVM) was labelled with a lanthanide Eu3+ and MAb to potato virus X (PVX) with another lanthanide Sm3+. A mixture of the labelled MAbs was used as a tracer. After performing the immunoreactions, the fluorescence of the dissociated lanthanides was measured at different wave-lengths in a time-resolved fluorometer to quantificate the PVX and PVM amount in a sample. Double-label time-resolved fluoroimmunoassay (TR-FIA) detected 1 ng/ml of each virus and was therefore more sensitive for simultaneous detection of PVX and PVM than reported for a single virus detection with double antibody sandwich ELISA (DAS-ELISA).

Changes in chromosome complement in long-term pea callus cultures.

A prolonged callus culture from pea (Pisum sativum L. var. Kiir) cotyledons subcultured for 7.5 years on Torrey's solid medium was examined cytologically. The initially (and up to 3.5-year cultivation) predominantly diploid pea callus strain changed into triploid. The frequency of aberrant ana- and telophases increased during 5 year cultivation from 9 to 40 per cent and thereafter returned to the initial rate maintaining it at least for one year of subculturing. Some possible mechanisms of chromosomal variation tendencies in vitro are discussed.