Epstein-Barr virus stimulates torque teno virus replication: a possible relationship to multiple sclerosis

Viral infections have been implicated in the pathogenesis of multiple sclerosis. Epstein-Barr virus (EBV) has frequently been investigated as a possible candidate and torque teno virus (TTV) has also been discussed in this context. Nevertheless, mechanistic aspects remain unresolved. We report viral replication, as measured by genome amplification, as well as quantitative PCR of two TTV-HD14 isolates isolated from multiple sclerosis brain in a series of EBV-positive and -negative lymphoblastoid and Burkitt's lymphoma cell lines. Our results demonstrate the replication of both transfected TTV genomes up to day 21 post transfection in all the evaluated cell lines. Quantitative amplification indicates statistically significant enhanced TTV replication in the EBV-positive cell lines, including the EBV-converted BJAB line, in comparison to the EBV-negative Burkitt's lymphoma cell line BJAB. This suggests a helper effect of EBV infections in the replication of TTV. The present study provides information on a possible interaction of EBV and TTV in the etiology and progression of multiple sclerosis.     

Comparison of diversity of torque teno virus 1 in different mucosal tissues and disorders

Diversity of TTV1 was assessed in the head and neck region in patients with potentially malignant (oral lichen planus, oral leukoplakia) and malignant lesions (oral and laryngeal squamous cell cancers) and was compared to that found in the uterine cervix (cervical atypia and cervical cancer) by directly sequencing the NG061-063 segment of ORF1. These sequences were classified by the formerly used genogroup-genotype system as well as by the newly accepted species classification by aligning with the corresponding region of the type sequences of the 29 TTV species. All sequences obtained during the study clustered together with the TTV1 type sequence; to express diversity within TTV1, genotypes and subtypes of the former classification were used.The commonest subtypes were 2c followed by 2b, 1a and 1b. Subtypes 2b and 2c were evenly distributed among cervical samples; subtype 1a was more frequent in patients with cervical atypia or cancer. Subtypes 2c was more frequent than 2b in head and neck lesions. In conclusion, genotype and even subtype distribution may be important in association with diseases, therefore using this classification for characterization of intraspecies diversity of TTV1 is proposed.     

Application of cation-coated filter method to detection of noroviruses, enteroviruses, adenoviruses, and torque teno viruses in the Tamagawa River in Japan

The occurrence of human enteric viruses in surface water in the Tamagawa River, Japan, was surveyed for 1 year, from April 2003 to March 2004. Sixty-four samples were collected from six sites along the river, and 500 ml of the sample was concentrated using the cation-coated filter method, which was developed in our previous study. This method showed recovery yields of 56% +/- 32% (n = 37) for surface water samples inoculated with polioviruses. More than one kind of tested virus was detected in 43 (67%) of 64 samples by TaqMan PCR. Noroviruses and adenoviruses were detected in a high positive ratio; 34 (53%), 28 (44%), and 29 (45%) of 64 samples were positive for norovirus genotype 1 and genotype 2 and adenoviruses, respectively. The mean concentrations of norovirus genotype 1 or genotype 2 determined by real-time PCR were 0.087 and 0.61 genome/ml, respectively, showing much higher values in winter (0.21 genome/ml for genotype 1 and 2.3 genomes/ml for genotype 2). Enteroviruses were detected by both direct PCR (6 of 64 samples; 9%) and cell culture PCR (2 of 64 samples; 3%). Torque teno viruses, emerging hepatitis viruses, were also isolated in three samples (5%). The concentration of total coliforms and the presence of F-specific phages showed a high correlation with the presence of viruses, which suggested that the simultaneous use of total coliforms and F-specific phages as indicators of surface water may work to monitor viral contamination.     

Hyperinitiation of DNA replication in Escherichia coli leads to replication fork collapse and inviability

Elevated dnaA expression from a multicopy plasmid induces more frequent initiation from the Escherichia coli replication origin, oriC, but viability is maintained. In comparison, chromosomally encoded dnaAcos also stimulates initiation, but this is lethal. By quantitative methods, we show that the level of initiation induced by elevated dnaA expression leads to collapsed replication forks that are mostly within 10 map units of oriC. Because forks collapse randomly, nucleoprotein complexes at specific sites such as datA are not the cause. When replication restart is blocked by a mutation in recB or priA, the increased initiations via elevated dnaA expression causes inviability. The amount of collapsed forks is substantially higher under elevated expression of dnaAcos compared to that of dnaA. We propose that the lethal phenotype of chromosomally encoded dnaAcos is a result of hyperinitiation that overwhelms the repair capacity of the cell.     

Prevalence and age distribution of porcine torque teno sus virus (TTSuV) in the Czech Republic

Torque teno sus viruses (TTSuV) were detected in the pig population in the Czech Republic by a nested PCR technique. The prevalence of individual TTSuV was found to be 42.9% (TTSuV1) and 46.7% (TTSuV2). The prevalence for TTSuV 1 and TTSuV2 was determined to be 52.7% and 60.6% in piglets at weaning, 90.9% and 63.6% in gilts, and 75% and 87.5% in sows, respectively. Both virus species were detected in 12% of newborn piglets, which suggests possible transplacental transmission. Sequencing of several virus strains showed that the sequenced TTSuV strains belong to two different species of viruses. The average similarity on the nucleotide levels between these two species was 43.3%.     

Dominance and diversity in the primary human CD4 T cell response to replication-competent vaccinia virus

Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication-incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relationship between Ab and CD4 T cell specificity for complex pathogens.     

Extending the cytoplasmic tail of the influenza a virus M2 protein leads to reduced virus replication in vivo but not in vitro

A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.     

Evolution of the rat kallikrein gene family: Gene conversion leads to functional diversity

Summary Kallikrein-like simple serine proteases are encoded by closely related members of a gene family in several mammalian species. Molecular cloning and genomic Southern blot analysis after conventional and pulsed-field gel electrophoresis indicate that the rat kallikrein gene family comprises 15–20 members, probably closely linked at a single locus. Determination of the nucleotide sequences of the rGK-3,-4, and-6 genes here completes sequence data for a total of nine rat kallikrein family members. Comparison of the rat gene sequences to each other and to those of human and mouse kallikrein family genes reveals patterns of relatedness indicative of concerted evolution. Analysis of nucleotide sequence variants in kallikrein family members shows that most sequence variants are shared by multiple family members; the patterns of shared variants are complex and indicate multiple short gene conversions between family members. Sequence exchanges between family members generate novel assortments of variants in amino acid coding regions that may affect substrate specificity and thereby contribute to the diversity of enzyme activity. Furthermore, small sequence exchanges also may play a role in generating the diverse patterns of tissue-specific expression of rat family members. These analyses indicate an important role for gene conversion in the evolution of the functional diversity of these duplicated genes.     

The Staphylococcus aureus mutation pcrA3 leads to the accumulation of pT181 replication initiation complexes

In Staphylococcus aureus cells carrying the pcrA3 chromosomal mutation, plasmid pT181 and its derivatives were maintained at a reduced copy number. A significant proportion of their DNA migrated during agarose gel electrophoresis as nicked DNA. The results obtained in the characterization of this plasmid DNA species show that it represents replication initiation complexes. Such complexes could not be detected in a wild-type host. The replication initiation complexes present in pcrA3 cells could resume replication after a lag. It was concluded from these results that the pcrA3 host mutation affected a step in plasmid pT181 replication immediately following the formation of the replication initiation complex, and that in pcrA3 this step became rate-limiting for plasmid pT181 replication.     

The resonance approach to the formation and structure of atoms and molecules

As shown recently, the set of electronic configurations of all chemical elements in the subshell partitioning has a four-dimensional structure. Hence, all elements can be subdivided into cycles and supercycles rather than into periods of the conventional Periodic Table. Moreover, in the plane of the principal and orbital quantum numbers there is an obvious symmetry between the hydrogen-atom spectrum (one-electron problem) and the electronic configurations of all atoms (multi-electron problem). In order to explain these phenomena, it is hypothesized that between shell electrons and nuclei protons there exists a reversible quantum-field interaction (electron + proton ↔ neutron + neutrino resonance). Even if this interaction were a rare event, it can explain the existence of 4D symmetries mentioned above. However, the model considered here is based on frequent epn interactions, taking place more often than 10¹⁵ times per second. In this case, epn resonance and resonance between such resonances are supposed to underlie the structure of all atoms and molecules. In particular, the functioning of all biological molecules, including electron transfer and enzymatic activity, is determined by resonance, resonance between resonances, hierarchy of resonances, and networks of resonances. The text was submittred by the author in english.     

The contribution of the calcium-dependent interaction of aggregation factor molecules to recognition: A system providing additional specificity forces?

Cells from the sponge Microciona prolifera display on their surfaces large but defined proteoglycan complexes (Microciona aggregation factor = MAF) that mediate species-specific cell aggregation by a process requiring high calcium ion concentrations. An analysis of MAF-MAF interactions based on binding studies of MAF to glutaraldehyde-fixed sponge cells and MAF-derivatized beads demonstrates that the requirement for high calcium concentrations can be overcome by extremely small amounts of certain polycations such as polybrene, polylysine, or histones. For measurements of the affinity of these substances to MAF, a method was adopted that partitions ¹²⁵I-labeled MAF between dextran and polyethyleneglycol in an aqueous two-phase polymer system depending on the net charge of the complex formed. Since only polymers of positive charges affect binding and partitioning at low concentrations, large areas of interaction similar to those found in glycosaminoglycans are proposed for MAF. Through a multitude of appropriately spaced interaction sites, the rather weak selectivity of single charged sites could in such a system still provide strong enough specificities to explain species-specific cell sorting. The biological significance of naturally occurring polycations as well as extracellular calcium includes their role in cell recognition, sorting out as well as the ordered and continual streaming movements of groups of cells seen in the mesohyl of live sponges.     

Carbonylation of the cytoskeletal protein actin leads to aggregate formation

Protein carbonylation is a common feature in cells exposed to oxidants, leading to protein dysfunction and protein aggregates. Actin, which is involved in manifold cellular processes, is a sensitive target protein to this oxidative modification. T-cell proteins have been widely described to be sensitive targets to oxidative modifications. The aim of this work was to test whether the formation of protein aggregates contributes to the impaired proliferation of Jurkat cells after oxidative stress and to test whether actin as a major oxidation-prone cytoskeletal protein is an integral part of such protein aggregates. We used Jurkat cells, an established T-cell model, showing the formation of actin aggregates along with the decrease of proteasome activity. The presence of these protein aggregates inhibits Jurkat proliferation even under conditions not influencing viability. As a conclusion, we propose that an oxidative environment leads to actin aggregates contributing to T-cell cellular functional impairment.     

In vivo formation and in vitro replication of a guanine-thymine intrastrand cross-link lesion

G[8-5m]T, a guanine-thymine intrastrand cross-link lesion where the C8 of guanine is covalently bonded to the neighboring 3'-thymine through its methyl carbon, was previously shown to form in an aqueous solution of duplex DNA upon exposure to gamma- or X-rays and in calf thymus DNA treated with Fenton reagents. Here, we employed LC-MS/MS and demonstrated for the first time that this lesion could be induced in a dose-dependent manner in human Hela-S3 cells upon exposure to gamma-rays. We further carried out in vitro replication studies on a substrate containing a site-specifically incorporated G[8-5m]T, and our results showed that the Klenow fragment of Escherichia coli DNA polymerase I stopped synthesis mostly after incorporating the correct nucleotide dAMP opposite the 3'-thymine moiety of the lesion. On the other hand, yeast Saccharomyces cerevisiae DNA polymerase eta (pol eta) was able to replicate past the cross-link lesion, but with markedly reduced efficiency in nucleotide incorporation opposite the 5'-guanine of the lesion. Steady-state kinetic analyses for nucleotide incorporation by yeast pol eta showed that the 5'-guanine portion of the lesion also decreased pronouncedly the fidelity of nucleotide incorporation; the insertion of dAMP and dGMP was favored over that of the correct nucleotide, dCMP. The above results support the conclusion that oxidative intrastrand cross-link lesions, if not repaired, can be cytotoxic and mutagenic.     

Evidence for additional genus-level diversity of Chlamydiales in the environment

The medically important order Chlamydiales has long been considered to contain a few closely related bacteria which occur exclusively in animals and humans. This perception of diversity and habitat had to be revised with the recent identification of the genera Simkania, Waddlia, Parachlamydia, and Neochlamydia with the latter two comprising endosymbionts of amoebae. Application of a newly developed PCR assay for the specific amplification of a near full length 16S rDNA fragment of these novel Chlamydia-related bacteria on activated sludge samples revealed the existence of at least four additional, previously unknown evolutionary lineages of Chlamydiales (each showing less than 92% 16S rRNA sequence similarity with all recognized members of this order). These findings suggest that some waste water treatment plants represent reservoirs for a diverse assemblage of environmental chlamydiae, a discovery which might also be of relevance from the viewpoint of human public health.     

Inactivation of the DnaB helicase leads to the collapse and degradation of the replication fork: a comparison to UV-induced arrest

Replication forks face a variety of structurally diverse impediments that can prevent them from completing their task. The mechanism by which cells overcome these hurdles is likely to vary depending on the nature of the obstacle and the strand in which the impediment is encountered. Both UV-induced DNA damage and thermosensitive replication proteins have been used in model systems to inhibit DNA replication and characterize the mechanism by which it recovers. In this study, we examined the molecular events that occur at replication forks following inactivation of a thermosensitive DnaB helicase and found that they are distinct from those that occur following arrest at UV-induced DNA damage. Following UV-induced DNA damage, the integrity of replication forks is maintained and protected from extensive degradation by RecA, RecF, RecO, and RecR until replication can resume. By contrast, inactivation of DnaB results in extensive degradation of the nascent and leading-strand template DNA and a loss of replication fork integrity as monitored by two-dimensional agarose gel analysis. The degradation that occurs following DnaB inactivation partially depends on several genes, including recF, recO, recR, recJ, recG, and xonA. Furthermore, the thermosensitive DnaB allele prevents UV-induced DNA degradation from occurring following arrest even at the permissive temperature, suggesting a role for DnaB prior to loading of the RecFOR proteins. We discuss these observations in relation to potential models for both UV-induced and DnaB(Ts)-mediated replication inhibition.     

Optimization of conditions for the in vitro formation of hybrid DNA molecules by DNA ligase.

Conditions for the ligation with T4 induced DNA ligase of two DNA molecules via their complementary sticky ends have been established which lead preferentially to the formation of hybrid molecules. This is demonstrated with two combinations of parent molecules varying greatly in their relative molecular weights. In one case the intact hybrid molecule could be directly isolated. In addition a DNA dependent quantitative electrophoretic assay for DNA ligase activity is described which does not need a radioactively labeled substrate. The ligation procedure has been shown to be useful in molecular cloning experiments.