A universal BMV-based RNA recombination system—how to search for general rules in RNA recombination

At present, there is no doubt that RNA recombination is one of the major factors responsible for the generation of new RNA viruses and retroviruses. Numerous experimental systems have been created to investigate this complex phenomenon. Consequently, specific RNA structural motifs mediating recombination have been identified in several viruses. Unfortunately, up till now a unified model of genetic RNA recombination has not been formulated, mainly due to difficulties with the direct comparison of data obtained for different RNA-based viruses. To solve this problem, we have attempted to construct a universal system in which the recombination activity of various RNA sequences could be tested. To this end, we have used brome mosaic virus, a model (+)RNA virus of plants, for which the structural requirements of RNA recombination are well defined. The effectiveness of the new homomolecular system has been proven in an experiment involving two RNA sequences derived from the hepatitis C virus genome. In addition, comparison of the data obtained with the homomolecular system with those generated earlier using the heteromolecular one has provided new evidence that the mechanisms of homologous and non-homologous recombination are different and depend on the virus' mode of replication

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

Screening the Expression of ABCB6 in Erythrocytes Reveals an Unexpectedly High Frequency of Lan Mutations in Healthy Individuals

Lan is a high-incidence blood group antigen expressed in more than 99.9% of the population. Identification of the human ABC transporter ABCB6 as the molecular basis of Lan has opened the way for studies assessing the relation of ABCB6 function and expression to health and disease. To date, 34 ABCB6 sequence variants have been described in association with reduced ABCB6 expression based on the genotyping of stored blood showing weak or no reactivity with anti-Lan antibodies. In the present study we examined the red blood cell (RBC) surface expression of ABCB6 by quantitative flow cytometry in a cohort of 47 healthy individuals. Sequencing of the entire coding region of the ABCB6 gene in low RBC ABCB6 expressors identified a new allele (IVS9+1G>A, affecting a putative splice site at the boundary of exon 9) and two nonsynonymous SNPs listed in the SNP database (R192Q (rs150221689) and G588 S (rs145526996)). The R192Q mutation showed co-segregation with reduced RBC ABCB6 expression in a family, and we found the G588 S mutation in a compound heterozygous individual with undetectable ABCB6 expression, suggesting that both mutations result in weak or no expression of ABCB6 on RBCs. Analysis of the intracellular expression pattern in HeLa cells by confocal microscopy indicated that these mutations do not compromise overall expression or the endolysosomal localization of ABCB6. Genotyping of two large cohorts, containing 235 and 1039 unrelated volunteers, confirmed the high allele frequency of Lan-mutations. Our results suggest that genetic variants linked to lower or absent cell surface expression of ABCB6/Langereis may be more common than previously thought.This work was supported by the Lendulet Program of the Hungarian Academy of Sciences (GS), OTKA 83533 and by the Polish POIG grant 01.01.02-10-005/08 TESTOPLEK, supported by the EU through the European Regional Development Fund. Hajnalka Andrikovics is a recipient of the Janos Bolyai Research Scholarship from the Hungarian Academy of Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. We thank Dr. Camilo Toro and Dr. William Gahl of the NIH Undiagnosed Diseases Program for an affected patient specimen; that work was supported by the Intramural Research Program of the National Human Genome Research Institute and the Office of the Director of the NIH. We thank Lionel Arnaud (National Institute of Blood Transfusion (INTS), Paris, France) for helpful discussions

Low recombination activity of R region located at both ends of the HIV-1 genome

Although two strand transfer events are indispensable for the synthesis of double-stranded DNA and establishing HIV-1 infection, the molecular basis of these phenomena is still unclear. The first obligatory template switching event occurs just at the beginning of the virus replication cycle and involves two copies of the 97-nucleotide long R region, located one each at the both ends of the HIV-1 genome (HIV-1 R). Thus, one can expect that the molecular mechanism of this process is similar to the mechanism of homologous recombination which operates in RNA viruses. To verify the above-mentioned hypothesis, we attempted to assess the recombination activity of HIV-1 R. To this end, we tested in vitro, how effectively it induces template switching by HIV-1 RT in comparison with another well-characterized sequence supporting frequent homologous crossovers in an unrelated virus (R region derived from Brome mosaic virus - BMV R). We also examined if the RNA sequences neighboring HIV-1 R influence its recombination activity. Finally, we tested if HIV-1 R could cause BMV polymerase complex to switch between RNA templates in vivo. Overall, our results have revealed a relatively low recombination activity of HIV-1 R as compared to BMV R. This observation suggests that different factors modulate the efficiency of the first obligatory strand transfer in HIV-1 and the homology-driven recombination in RNA viruses