Independent loss events of a functional tetherin gene in galliform birds.

IMPORTANCE: Birds represent important hosts for numerous viruses, including zoonotic viruses and pathogens with the potential to cause major economic losses to the poultry industry. Viral replication and transmission can be inhibited or blocked by the action of antiviral restriction factors (RFs) encoded by the host. One well-characterized RF is tetherin, a protein that directly blocks the release of newly formed viral particles from infected cells. Here, we describe the evolutionary loss of a functional tetherin gene in two galliform birds, turkey (Meleagris gallopavo) and Mikado pheasant (Syrmaticus mikado). Moreover, we demonstrate that the structurally related protein TMCC(aT) exerts antiviral activity in several birds, albeit by a mechanism different from that of tetherin. The evolutionary scenario described here represents the first documented loss-of-tetherin cases in vertebrates.

Heterologous avian system for quantitative analysis of Syncytin-1 interaction with ASCT2 receptor.

BACKGROUND: Human Syncytin-1 is a placentally-expressed cell surface glycoprotein of retroviral origin. After interaction with ASCT2, its cellular receptor, Syncytin-1 triggers cell-cell fusion and formation of a multinuclear syncytiotrophoblast layer of the placenta. The ASCT2 receptor is a multi-spanning membrane protein containing a protruding extracellular part called region C, which has been suggested to be a retrovirus docking site. Precise identification of the interaction site between ASCT2 and Syncytin-1 is challenging due to the complex structure of ASCT2 protein and the background of endogenous ASCT2 gene in the mammalian genome. Chicken cells lack the endogenous background and, therefore, can be used to set up a system with surrogate expression of the ASCT2 receptor. RESULTS: We have established a retroviral heterologous chicken system for rapid and reliable assessment of ectopic human ASCT2 protein expression. Our dual-fluorescence system proved successful for large-scale screening of mutant ASCT2 proteins. Using this system, we demonstrated that progressive deletion of region C substantially decreased the amount of ASCT2 protein. In addition, we implemented quantitative assays to determine the interaction of ASCT2 with Syncytin-1 at multiple levels, which included binding of the soluble form of Syncytin-1 to ASCT2 on the cell surface and a luciferase-based assay to evaluate cell-cell fusions that were triggered by Syncytin-1. Finally, we restored the envelope function of Syncytin-1 in a replication-competent retrovirus and assessed the infection of chicken cells expressing human ASCT2 by chimeric Syncytin-1-enveloped virus. The results of the quantitative assays showed that deletion of the protruding region C did not abolish the interaction of ASCT2 with Syncytin-1. CONCLUSIONS: We present here a heterologous chicken system for effective assessment of the expression of transmembrane ASCT2 protein and its interaction with Syncytin-1. The system profits from the absence of endogenous ASCT2 background and implements the quantitative assays to determine the ASCT2-Syncytin-1 interaction at several levels. Using this system, we demonstrated that the protruding region C was essential for ASCT2 protein expression, but surprisingly, not for the interaction with Syncytin-1 glycoprotein.

Topologically Associated Domains Delineate Susceptibility to Somatic Hypermutation.

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes but also causes mutations in other parts of the genome. We have used lentiviral SHM reporter vectors to identify regions of the genome that are susceptible ("hot") and resistant ("cold") to SHM, revealing that SHM susceptibility and resistance are often properties of entire topologically associated domains (TADs). Comparison of hot and cold TADs reveals that while levels of transcription are equivalent, hot TADs are enriched for the cohesin loader NIPBL, super-enhancers, markers of paused/stalled RNA polymerase 2, and multiple important B cell transcription factors. We demonstrate that at least some hot TADs contain enhancers that possess SHM targeting activity and that insertion of a strong Ig SHM-targeting element into a cold TAD renders it hot. Our findings lead to a model for SHM susceptibility involving the cooperative action of cis-acting SHM targeting elements and the dynamic and architectural properties of TADs.

Epigenetic regulation of transcription and splicing of syncytins, fusogenic glycoproteins of retroviral origin.

Syncytin-1 and -2, human fusogenic glycoproteins encoded by the env genes of the endogenous retroviral loci ERVWE1 and ERVFRDE1, respectively, contribute to the differentiation of multinucleated syncytiotrophoblast in chorionic villi. In non-trophoblastic cells, however, the expression of syncytins has to be suppressed to avoid potential pathogenic effects. We studied the epigenetic suppression of ERVWE1 and ERVFRDE1 5'-long terminal repeats by DNA methylation and chromatin modifications. Immunoprecipitation of the provirus-associated chromatin revealed the H3K9 trimethylation at transcriptionally inactivated syncytins in HeLa cells. qRT-PCR analysis of non-spliced ERVWE1 and ERVFRDE1 mRNAs and respective env mRNAs detected efficient splicing of endogenously expressed RNAs in trophoblastic but not in non-placental cells. Pointing to the pathogenic potential of aberrantly expressed syncytin-1, we have found deregulation of transcription and splicing of the ERVWE1 in biopsies of testicular seminomas. Finally, ectopic expression experiments suggest the importance of proper chromatin context for the ERVWE1 splicing. Our results thus demonstrate that cell-specific retroviral splicing represents an additional epigenetic level controling the expression of endogenous retroviruses.

OmniPlex--a new QF-PCR assay for prenatal diagnosis of common aneuploidies based on evaluation of the heterozygosity of short tandem repeat loci in the Czech population.

OBJECTIVES: The aim of our study was to assess the utility of commonly used multiplex assays of short tandem repeat markers used for quantitative fluorescent polymerase chain reaction (QF-PCR) for prenatal rapid aneuploidy detection (RAD) in routine prenatal diagnosis in the Czech population. METHODS: Two previously published RAD multiplexes (2M test) were tested on 2906 local prenatal samples and used to calculate the rates of heterozygosity within this population. Most of the markers used in both multiplexes were highly informative. However, some had little utility, either due to a low heterozygosity rate (D21S499, D18S978 and P39) or because they were difficult to evaluate (DXS1283E). RESULTS: After evaluation of the 2M test results, a new multiplex assay (OmniPlex) was designed, developed and tested on 960 samples. This new assay was evaluated for heterozygosity rates and for the probability of having two or more informative markers on each chromosome. CONCLUSIONS: OmniPlex assay significantly improved the QF-PCR methodology for rapid prenatal aneuploidy detection in the Czech population. Based on detected heterozygosity of markers used for QF-PCR in this population, OmniPlex is a robust assay for the detection of chromosomes 13, 18, 21, X and Y in a single reaction.

QF-PCR-based prenatal detection of common aneuploidies in the Czech population: five years of experience.

We present the results from the largest clinical application of QF-PCR for antenatal rapid aneuploidy detection (RAD) in routine prenatal diagnosis in the Czech Republic. QF-PCR was performed in addition to karyotyping (dual testing) in two settings: the first was a single multiplex reaction testing only trisomy 21 and amelogenin X/Y alleles in the second trimester screened positive cases (T21 test), and the second setting consisted of two multiplexes (2M test) for common aneuploidies (13, 18, 21, X and Y) in cases with other RAD indications such as ultrasound findings, late booking or maternal anxiety. Dual testing was performed in 6349/12,778 (49.7%) of prenatal samples using either T21 or 2M test between 2002 and 2007. The clinical acceptability of our dual testing policy, methodological efficiency of RAD and residual risks of other chromosomal aberrations (CHAs) were evaluated. QF-PCR detected 92% (175/190) of significant CHAs. The 2M test identified 93.5% and the T21 test identified 87.5% of the significant CHAs with complete specificity. The residual risk of significant CHA was 1/231 in the 2M test and 1/565 in the T21 test. If RAD for all common aneuploidies is used as the sole prenatal diagnosis method, the odds of missing a CHA of any type are 1:90 and the odds of missing significant CHA with no ultrasound findings are 1:1513. If prenatal karyotyping were used as an additional procedure to RAD in cases only with ultrasound findings, 186/190 (97.8%) of the significant CHAs would be detected when 15.7% cases were karyotyped, according to our data. We consider RAD directed towards trisomy 21 alone (our T21 test) as an economically and clinically acceptable part of second trimester screening for Down syndrome. Both RAD tests allow fast alleviation of maternal anxiety with low residual risk when the test results are negative, and allow fast decision making if the results are positive. However, replacement of dual testing with only the RAD procedure in specific indications accepted in some countries (Great Britain) remains in the Czech Republic a theme for debate.