CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation. C ytosine methylation in CpG dinucleotides is an important mechanism of transcriptional regulation in vertebrates. Especially in the genome of mammalian somatic cells, the distribution of CpG dinucleotides, and the pattern of methylation are bimodal. In the major part of the genome, CpGs are underrepresented, dispersed, and predominantly methylated. Approximately 1-2% of the genome consists of nonmethylated CpG-rich stretches (CpG islands) that are typically 0.5-2 kb in length and usually associated with housekeeping genes (1, 2). This bimodal somatic methylation pattern is probably established by general de novo methylation, which skips CpG islands and adjacent gene promoters and leaves them unmethylated, and͞or by active demethylation of these sequences. Sp1 sites within the core sequence of CpG islands were shown to be required to prevent methylation (3, 4).Rous sarcoma virus (RSV) is an avian retrovirus that does not replicate in mammalian host cells. The nonpermissiveness of heterologous hosts is caused by several blocks of the replication cycle. Mammalian cells lack specific receptors for virus entry and do not promote correct splicing of retroviral mRNA, cleavage of viral polyproteins, or assembly of infectious virus particles on the inner surface of the cell membrane (reviewed in ref. 5). In addition to these obstacles, RSV proviral DNA is usually transcriptionally suppressed after integration into the mammalian genome. Fewer than 0.1% of RSV provirus-containing mammalian cells displays morphological transformation by virtue of the v-src oncogene (6, 7). The vast majority of RSV-infected cells harbors transcriptionally silent proviruses with undetectable amount of viral RNA (7, 8). Even in transformed mammalian cells, the proviral expression often tends to spontaneou...
The J subgroup of avian leukosis virus (ALV-J) infects domestic chickens, jungle fowl, and turkeys. This virus enters the host cell through a receptor encoded by the tvj locus and identified as Na ϩ /H ϩ exchanger 1. The resistance to avian leukosis virus subgroup J in a great majority of galliform species has been explained by deletions or substitutions of the critical tryptophan 38 in the first extracellular loop of Na ϩ /H ϩ exchanger 1. Because there are concerns of transspecies virus transmission, we studied natural polymorphisms and susceptibility/resistance in wild galliforms and found the presence of tryptophan 38 in four species of New World quails. The embryo fibroblasts of New World quails are susceptible to infection with avian leukosis virus subgroup J, and the cloned Na ϩ /H ϩ exchanger 1 confers susceptibility on the otherwise resistant host. New World quails are also susceptible to new avian leukosis virus subgroup J variants but resistant to subgroups A and B and weakly susceptible to subgroups C and D of avian sarcoma/leukosis virus due to obvious defects of the respective receptors. Our results suggest that the avian leukosis virus subgroup J could be transmitted to New World quails and establish a natural reservoir of circulating virus with a potential for further evolution.IMPORTANCE Since its spread in broiler chickens in China and Southeast Asia in 2000, ALV-J remains a major enzootic challenge for the poultry industry. Although the virus diversifies rapidly in the poultry, its spillover and circulation in wild bird species has been prevented by the resistance of most species to ALV-J. It is, nevertheless, important to understand the evolution of the virus and its potential host range in wild birds. Because resistance to avian retroviruses is due particularly to receptor incompatibility, we studied Na ϩ /H ϩ exchanger 1, the receptor for ALV-J. In New World quails, we found a receptor compatible with virus entry, and we confirmed the susceptibilities of four New World quail species in vitro. We propose that a prospective molecular epidemiology study be conducted to identify species with the potential to become reservoirs for ALV-J.KEYWORDS ALV-J, antiretroviral resistance, Na ϩ /H ϩ exchanger, New World quail, retroviral receptor T he range of hosts susceptible to a given retrovirus results from the process of coevolution between the viral envelope glycoproteins and host cell receptors. Selection forces imposed by the retrovirus drive the positive selection of receptors toward variants with decreased or even abrogated binding to retroviral envelopes. Vice versa, the highly error prone replication of retroviruses enables the rapid evolution of new strains with the capacity to bind to the variant receptors or even to quite new cell surface molecules. The results of these processes acting over evolutionary time are visible particularly in avian sarcoma/leukosis viruses (ASLVs), murine leukemia viruses (MLV), and feline leukemia viruses, where closely related virus subgroups differ in
The breeding history of the first inbred strain of Khaki Campbell ducks is presented. The genetic homogeneity of this strain was tested on the basis of serum amyloid A (SAA) polymorphism and it was established that it harbours only the SAA allele A, which is expressed in liver, lung and bursa of Fabricius tissues. Pathogenic changes in control and avian leukosis virus-C (ALV-C) persistently infected ducks were evaluated during the period spanning 1 to 10 months after hatching. In both groups, AA amyloidosis was revealed and characterized. In spite of the inbred nature of animals, the incidence of amyloid A deposition varied among experiments, suggesting that additional non-genetic factors are involved. Similar variation was found in ALV-C persistently infected ducks, where only in one out of three experiments was the incidence of AA amyloidosis significantly higher than in controls.
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.
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