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We sequenced unselected integration sites of subgroup C Rous sarcoma virus from infected chicken cells and mapped them on the chicken draft genome assembly. Our genome-wide analysis demonstrates the non-random character of subgroup C Rous sarcoma virus integration into genes, gene-rich regions and GC-rich regions. Within genes, there is no significant integration bias in favor of transcription start sites. Integration sites are underrepresented on microchromo- somes. These results may be important for the development of gene transfer vectors and gene therapy strategies based on avian sarcoma and leukosis viruses.

The avian sarcoma and leukosis virus (ASLV) family of retroviruses contains five highly related envelope subgroups (A to E) thought to have evolved from a common viral ancestor in the chicken population. Three genetic loci in chickens determine the susceptibility or resistance of cells to infection by the subgroup A to E ASLVs. Some inbred lines of chickens display phenotypes that are somewhere in between either efficiently susceptible or resistant to infection by specific subgroups of ASLV. The tvb gene encodes the receptor for subgroups B, D, and E ASLVs. The wild-type Tvb S1 receptor confers susceptibility to subgroups B, D, and E ASLVs. In this study, the genetic defect that accounts for the altered susceptibility of an inbred chicken line, line M, to infection by ASLV(B), ASLV(D), and ASLV(E) was identified. The tvb gene in line M, tvb r2 , encodes a mutant Tvb S1 receptor protein with a substitution of a serine for a cysteine at position 125 (C125S). Here, we show that the C125S substitution in Tvb S1 significantly reduces the susceptibility of line M cells to infection by ASLV(B) and ASLV(D) and virtually eliminates susceptibility to ASLV(E) infection both in cultured cells and in the incidence and growth of avian sarcoma virus-induced sarcomas in chickens. The C125S substitution significantly reduces the binding affinity of the Tvb S1 receptor for the subgroup B, D, and E ASLV envelope glycoproteins. These are the first results that demonstrate a possible role of the cysteine-rich domain 3 in the function of the Tvb receptors.

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.

Subgroup J avian leukosis virus (ALV-J) is unique among the avian sarcoma and leukosis viruses in using the multimembrane-spanning cell surface protein Na + /H + exchanger type 1 (NHE1) as a receptor. The precise localization of amino acids critical for NHE1 receptor activity is key in understanding the virus-receptor interaction and potential interference with virus entry. Because no resistant chicken lines have been described until now, we compared the NHE1 amino acid sequences from permissive and resistant galliform species. In all resistant species, the deletion or substitution of W38 within the first extracellular loop was observed either alone or in the presence of other incidental amino acid changes. Using the ectopic expression of wild-type or mutated chicken NHE1 in resistant cells and infection with a reporter recombinant retrovirus of subgroup J specificity, we studied the effect of individual mutations on the NHE1 receptor capacity. We suggest that the absence of W38 abrogates binding of the subgroup J envelope glycoprotein to ALV-J-resistant cells. Altogether, we describe the functional importance of W38 for virus entry and conclude that natural polymorphisms in NHE1 can be a source of host resistance to ALV-J.

Background The administration of anti-trypanosome nitroderivatives curtails Trypanosoma cruzi infection in Chagas disease patients, but does not prevent destructive lesions in the heart. This observation suggests that an effective treatment for the disease requires understanding its pathogenesis. Methodology/Principal Findings To understand the origin of clinical manifestations of the heart disease we used a chicken model system in which infection can be initiated in the egg, but parasite persistence is precluded. T. cruzi inoculation into the air chamber of embryonated chicken eggs generated chicks that retained only the parasite mitochondrial kinetoplast DNA minicircle in their genome after eight days of gestation. Crossbreeding showed that minicircles were transferred vertically via the germ line to chicken progeny. Minicircle integration in coding regions was shown by targeted-primer thermal asymmetric interlaced PCR, and detected by direct genomic analysis. The kDNA-mutated chickens died with arrhythmias, shortness of breath, cyanosis and heart failure. These chickens with cardiomyopathy had rupture of the dystrophin and other genes that regulate cell growth and differentiation. Tissue pathology revealed inflammatory dilated cardiomegaly whereby immune system mononuclear cells lyse parasite-free target heart fibers. The heart cell destruction implicated a thymus-dependent, autoimmune; self-tissue rejection carried out by CD45+, CD8γδ+, and CD8α lymphocytes. Conclusions/Significance These results suggest that genetic alterations resulting from kDNA integration in the host genome lead to autoimmune-mediated destruction of heart tissue in the absence of T. cruzi parasites., Author Summary The Trypanosoma cruzi acute infections can be asymptomatic but approximately one third of the chronically infected cases may present Chagas disease. Parasite persistence and autoimmunity are theories trying to explain the clinical and pathological manifestations of Chagas disease in the heart and the digestive system. To clearly demonstrate roles played by parasite persistence and autoimmunity in Chagas disease we used a chicken model refractory to the T. cruzi. In this study we inoculated the invasive T. cruzi in the air chamber of embryonated eggs. The infection was eradicated by the innate immunity and the chicks were parasite-free at hatching, but they retained the parasitic mitochondrial kinetoplast DNA minicircle in their genome. We documented the kDNA minicircle integrated in the chicken genome by a targeted prime TAIL-PCR, Southern hybridizations, cloning and sequencing. The kDNA minicircles integrated in coding regions of various chromosomes, and mutated chickens developed an inflammatory cardiomyopathy hallmark of Chagas disease, whereby immune system mononuclear cells lyse parasite-free target heart fibers. Genotype alterations resulting from transfers of the parasitic DNA were associated with the tissue destruction carried out by effectors CD45+, CD8γδ+, CD8α lymphocytes. This research provides insights about a protozoan infection that can induce genetically driven autoimmune disease.

Integration of retroviruses occurs in most genomic regions with weak but statistical significant preferences for target site sequences. HIV-1 preferentially targets genes, particularly the transcriptionally active ones. Avian sarcoma and leukosis viruses (ASLV) integrate with only slight preference for genes while murine leukemia virus uniquely favors integration in close proximity to upstream or downstream transcription start sites. Genomic features as well as the “epigenetic landscape“ at the site of integration are crucial for the outcome of each integration event, i. e. stable long terminal repeat-driven expression of the provirus or silencing and transcriptional suppression. The current availability of assembled genome sequences with epigenomic characteristics and reliable cloning of retrovirus integration sites enables to study the interplay between transcription signals of the retrovirus and enhancing or suppressive influences of the adjacent cellular DNA. We studied the integration sites of ASLV-derived vectors integrated into normal or DNA methylation-deficient human cells. Integration sites from cell clones containing single provirus were cloned and their genomic features as well as epigenetic landscape were correlated with expression or silencing of the provirus. Dnmt3b turned out to be the major player in silencing the long terminal repeat-driven transcription. Proviruses integrated into H3K4me3-rich CpG islands associated with promoters of active genes display long-term stability of expression and are resistant to the transcriptional silencing after over expression of Dnmt3b. Stability of expression and resistance to the silencing decreases with the distance from transcription start site. By contrast, proviruses integrated into the intergenic regions tend to the spontaneous transcriptional silencing even in DNA methyltransferase-deficient cells.