Genetic and Biochemical Analysis of the Bryan Strain of Rous Sarcoma Virus

Integration of Rous sarcoma virus DNA into its host genome was analyzed under conditions where secondary integration via virus spread was inhibited. This was accomplished by using the noninfectious pol- ,env- alpha variant of the Bryan high titer strain of Rous sarcoma virus (BH-RSV). Twelve independent BHRSV- transformed chicken embryo fibroblast clones were obtained and the provirus-cell junction fragments were mapped by restriction endonuclease cleavage and Southern blotting analyses. We found that expression of the viral genes could occur after proviral integration at many sites on the chicken genome and that there was no apparent preference for specific integration sites. BH-RSV DNA was further analyzed in order to precisely determine the defects in this strain. A 5kb EcoRI fragment which contained the entire pol and src genes was molecularly cloned from integrated proviruses of BH-RSV alpha and its pol+ parent BH-RSV beta. DNA sequencing of the pol-src junction of BH-RSV revealed that the env sequence was almost entirely absent; only 6 bp following the pol stop codon remained. Starting at position 7 (relative to the end of pol), a 91 bp sequence identical to the 91 bp immediately upstream from src in other strains of Rous sarcoma virus (RSV) was found. This was followed by the src coding sequence. It appears remarkable that in all RSV strains, including this defective BH-RSV, as well as in cellular-src DNA, these 91 bp are conserved. The helper virus-related sequence of about 100 bp, which is present as a direct repeat in the 5\u27 and 3\u27 sides of src in other RSVs, was present only on the 3\u27 side of src in BH-RSV. The sequence of about 100 nucleotides immediately following src in BH-, PR-, and SR-RSV seems to be completely unconserved. Restriction enzyme mapping analysis showed the structure of the BH-RSV alpha pol gene to be basically unchanged from that of BH-RSV beta. Using cloned DNAs, we have constructeds molecular clones in which the pol gene of non-defective RSV was replaced with the pol gene of BH-RSV alpha or BH-RSV beta. The BH-RSV alpha pol gene was found to be biologically inactive in a transfection assay. Further in vitro recombination localized the lesion to an 859 bp Xbal-Bglll fragment in the second third of the pol gene. Analysis of the proteins synthesized in BH-RSV alpha infected cells revealed the fact that BH-RSV alpha directed the synthesis of a full-sized Prl80 gag-pol precursor, yet no polymerase related proteins were found in BH-RSV alpha virion particles. The pol lesion in the BH-RSV alpha genome appears to cause a defect in the processing or packaging of reverse transcriptase

The Oncogene of Avian Sarcoma Virus UR2 and its Cellular Homologue: Structure, Sequence and Expression

Avian sarcoma virus UR2 is a replication-defective virus that can induce sarcomas in vivo and transforms chicken embryo fibroblasts in culture to a characteristic, extremely elongated morphology. The genome of UR2 contains a 1.2 kb transformation-specific sequence, v-ros, which has a homologous counterpart, cros, in normal chicken cellular DNA. The 5\u27 end of v-ros is fused to the helper virus UR2AV-related sequencing coding for one of the viral gag proteins, p19. The fused p19 and ros sequences in UR2 code for a polyprotein of 68 kd, P68gag-ros, which has an associated tyrosine kinase activity. To elucidate the basis of the functional conservation as well as differences between ros and other oncogenes, I sequenced the entire genome of UR2 and compared the predicted amino acid sequence of P68 with other members of the tyrosine kinase family. The results show that ros is 1273 nucleotides in length, including a 65 bp 3\u27 noncoding stretch. ros is joined at its 5\u27 and 3\u27 ends to the 3\u27 region of p19, and the 3\u27 region of gp37, respectively, and replaces the sequences of UR2AV in between. The deduced amino acid sequence for P68 gives a molecular weight of 61,113 daltons and shows that it is closely related to the oncogene family coding for tyrosine protein kinases. However, P68 contains two distinctive hydrophobic regions that are absent in most of the other tyrosine kinases and it has unique amino acid changes and insertions within the conserved domain of the kinases. I also determined the sequence of cellular ros and compared it to viral ros to determine the changes between them that may be responsible for their differential oncogenicity; in addition I have analysed the expression of the cellular gene in both embryonic and adult chickens. The 1.2 kb v-ros sequence is remarkably well conserved when compared with the corresponding region of c-ros. The v-ros sequences are distributed in nine exons of c-ros over a. range of 12 kb of DNA

Oncogenes and human cancer

The first demonstrations that cancer could have an infectious nature was by Ellerman and Bang (1) ~ who showed that leukemia in chickens was transmissible with cell-free extracts and by Rous (2), who found in a similar fashion that naturally occurring chicken sarcomas were transmissible. Although they were able to show that these cell-free extracts contained a transmissible agent~ the idea that this induced cancer was received by the scientific world at that time with great skepticism. The interest in oncogenic viruses was strongly enhanced in the early 60's by the isolation of mammalian tumor viruses and the general acceptance that at least some of these viruses were tumorigenic. The discovery of the reverse transcriptase enzyme in RNA tumor viruses (3,4), gave a logical explanation for how these viruses became integrated in the chromosomes of eukaryotic cells. Taxonomically, oncogenic viruses are members of diverse families. DNA viruses (herpes-, adeno- and papovaviruses) as well as many members of the retrovirus family (containing RNA such as the type C RNA viruses) are capable of inducing tumors. For the retroviruses two different routes to become transforming (oncogenic) have become clear. The majority of these viruses (the acute type C RNA transforming viruses) 11 acquire11 certain genetic sequences (oncogenes) from their host, which are necessary to initiate and maintain the malignant transformation of the cell by the virus. Other retroviruses integrate their genome nearby oncogenic sequences in the chromosome of their host. Independent of the exact mechanism, these viruses share the capability of inducing tumorigenesis by triggering the transcription of certain sequences, and it is the proteins encoded by these sequences which are necessary to maintain the neoplastic phenotype of the infected cell The accumulating number of independent isolates of tumorigenic retroviruses induced in the mid-70's a worldwide search for these viruses in humans. Only very recently the isolation of a human tumor T-cell leukemia retrovirus (HTLV) was reported (5,6). Another approach was initiated in the beginning of the 80's, with the finding that the acquired sequences of retroviruses are strongly conserved among species. In general, the cellular homologs of these sequences were easily detectable and could be studied in more detail by molecular cloning, using the oncogenic acquired sequences of retroviruses as probes. This approach seems to be very fruitful and will be discussed in more detail below. Although the oncogenic potential of the acquired sequences in a number of these viruses in vertebrates is well estabished, the involvement of their human cellular homologs in human tumorigenesis has been and will be a rich source for discussion. However, at the moment they provide us the best available model for the induction of human cancer at the molecular leve

Identification of Biomarkers Associated with Rous Sarcoma Virus-induced Tumors in Two Divergently Selected Chicken Lines

Poultry has become especially important to genetic research due to breeding feasibility, short generation turnover, and ease of maintaining large populations. The discovery of virus induced cancer has paved the way for further genetic studies. Rous Sarcoma Virus (RSV) is a tumor-causing virus that infects poultry. While not prevalent today, it can serve as a model for virus-induced cancer in humans and create additional insight to marker assisted selection in poultry. Genetically selected Arkansas Progressor (AP) and Arkansas Regressor (AR) chicken lines have been established and maintained at the Arkansas Experimental Station (AES) in Fayetteville, AR. Previous research has investigated the immunological and genetic characteristics of virus induced tumors. Publication of the Red Jungle Fowl genome has made it possible to perform genome wide studies to identify biomarkers associated with the susceptibility to RSV induced tumors. In this study, whole genomes of AP and AR lines were sequenced and analyzed using Illumina platform next generation sequencing. Over 9,000,000 SNPs were identified against the reference genome and 12,000 were classified as unique SNPs between the AR and AP line. Most SNPs were found in intergenic regions, while few were in protein coding regions. Unique SNPs were categorized by the following mutations: Frameshift (134), No Start (25), No Stop (7), Non-synonymous (6884), and Nonsense (112). Unique SNPs were characterized as occurring in only one line. Nine SNPs were chosen after genomic analyses for testing using PCR and Sanger sequencing in larger populations of AR and AP birds followed by validation in unrelated populations. No statistical correlations were found between the 9 SNPs in the unrelated populations, Giant Jungle Fowl (GJF) and White Leghorn (WL). No genetic difference was detected between birds of differing phenotypes within the GJF and WL lines. SNP frequencies were the same for both birds that regressed tumors and those that progressed tumors. Interestingly, it was observed that the GJF line response to RSV resembled the AR line and the WL line resembled the AP line, and GJF and WL exhibited differing phenotypes, indicating the potential to find biomarkers in larger population sizes

Cytokines of Birds: Conserved Functions

Targeted disruptions of the mouse genes for cytokines, cytokine receptors, or components of cytokine signaling cascades convincingly revealed the important roles of these molecules in immunologic processes. Cytokines are used at present as drugs to fight chronic microbial infections and cancer in humans, and they are being evaluated as immune response modifiers to improve vaccines. Until recently, only a few avian cytokines have been characterized, and potential applications thus have remained limited to mammals. Classic approaches to identify cytokine genes in birds proved difficult because sequence conservation is generally low. As new technology and high throughput sequencing became available, this situation changed quickly. We review here recent work that led to the identification of genes for the avian homologs of interferon-a/b (IFNa/b) and IFN-g, various interleukins (IL), and several chemokines. From the initial data on the biochemical properties of these molecules, a picture is emerging that shows that avian and mammalian cytokines may perform similar tasks, although their primary structures in most cases are remarkably different

Modulation of Functional Activities of Chicken Heterophils by Recombinant Chicken IFN-γ

The objective of the present studies was to examine the in vitro effects of recombinant chicken interferon-γ (rChIFN-γ) on shape change, phagocytosis, and the oxidative/nonoxidative killing activities of day-old chicken heterophils. Heterophils (4 × 106/ml) were incubated with various concentrations of recombinant ChIFN-γ from both Escherichia coli and transfected Cos cells for 2 h at 39°C. The incubation of the neonatal heterophils with rChIFN-γ resulted in significantly greater numbers of cells with membrane shape change when compared with the mock-treated heterophils. Both Cos cell-derived and E. coli-derived ChIFN-γ significantly increased (p < 0.01) the phagocytosis of opsonized or nonopsonized Salmonella enteritidis by the neonatal heterophils in a concentration-dependent manner. Incubation with ChIFN-γ induced no direct stimulation of the respiratory burst by the chicken heterophils but did prime the heterophils for a significantly strengthened respiratory burst to subsequent stimulation with opsonized zymosan (OZ). Lastly, incubation of the heterophils with ChIFN-γ primed the cells for a significant increase in the release of β-D-glucuronidase following stimulation with OZ. These results show that neonatal avian heterophils can respond to cytokine modulation with enhanced functional competence, suggesting that ChIFN-γ can enhance the immune competence of the innate defenses of chickens during the first week of life

Article epigenetic silencing of microrna-126 promotes cell growth in marek’s disease

During latency, herpesvirus infection results in the establishment of a dormant state in which a restricted set of viral genes are expressed. Together with alterations of the viral genome, several host genes undergo epigenetic silencing during latency. These epigenetic dysregulations of cellular genes might be involved in the development of cancer. In this context, Gallid alphaherpesvirus 2 (GaHV-2), causing Marek’s disease (MD) in susceptible chicken, was shown to impair the expression of several cellular microRNAs (miRNAs). We decided to focus on gga-miR-126, a host miRNA considered a tumor suppressor through signaling pathways controlling cell proliferation. Our objectives were to analyze the cause and the impact of miR-126 silencing during GaHV-2 infection. This cellular miRNA was found to be repressed at crucial steps of the viral infection. In order to determine whether miR-126 low expression level was associated with specific epigenetic signatures, DNA methylation patterns were established in the miR-126 gene promoter. Repression was associated with hypermethylation at a CpG island located in the miR-126 host gene epidermal growth factor like-7 (EGFL-7). A strategy was developed to conditionally overexpress miR-126 and control miRNAs in transformed CD4+ T cells propagated from Marek’s disease (MD) lymphoma. This functional assay showed that miR-126 restoration specifically diminishes cell proliferation. We identified CT10 regulator of kinase (CRK), an adaptor protein dysregulated in several human malignancies, as a candidate target gene. Indeed, CRK protein levels were markedly reduced by the miR-126 restoration