Identification of key residues in subgroup A avian leukosis virus envelope determining receptor binding affinity and infectivity of cells expressing chicken or quail Tva receptor. (1/162)

To better understand retroviral entry, we have characterized the interactions between subgroup A avian leukosis virus [ALV(A)] envelope glycoproteins and Tva, the receptor for ALV(A), that result in receptor interference. We have recently shown that soluble forms of the chicken and quail Tva receptor (sTva), expressed from genes delivered by retroviral vectors, block ALV(A) infection of cultured chicken cells ( approximately 200-fold antiviral effect) and chickens (>98% of the birds were not infected). We hypothesized that inhibition of viral replication by sTva would select virus variants with mutations in the surface glycoprotein (SU) that altered the binding affinity of the subgroup A SU for the sTva protein and/or altered the normal receptor usage of the virus. Virus propagation in the presence of quail sTva-mIgG, the quail Tva extracellular region fused to the constant region of the mouse immunoglobulin G (IgG) protein, identified viruses with three mutations in the subgroup A hr1 region of SU, E149K, Y142N, and Y142N/E149K. These mutations reduced the binding affinity of the subgroup A envelope glycoproteins for quail sTva-mIgG (32-, 324-, and 4,739-fold, respectively) but did not alter their binding affinity for chicken sTva-mIgG. The ALV(A) mutants efficiently infected cells expressing the chicken Tva receptor but were 2-fold (E149K), 10-fold (Y142N), and 600-fold (Y142N/E149K) less efficient at infecting cells expressing the quail Tva receptor. These mutations identify key determinants of the interaction between the ALV(A) glycoproteins and the Tva receptor. We also conclude from these results that, at least for the wild-type and variant ALV(A)s tested, the receptor binding affinity was directly related to infection efficiency.  (+info)

Role of calcium in protein folding and function of Tva, the receptor of subgroup A avian sarcoma and leukosis virus. (2/162)

Tva is the cellular receptor for subgroup A avian sarcoma and leukosis virus (ASLV-A). The viral receptor function of Tva is determined by a 40-residue cysteine-rich motif called the LDL-A module. In this study, we expressed and purified the wild-type (wt) Tva LDL-A module as well as several mutants and examined their in vitro folding properties. We found that, as for other LDL-A modules, correct folding and structure of the Tva LDL-A module is Ca2+ dependent. When calcium was present during in vitro protein folding, the wt module was eluted as a single peak by reverse-phase high-pressure liquid chromatography. Furthermore, two-dimensional nuclear magnetic resonance (NMR) spectroscopy gave well-dispersed spectra in the presence of calcium. In contrast, the same protein folded in vitro in the absence of calcium was eluted as multiple broad peaks and gave a poorly dispersed NMR spectrum in the presence of calcium. The calcium affinity (Kd) of the Tva LDL-A module, determined by isothermal titration calorimetry, is approximately 40 microM. Characterization of several Tva mutants provided further evidence that calcium is important in protein folding and function of Tva. Mutations of the Ca2+-binding residues (D46A and E47A) completely abrogated the Ca2+-binding ability of Tva, and the proteins were not correctly folded. Interestingly, mutations of two non-calcium-binding residues (W48A and L34A) also exerted adverse effect on Ca2+-dependent folding, albeit to a much less extent. Our results provide new insights regarding the structure and function of Tva in ASLV-A entry.  (+info)

Isolation and characterization of nuclear RNA polymerase II from chicken myeloblastosis cells. (3/162)

Nuclear RNA polymerases of chicken myeloblastosis cells were solubilized and fractionated by diethylaminoethyl-Sephades A25 column chromatography. Both alpha-amanitin-insenstitive (polymerase I) and- sensitive (polymerase II) species were isolated. Polymerase activity, contained two peaks of enzyme (IIa and IIb), which were further purified by glycerol gradient centrifugation. The partially purified enzymes were characterized by their requirement of four nucleoside triphosphates and metal ions and by their sensitivity to several inhibitors. The enzymes were compared with RNA polymearases derived from normal chickent bone marrow cells,and the total extractable myeloblastosis than in bone marrow cells. Polymearse II from both cell types was shown to be sensitive to cytosine arabinoside triphosphate inhibiton.  (+info)

Avian erythroleukemia: a model for corepressor function in cancer. (4/162)

Transcriptional regulation at the level of chromatin plays crucial roles during eukaryotic development and differentiation. A plethora of studies revealed that the acetylation status of histones is controlled by multi-protein complexes containing (de)acetylase activities. In the current model, histone deacetylases and acetyltransferases are recruited to chromatin by DNA-bound repressors and activators, respectively. Shifting the balance between deacetylation, i.e. repressive chromatin and acetylation, i.e. active chromatin can lead to aberrant gene transcription and cancer. In human acute promyelocytic leukemia (APL) and avian erythroleukemia (AEL), chromosomal translocations and/or mutations in nuclear hormone receptors, RARalpha [NR1B1] and TRalpha [NR1A1], yielded oncoproteins that deregulate transcription and alter chromatin structure. The oncogenic receptors are locked in their 'off' mode thereby constitutively repressing transcription of genes that are critical for differentiation of hematopoietic cells. AEL involves an oncogenic version of the chicken TRalpha, v-ErbA. Apart from repression by v-ErbA via recruitment of corepressor complexes, other repressors and corepressors appear to be involved in repression of v-ErbA target genes, such as carbonic anhydrase II (CAII). Reactivation of repressed genes in APL and AEL by chromatin modifying agents such as inhibitors of histone deacetylase or of methylation provides new therapeutic strategies in the treatment of acute myeloid leukemia.  (+info)

Heterophil function and resistance to staphylococcal challenge in broiler chickens naturally infected with avian leukosis virus subgroup J. (5/162)

Avian leukosis virus subgroup J has a high tropism for myeloid lineage cells and frequently induces neoplastic transformation of myelocytes. The impact of congenital avian leukosis virus subgroup J infection on the function of circulating heterophils and susceptibility to staphylococcal infection was investigated. Six-week-old broiler chickens negative for exogenous avian leukosis viruses or congenitally infected with avian leukosis virus subgroup J were inoculated intravenously with 10(6) colony-forming units of Staphylococcus aureus, and pre- and postinoculation heterophil function was assessed. All chickens developed a leukocytosis with heterophilia after inoculation, but total leukocyte and heterophil counts were significantly higher in leukosis-negative chickens than in virus-infected chickens. Tenosynovitis was more severe in leukosis-negative chickens, and 2/10 (20%) of the virus-infected chickens had no histologic evidence of tenosynovitis. Osteomyelitis in the tibiotarsus or tarsometatarsus developed in 5/10 (50%) of the chickens in each group. S. aureus was recovered from the hock joint of 6/10 (60%) of the chickens in each group. Heterophils from all chickens exhibited similar phagocytic ability pre- and postinoculation. Heterophils from virus-infected chickens exhibited less bactericidal ability preinoculation than did heterophils from leukosis-negative chickens. However, postinoculation bactericidal ability was similar in both groups. Avian leukosis virus subgroup J provirus was present in heterophils isolated from congenitally infected chickens. Heterophils isolated from broiler chickens congenitally infected with avian leukosis virus subgroup J exhibit no significant functional deficits, and infected and uninfected chickens exhibit similar susceptibility to staphylococcal infection.  (+info)

Localization of avian leukosis virus subgroup J in naturally infected chickens by RNA in situ hybridization. (6/162)

The novel subgroup J of avian leukosis virus (ALV-J) has emerged as a significant cause of myeloid neoplasia and weight suppression in broiler chickens. We investigated viral tropism using RNA in situ hybridization (ISH) in naturally infected chickens. Formalin-fixed tissues were collected from 12-day-old embryos (seven infected, two control) and from 0-week-old (four infected, one control), 3-week-old (five infected, one control), 6-week-old (five infected, one control), and 9-week-old (10 infected, two control) chickens naturally infected with ALV-J in ovo. A 636-base antisense riboprobe complementary to the 3' and 5' ends of the pol and env viral genes, respectively, was constructed. Strong positive staining was present in cardiac myocytes, Purkinje fibers, vascular and pulmonary smooth muscle, renal glomeruli, distal tubules, and pituitary glands. Light staining was present in gastrointestinal smooth muscle, thyroid and adrenal glands, and follicular medullae in the cloacal bursa. Staining was not present in any hematopoietic precursors. Tissues from newly hatched chicks exhibited the strongest and most consistent staining, whereas staining in embryos was minimal. RNA ISH confirmed the presence of ALV-J-specific nucleic acid within cytoplasmic inclusions in cardiac myocytes, Purkinje fibers, pituitary glands, and renal glomeruli. Viral tropism for cardiac myocytes and Purkinje fibers may relate pathogenetically to the cardiomyopathy and congestive heart failure described in index chicken flocks infected with ALV-J. Viral tropism for endocrine organs may relate pathogenetically to the weight suppression associated with infection.  (+info)

Avian bic, a gene isolated from a common retroviral site in avian leukosis virus-induced lymphomas that encodes a noncoding RNA, cooperates with c-myc in lymphomagenesis and erythroleukemogenesis. (7/162)

bic is a novel gene identified at a common retroviral integration site in avian leukosis virus-induced lymphomas and has been implicated as a collaborator with c-myc in B lymphomagenesis. It lacks an extensive open reading frame and is believed to function as an untranslated RNA (W. Tam, Gene 274:157-167, 2001; W. Tam, D. Ben-Yehuda, and W. S. Hayward, Mol. Cell. Biol. 17:1490-1502, 1997). The oncogenic potential of bic, particularly its ability to cooperate with c-myc in oncogenesis, was tested directly by expressing c-myc and bic, either singly or in pairwise combination, in cultured chicken embryo fibroblasts (CEFs) and in chickens using replication-competent retrovirus vectors. Coexpression of c-myc and bic in CEFs caused growth enhancement of cells. Most importantly, chick oncogenicity assays demonstrated that bic can cooperate with c-myc in lymphomagenesis and erythroleukemogenesis. The present study provides direct evidence for the involvement of untranslated RNAs in oncogenesis and provides further support for the role of noncoding RNAs as riboregulators.  (+info)

Cardiomyopathy in broiler chickens congenitally infected with avian leukosis virus subgroup J. (8/162)

Dilated cardiomyopathy and ascites in broiler chickens are frequently associated with rapid growth and pulmonary hypertension, but can be associated with some avian leukosis virus (ALV) infections. The novel subgroup J of ALV has a high cardiac tropism, but dilated cardiomyopathy has not been reported previously. We report a dilated cardiomyopathy incidence of 11.1% in broiler chickens congenitally infected with ALV subgroup J (ALV-J). Gross lesions included severe body weight suppression, cardiomegaly with biventricular dilation, right ventricular hypertrophy, visceral congestion, and ascites. Cardiac myocytes and Purkinje fibers contained 2- to 10-microm intracytoplasmic magenta inclusions that contained ALV-J-specific nucleic acid. Ultrastructurally, inclusions contained ribosomes and immature virions and were associated with myofibril disruption and disarray. Peracute centrilobular hepatic necrosis was present in most cases. ALV-J-associated cardiomyopathy may involve a direct viral effect on cardiac myocytes and Purkinje fibers.  (+info)

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