3'UTR of ALV-J can affect viral replication through promoting transcription and mRNA nuclear export.

IMPORTANCE: 3'UTRs can affect gene transcription and post-transcriptional regulation in multiple ways, further influencing the function of proteins in a unique manner. Recently, ALV-J has been mutating and evolving rapidly, especially the 3'UTR of viral genome. Meanwhile, clinical symptoms caused by ALV-J have changed significantly. In this study, we found that the ALV-J strains containing △-r-TM-type 3'UTR are the most abundant. By constructing ALV-J infectious clones and subgenomic vectors containing different 3'UTRs, we prove that 3'UTRs directly affect viral tissue preference and can promote virus replication as an enhancer. ALV-J strain containing 3'UTR of △-r-TM proliferated fastest in primary cells. All five forms of 3'UTRs can assist intron-containing viral mRNA nuclear export, with similar efficiency. ALV-J mRNA half-life is not influenced by different 3'UTRs. Our results dissect the roles of 3'UTR on regulating viral replication and pathogenicity, providing novel insights into potential anti-viral strategies.

Precise CRISPR/Cas9 editing of the NHE1 gene renders chickens resistant to the J subgroup of avian leukosis virus.

Avian leukosis virus subgroup J (ALV-J) is an important concern for the poultry industry. Replication of ALV-J depends on a functional cellular receptor, the chicken Na+/H+ exchanger type 1 (chNHE1). Tryptophan residue number 38 of chNHE1 (W38) in the extracellular portion of this molecule is a critical amino acid for virus entry. We describe a CRISPR/Cas9-mediated deletion of W38 in chicken primordial germ cells and the successful production of the gene-edited birds. The resistance to ALV-J was examined both in vitro and in vivo, and the ΔW38 homozygous chickens tested ALV-J-resistant, in contrast to ΔW38 heterozygotes and wild-type birds, which were ALV-J-susceptible. Deletion of W38 did not manifest any visible side effect. Our data clearly demonstrate the antiviral resistance conferred by precise CRISPR/Cas9 gene editing in the chicken. Furthermore, our highly efficient CRISPR/Cas9 gene editing in primordial germ cells represents a substantial addition to genotechnology in the chicken, an important food source and research model.

TMT-based proteomic analysis reveals integrins involved in the synergistic infection of reticuloendotheliosis virus and avian leukosis virus subgroup J.

BACKGROUND: Co-infection with the avian leukosis virus subgroup J (ALV-J) and the reticuloendotheliosis virus (REV) increases mutual viral replication, causing a more serious pathogenic effect by accelerating the progression of neoplasia and extending the tumor spectrum. However, the molecular mechanism underlying the synergistic replication of ALV-J and REV remains unclear. RESULTS: Here, we performed this study to compare the differentially expressed proteins among CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time using TMT-based quantitative proteomics. We identified a total of 719 (292 upregulated and 427 downregulated) and 64 (35 upregulated and 29 downregulated) proteins by comparing co-infecting both viruses with monoinfecting ALV-J and REV, respectively. GO annotation and KEGG pathway analysis showed the differentially expressed proteins participated in virus-vector interaction, biological adhesion and immune response pathways in the synergistic actions of ALV-J and REV at the protein levels. Among the differentially expressed proteins, a large number of integrins were inhibited or increased in the co-infection group. Further, eight integrins, including ITGα1, ITGα3, ITGα5, ITGα6, ITGα8, ITGα9, ITGα11 and ITGß3, were validated in CEF cells by qRT-PCR or western blot. CONCLUSIONS: These findings proved that integrins may be key regulators in the mechanism of synergistic infection of REV and ALV-J, which will provide more insight into the pathogenesis of synergism of REV and ALV-J at protein level.

The Bipartite Sequence Motif in the N and C Termini of gp85 of Subgroup J Avian Leukosis Virus Plays a Crucial Role in Receptor Binding and Viral Entry.

Subgroup J avian leukemia virus (ALV-J), belonging to the genus Alpharetrovirus, enters cells through its envelope surface unit (gp85) via specifically recognizing the cellular receptor chicken Na+/H+ exchanger type I (chNHE1), the 28 to 39 N-terminal residues of which were characterized as the minimal receptor functional domain in our previous studies. In this study, to further clarify the precise organization and properties of the interaction between ALV-J gp85 and chNHE1, we identified the chNHE1-binding domain of ALV-J gp85 using a series of gp85 mutants with segment substitutions and evaluating their effects on chNHE1 binding in protein-cell binding assays. Our results showed that hemagglutinin (HA) substitutions of amino acids (aa) 38 to 131 (N terminus of gp85) and aa 159 to 283 (C terminus of gp85) significantly inhibited the interaction between gp85 and chNHE1/chNHE1 loop 1. In addition, these HA-substituted chimeric gp85 proteins could not effectively block the entry of ALV-J into chNHE1-expressing cells. Furthermore, analysis of various N-linked glycosylation sites and cysteine mutants in gp85 revealed that glycosylation sites (N6 and N11) and cysteines (C3 and C9) were directly involved in receptor-gp85 binding and important for the entry of ALV-J into cells. Taken together, our findings indicated that the bipartite sequence motif, spanning aa 38 to 131 and aa 159 to 283, of ALV-J gp85 was essential for binding to chNHE1, with its two N-linked glycosylation sites and two cysteines being important for its receptor-binding function and subsequent viral infection steps.IMPORTANCE Infection of a cell by retroviruses requires the attachment and fusion of the host and viral membranes. The specific adsorption of envelope (Env) surface proteins to cell receptors is a key step in triggering infections and has been the target of antiviral drug screening. ALV-J is an economically important avian pathogen that belongs to the genus Alpharetrovirus and has a wider host range than other ALV subgroups. Our results showed that the amino acids 38 to 131 of the N terminus and 159 to 283 of the C terminus of ALV-J gp85 controlled the efficiency of gp85 binding to chNHE1 and were critical for viral infection. In addition, the glycosylation sites (N6 and N11) and cysteines (C3 and C9) of gp85 played a crucial role in the receptor binding and viral entry. These findings might help elucidate the mechanism of the entry of ALV-J into host cells and provide antiviral targets for the control of ALV-J.

Mutual regulation between chicken telomerase reverse transcriptase and the Wnt/ß-catenin signalling pathway inhibits apoptosis and promotes the replication of ALV-J in LMH cells.

This study aimed to explore the mutual regulation between chicken telomerase reverse transcriptase (chTERT) and the Wnt/ß-catenin signalling pathway and its effects on cell growth and avian leukosis virus subgroup J (ALV-J) replication in LMH cells. First, LMH cells stably overexpressing the chTERT gene (LMH-chTERT cells) and corresponding control cells (LMH-NC cells) were successfully constructed with a lentiviral vector expression system. The results showed that chTERT upregulated the expression of ß-catenin, Cyclin D1, TCF4 and c-Myc. chTERT expression level and telomerase activity were increased when cells were treated with LiCl. When the cells were treated with ICG001 or IWP-2, the activity of the Wnt/ß-catenin signalling pathway was significantly inhibited, and chTERT expression and telomerase activity were also inhibited. However, when the ß-catenin gene was knocked down by small interfering RNA (siRNA), the changes in chTERT expression and telomerase activity were consistent with those in cells treated with ICG001 or IWP-2. These results indicated that chTERT and the Wnt/ß-catenin signalling pathway can be mutually regulated. Subsequently, we found that chTERT not only shortened the cell cycle to promote proliferation but also inhibited apoptosis by downregulating the expression of Caspase 3, Caspase 9 and BAX; upregulating BCL-2 and BCL-X expression; and promoting autophagy. Moreover, chTERT significantly enhanced the migration ability of LMH cells, upregulated the protein and mRNA expression of ALV-J and increased the virus titre. ALV-J replication promoted chTERT expression and telomerase activity.

The Novel Avian Leukosis Virus Subgroup K Shares Its Cellular Receptor with Subgroup A.

Avian leukosis virus subgroup K (ALV-K) is composed of newly emerging isolates, which, in sequence analyses, cluster separately from the well-characterized subgroups A, B, C, D, E, and J. However, it remains unclear whether ALV-K represents an independent ALV subgroup with regard to receptor usage, host range, and superinfection interference. In the present study, we examined the host range of the Chinese infectious isolate JS11C1, an ALV-K prototype, and we found substantial overlap of species that were either resistant or susceptible to ALV-A and JS11C1. Ectopic expression of the chicken tva gene in mammalian cells conferred susceptibility to JS11C1, while genetic ablation of the tva gene rendered chicken DF-1 cells resistant to infection by JS11C1. Thus, tva expression is both sufficient and necessary for JS11C1 entry. Receptor sharing was also manifested in superinfection interference, with preinfection of cells with ALV-A, but not ALV-B or ALV-J, blocking subsequent JS11C1 infection. Finally, direct binding of JS11C1 and Tva was demonstrated by preincubation of the virus with soluble Tva, which substantially decreased viral infectivity in susceptible chicken cells. Collectively, these findings indicate that JS11C1 represents a new and bona fide ALV subgroup that utilizes Tva for cell entry and binds to a site other than that for ALV-A.IMPORTANCE ALV consists of several subgroups that are particularly characterized by their receptor usage, which subsequently dictates the host range and tropism of the virus. A few newly emerging and highly pathogenic Chinese ALV strains have recently been suggested to be an independent subgroup, ALV-K, based solely on their genomic sequences. Here, we performed a series of experiments with the ALV-K strain JS11C1, which showed its dependence on the Tva cell surface receptor. Due to the sharing of this receptor with ALV-A, both subgroups were able to interfere with superinfection. Because ALV-K could become an important pathogen and a significant threat to the poultry industry in Asia, the identification of a specific receptor could help in the breeding of resistant chicken lines with receptor variants with decreased susceptibility to the virus.

Regulatory effects of chicken TRIM25 on the replication of ALV-A and the MDA5-mediated type I interferon response.

This study focuses on the immunoregulatory effects of chicken TRIM25 on the replication of subgroup A of avian leukosis virus (ALV-A) and the MDA5-mediated type I interferon response. The ALV-A-SDAU09C1 strain was inoculated into DF1 cells and 1-day-old SPF chickens, and the expression of TRIM25 was detected at different time points after inoculation. A recombinant overexpression plasmid containing the chicken TRIM25 gene (TRIM25-GFP) was constructed and transfected into DF1 cells to analyse the effects of the overexpression of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-ß. A small interfering RNA targeting chicken TRIM25 (TRIM25-siRNA) was prepared and transfected into DF1 cells to assess the effects of the knockdown of chicken TRIM25 on the replication of ALV-A and the expression of MDA5, MAVS and IFN-ß. The results showed that chicken TRIM25 was significantly upregulated at all time points both in ALV-A-infected cells and in ALV-A-infected chickens. Overexpression of chicken TRIM25 in DF1 cells dramatically decreased the antigenic titres of ALV-A in the cell supernatant and upregulated the relative expression of MDA5, MAVS and IFN-ß induced by ALV-A or by poly(I:C); in contrast, knockdown of chicken TRIM25 significantly increased the antigenic titres of ALV-A and downregulated the relative expression of MDA5, MAVS and IFN-ß. It can be concluded that chicken TRIM25 can inhibit the replication of ALV-A and upregulate the MDA5 receptor-mediated type I interferon response in chickens. This study can help improve the understanding of the antiviral activities of chicken TRIM25 and enrich the knowledge of antiviral responses in chickens.

A balanced game: chicken macrophage response to ALV-J infection.

Avian leukosis virus subgroup J (ALV-J) infection can cause tumors and immunosuppression in infected chickens. Macrophages play a central role in host defense against invading pathogens. In this study, we discovered an interesting phenomenon: ALV-J replication is weakened from 3 hours post-infection (hpi) to 36 hpi, which was verified using Western blotting and RT-PCR. To further investigate the interaction between ALV-J and macrophages, transcriptome analysis was performed to analyze the host genes' function in chicken primary monocyte-derived macrophages (MDM). Compared to the uninfected control, 624 up-regulated differentially expressed genes (DEG) and 341 down-regulated DEG at 3 hpi, and 174 up-regulated DEG and 87 down-regulated DEG at 36 hpi were identified in chicken MDM, respectively. ALV-J infection induced strong innate immune responses in chicken MDM at 3 hpi, instead of 36 hpi, according to the analysis results of Gene Ontology and KEGG pathway. Importantly, the host factors, such as up-regulated MIP-3α, IL-1ß, iNOS, K60, IRG1, CH25H, NFKBIZ, lysozyme and OASL were involved in the host defense response during the course of ALV-J infection. On the contrary, up-regulated EX-FABP, IL4I1, COX-2, NFKBIA, TNFAIP3 and the Jak STAT pathway inhibitors including CISH, SOCS1 and SOCS3 are beneficial to ALV-J survival in chicken macrophages. We speculated that ALV-J tropism for macrophages helps to establish a latent infection in chicken MDM from 6 to 36 hpi. The present study provides a comprehensive view of the interactions between macrophages and ALV-J. It suggests the mechanisms of defense of chicken macrophages against ALV-J invasion and how ALV-J escape the host innate immune responses.

Reticuloendotheliosis virus and avian leukosis virus subgroup J synergistically increase the accumulation of exosomal miRNAs.

BACKGROUND: Co-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown. RESULTS: In this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth. CONCLUSIONS: We demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.

Identification of New World Quails Susceptible to Infection with Avian Leukosis Virus Subgroup J.

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+/H+ 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+/H+ 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+/H+ 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+/H+ 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.

The construction and application of a cell line resistant to novel subgroup avian leukosis virus (ALV-K) infection.

A novel avian leukosis viruses (ALV) subgroup named ALV-K was recently isolated from Chinese indigenous chickens which is different from the subgroups (A to E and J) that have previously been reported to infect chickens. More and more ALV-K strains have recently been isolated from local breeds of Chinese chickens. However, there are no more effective diagnostic methods for ALV-K other than virus isolation followed by envelope gene sequencing and comparison. Viral infection can be blocked through expression of the viral receptor-binding protein. In this study, we have engineered a cell line, DF-1/K, that expresses ALV-K env protein and thereby confers resistance to ALV-K infection. DF-1/K can be used in combination with the ALV-K susceptible cell line DF-1 as a specific diagnostic tool for ALV-K and provides a good tool for further research into the molecular mechanisms of interaction between ALV-K env protein and the host cell receptor.

Ubiquitination of non-lysine residues in the retroviral integrase.

Retroviral integrase catalyzes the integration of retroviral genome into host chromosomal DNA, which is a prerequisite of effective viral replication and infection. The human immunodeficiency virus type 1 (HIV-1) integrase has previously been reported to be regulated by the ubiquitination, but the molecular characterization of integrase ubiquitination is still unclear. In this study, we analyzed the ubiquitination of avian leukosis virus (ALV) integrase in detail. The ubiquitination assay showed that, like HIV-1, ALV integrase could also be modified by ubiquitination when expressed in 293 T and DF-1 cells. Domain mapping analysis revealed that the ubiquitination of ALV integrase might mainly occurred in the catalytic core and the N-terminal zinc-binding domains. Both lysine and non-lysine residues within integrase of ALV and HIV-1 were responsible for the ubiquitin conjugation, and the N-terminal HHCC zinc-binding motif might play an important role in mediating integrase ubiquitination. Interestingly, mass spectrometry analysis identified the Thr10 and Cys37 residues in the HHCC zinc-binding motif as the ubiquitination sites, indicating that ubiquitin may be conjugated to ALV integrase through direct interaction with the non-lysine residues. These findings revealed the detailed features of retroviral integrase ubiquitination and found a novel mechanism of ubiquitination mediated by the non-lysine residues within the N-terminal zinc-binding domain of integrase.

Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.

J subgroup avian leukosis virus (ALV-J) is an exogenous retrovirus of avian. A key feature of ALV-J infection is leading to severe immunosuppressive characteristic of diseases. Viral components of retrovirus were reported closely associated with immunosuppression, and several similarities between exosomes and retrovirus preparations have lead to the hypotheses of retrovirus hijacker exosomes pathway. In this study, we purified exosomes from DF-1 cells infected and uninfected by ALV-J. Electron microscopy and mass spectrometry (MS) analysis showed that ALV-J not only increased the production of exosomes from ALV-J infected DF-1 cells (Exo-J) but also stimulated some proteins expression, especially ALV-J components secreted in exosomes. Immunosuppressive domain peptide (ISD) of envelope subunit transmembrane (TM) and gag of ALV-J were secreted in Exo-J. It has been reported that HIV gag was budded from endosome-like domains of the T cell plasma membrane. But env protein was first detected in exosomes from retrovirus infected cells. We found that Exo-J caused negative effects on splenocytes in a dose-dependant manner by flow cytometric analysis. And low dose of Exo-J activated immune activity of splenocytes, while high dose possessed immunosuppressive properties. Interestingly, Exo-J has no significant effects on the immunosuppression induced by ALV-J, and the immunosuppressive effects induced by Exo-J lower than that by ALV-J. Taken together, our data indicated that Exo-J supplied a microenvironment for the replication and transformation of ALV-J.

Growth retardation induced by avian leukosis virus subgroup J associated with down-regulated Wnt/ß-catenin pathway.

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces growth retardation and neoplasia in chickens, leading to enormous economic losses in poultry industry. Increasing evidences showed several signal pathways involved in ALV-J infection. However, what signaling pathway involved in growth retardation is largely unknown. To explore the possible signaling pathway, we tested the cell proliferation and associated miRNAs in ALV-J infected CEF cells by CCK-8 and Hiseq, respectively. The results showed that cell proliferation was significantly inhibited by ALV-J and three associated miRNAs were identified to target Wnt/ß-catenin pathway. To verify the Wnt/ß-catenin pathway involved in cell growth retardation, we analyzed the key molecules of Wnt pathway in ALV-J infected CEF cells. Our data demonstrated that protein expression of ß-catenin was decreased significantly post ALV-J infection compared with the normal (P < 0.05). The impact of this down-regulation caused low expression of known target genes (Axin2, CyclinD1, Tcf4 and Lef1). Further, to obtain in vivo evidence, we set up an ALV-J infection model. Post 7 weeks infection, ALV-J infected chickens showed significant growth retardation. Subsequent tests showed that the expression of ß-catenin, Tcf1, Tcf4, Lef1, Axin2 and CyclinD1 were down-regulated in muscles of growth retardation chickens. Taken together, all data demonstrated that chicken growth retardation caused by ALV-J associated with down-regulated Wnt/ß-catenin signaling pathway.

Inhibition of ERK/MAPK suppresses avian leukosis virus subgroup A and B replication.

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway contributes to subgroup J avian leukosis virus (ALV-J) replication and tumorigenicity. However, a role for ERK/MAPK signaling in ALV-A and ALV-B replication is unknown. In this study we successfully constructed and recovered a recombinant form of ALV-A strain GD13-1 which showed similarities in growth to the parental wild type virus in vitro. ALV subgroups J, A or B all triggered ERK2 activation in primary CEF cells. ERK/MAPK inhibition markedly suppressed ALV-A and ALV-B replication as shown by extremely low levels of viral transcription and virus protein production. This finding provides evidence that ERK/MAPK signaling responses play important roles in ALV replication and may represent novel drug targets for therapeutic intervention strategies.

Vertical transmission of avian leukosis virus subgroup J (ALV-J) from hens infected through artificial insemination with ALV-J infected semen.

BACKGROUND: Avian leukosis virus (ALV) is one of the main causes of tumour development within the poultry industry in China. The subgroup J avian leukosis viruses (ALV-J), which induce erythroblastosis and myelocytomatosis, have the greatest pathogenicity and transmission ability within this class of viruses. ALV can be transmitted both horizontally and vertically; however, the effects of ALV infection in chickens-especially roosters-during the propagation, on future generations is not clear. Knowing the role of the cock in the transmission of ALV from generation to generation might contribute to the eradication programs for ALV. RESULTS: The results showed that two hens inseminated with ALV-J-positive semen developed temporary antibody responses to ALV-J at 4-5 weeks post insemination. The p27 antigen was detected in cloacal swabs of six hens, and in 3 of 26 egg albumens at 1-6 weeks after insemination. Moreover, no viremia was detected at 6 weeks after insemination even when virus isolation had been conducted six times at weekly intervals for each of the 12 females. However, ALV-J was isolated from 1 of their 34 progeny chicks at 1 week of age, and its gp85 had 98.4%-99.2% sequence identity with the gp85 of ALV-J isolated from semen samples of the six cocks. CONCLUSIONS: Our findings indicated that females that were late horizontally infected with ALV-J by artificial insemination might transmit the virus to progeny through eggs, which amounts to vertical transmission.

Poly(ADP-ribose) polymerase-1 silences retroviruses independently of viral DNA integration or heterochromatin formation.

PARP-1 silences retrotransposons in Drosophila, through heterochromatin maintenance, and integrated retroviruses in chicken. Here, we determined the role of viral DNA integration and cellular heterochromatin in PARP-1-mediated retroviral silencing using HIV-1-derived lentiviral vectors and Rous-associated virus type 1 (RAV-1) as models. Analysis of the infection of PARP-1 knockout and control cells with HIV-1 harbouring WT integrase, in the presence or absence of an integrase inhibitor, or catalytic-dead mutant integrase indicated that silencing does not require viral DNA integration. The mechanism involves the catalytic activity of histone deacetylases but not that of PARP-1. In contrast to Drosophila, lack of PARP-1 in avian cells did not affect chromatin compaction globally or at the RAV-1 provirus, or the cellular levels of histone H3 N-terminal acetylated or Lys27 trimethylated, as indicated by micrococcal nuclease accessibility and immunoblot assays. Therefore, PARP-1 represses retroviruses prior to viral DNA integration by mechanisms involving histone deacetylases but not heterochromatin formation.

The MET gene is a common integration target in avian leukosis virus subgroup J-induced chicken hemangiomas.

UNLABELLED: Avian leukosis virus subgroup J (ALV-J) is a simple retrovirus that can cause hemangiomas and myeloid tumors in chickens and is currently a major economic problem in Asia. Here we characterize ALV-J strain PDRC-59831, a newly studied U.S. isolate of ALV-J. Five-day-old chicken embryos were infected with this virus, and the chickens developed myeloid leukosis and hemangiomas within 2 months after hatching. To investigate the mechanism of pathogenesis, we employed high-throughput sequencing to analyze proviral integration sites in these tumors. We found expanded clones with integrations in the MET gene in two of the five hemangiomas studied. This integration locus was not seen in previous work characterizing ALV-J-induced myeloid leukosis. MET is a known proto-oncogene that acts through a diverse set of signaling pathways and is involved in many neoplasms. We show that tumors harboring MET integrations exhibit strong overexpression of MET mRNA. IMPORTANCE: These data suggest that ALV-J induces oncogenesis by insertional mutagenesis, and integrations in the MET oncogene can drive the overexpression of MET and contribute to the development of hemangiomas.

Model of the TVA receptor determinants required for efficient infection by subgroup A avian sarcoma and leukosis viruses.

UNLABELLED: The study of the interactions of subgroup A avian sarcoma and leucosis viruses [ASLV(A)] with the TVA receptor required to infect cells offers a powerful experimental model of retroviral entry. Several regions and specific residues in the TVA receptor have previously been identified to be critical determinants of the binding affinity with ASLV(A) envelope glycoproteins and to mediate efficient infection. Two homologs of the TVA receptor have been cloned: the original quail TVA receptor, which has been the basis for most of the initial characterization of the ASLV(A) TVA, and the chicken TVA receptor, which is 65% identical to the quail receptor overall but identical in the region thought to be critical for infection. Our previous work characterized three mutant ASLV(A) isolates that could efficiently bind and infect cells using the chicken TVA receptor homolog but not using the quail TVA receptor homolog, with the infectivity of one mutant virus being >500-fold less with the quail TVA receptor. The mutant viruses contained mutations in the hr1 region of the surface glycoprotein. Using chimeras of the quail and chicken TVA receptors, we have identified new residues of TVA critical for the binding affinity and entry of ASLV(A) using the mutant glycoproteins and viruses to probe the function of those residues. The quail TVA receptor required changes at residues 10, 14, and 31 of the corresponding chicken TVA residues to bind wild-type and mutant ASLV(A) glycoproteins with a high affinity and recover the ability to mediate efficient infection of cells. A model of the TVA determinants critical for interacting with ASLV(A) glycoproteins is proposed. IMPORTANCE: A detailed understanding of how retroviruses enter cells, evolve to use new receptors, and maintain efficient entry is crucial for identifying new targets for combating retrovirus infection and pathogenesis, as well as for developing new approaches for targeted gene delivery. Since all retroviruses share an envelope glycoprotein organization, they likely share a mechanism of receptor triggering to begin the entry process. Multiple, noncontiguous interaction determinants located in the receptor and the surface (SU) glycoprotein hypervariable domains are required for binding affinity and to restrict or broaden receptor usage. In this study, further mechanistic details of the entry process were elucidated by characterizing the ASLV(A) glycoprotein interactions with the TVA receptor required for entry. The ASLV(A) envelope glycoproteins are organized into functional domains that allow changes in receptor choice to occur by mutation and/or recombination while maintaining a critical level of receptor binding affinity and an ability to trigger glycoprotein conformational changes.

Avian leukosis virus subgroup J induces its receptor--chNHE1 up-regulation.

BACKGROUND: Avian leukosis virus subgroup J (ALV-J) is an oncogenic retrovirus which causes immunosuppression and neoplasia in meat-type and egg-type chickens. ALV-J infects host cells via specific interaction between the viral Env and the cell surface receptor -chicken sodium hydrogen exchanger type 1 (chNHE1). NHE1 involved in altering the cellular pH and playing a critical role in tumorigenesis. However, little is known about the other relationship between ALV-J and chNHE1. METHODS AND RESULTS: In ALV-J infected DF-1 cells, the mRNA level of chNHE1 was up-regulated with time-dependent manner tested by real time PCR, and accordingly, intracellular pH was increased tested by spectrofluorometer. In vivo, the mRNA level of chNHE1 was determined by real time PCR in ALV-J infected experimental chickens and field cases. The result showed that the mRNA level of chNHE1 was up-regulated after virus shedding, especially in continuous viremic shedders (CS group). However, no significant difference was found between non-shedding group (NS group) and control group. In field cases, mRNA level of chNHE1 was positively correlated with increasing ALV-J load in tumor bearing and immune tolerance chickens. Furthermore, immunohistochemistry results showed that the protein expression of chNHE1 was up-regulated in different organs of both experimental chickens and tumor bearing chickens compared with the control. CONCLUSION: Taken together, we conclude that ALV-J induces chNHE1 up-regulation in viremia and neoplasia chickens.