Avian Leukosis-sarcoma Viruses Research Articles

The Use of Cell-penetrating Peptide for Delivery of Recombinant Transcription Factor DNA into Primary Human Fibroblast

Background: Reprogrammed cell therapy has not been applied for clinical purposes due to the malignancy issue. The aim of this study was to design the recombinant vector of the transcription factors and analyze the effectiveness of cell-penetrating peptide delivering system for human primary fibroblast transfection to avoid the malignancy issue.Materials and methods: The constructions of CCAT/enhancer binding protein alpha (CEBPA), hepatocyte nuclear factor 4 alpha (HNF4A), nuclear receptor subfamily 1 group I member 2 (NR1I2) were confirmed with DNA digestion and sequencing. Breast reduction (BRED) and palate (PAL) tissue were used as human primary fibroblast sources. The transcription factors were delivered into BRED and PAL with recombination of avian leukosis sarcoma virus (ALSV), human immunodeficiency virus (HIV) matrix, and regulator of expression of virion proteins (Rev) (ALMR), tagged with enhanced green fluorescence protein (eGFP). Post-transfection cells were then cultivated with optimized medium. Gene expression was measured with quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR).Results: Gene expression levels of CEBPA, HNF4A, NR1I2, glutamate-ammonia ligase (GLUL), albumin (ALB), and cytochrome P450 (CYP) were increased. Transfection with ALMR, which were more efficient in BRED than PAL fibroblasts may have the advantage in autologous cell therapy for elderly patients.Conclusion: Transfection of transcription factors to human primary fibroblast may be performed by using constructions of plasmid as designed in this study.Keywords: recombinant plasmid, hepatocyte-like cells, primary fibroblasts, recombinant peptide, cell reprogramming, autologous cells therapy

Industrial hygiene assessment of reticuloendotheliosis viruses exposure in the poultry industry

Reticuloendotheliosis viruses (REV) are a group of retroviruses like avian leukosis/sarcoma viruses (ALSV) that naturally infect and cause cancers in chickens. We recently found that ALSV antibody levels were associated with job tasks in the poultry industry. The objectives of this study are to examine whether a similar association can be found with REV antibody levels and to examine the correlation between REV and ALSV antibody levels. Relative risk was estimated comparing REV antibody levels of 45 poultry workers with those of 44 controls. The expected mean antibody level was predicted for the association with employment by a generalized linear model. Correlation coefficient was measured between ALSV and REV antibody levels. REV antibody levels were significantly higher in poultry workers than in control subjects and were associated with gender and employment conditions, especially employment duration. The relative risk was significantly higher for some job categories. A significant correlation was observed between REV and ALSV antibody levels, which was strong among poultry workers, but weak among the control subjects. Antibody levels can be validly used to identify certain job tasks associated with high risk of exposure to REV in the workplace, and the practical implication is recommendations for protection at these job tasks. Importantly, in situations where there is exposure to multiple pathogens in the workplace, the analysis of antibody levels of one pathogen may sufficiently represent exposure to the other correlated pathogens. This suggested exposure assessment may hold true for pathogens with a similar route of transmission.

Lentivirus-Mediated Oncogene Introduction into Mammary Cells In Vivo Induces Tumors

We recently reported the introduction of oncogene-expressing avian retroviruses into somatic mammary cells in mice susceptible to infection by transgenic expression of tva, encoding the receptor for subgroup A avian leukosis-sarcoma virus (ALSV). Because ALSV-based vectors poorly infect nondividing cells, they are inadequate for studying carcinogenesis initiated from nonproliferative cells (e.g., stem cells). Lentivirus pseudotyped with the envelope protein of ALSV infects nondividing TVA-producing cells in culture but has not previously been tested for introducing genes in vivo. Here, we demonstrate that these vectors infected mammary cells in vivo when injected into the mammary ductal lumen of mice expressing tva under the control of the keratin 19 promoter. Furthermore, intraductal injection of this lentiviral vector carrying the polyoma middle T antigen gene induced atypical ductal hyperplasia and ductal carcinoma in situ-like premalignant lesions in 30 days and palpable invasive tumors at a median latency of 3.3 months. Induced tumors were a mixed epithelial/myoepithelial histologic diagnosis, occasionally displayed squamous metaplasia, and were estrogen receptor-negative. This work demonstrates the first use of a lentiviral vector to introduce oncogenes for modeling cancer in mice, and this vector system may be especially suitable for introducing genetic alterations into quiescent cells in vivo.

Evolutionary pressure of a receptor competitor selects different subgroup a avian leukosis virus escape variants with altered receptor interactions.

A complex interaction between the retroviral envelope glycoproteins and a specific cell surface protein initiates viral entry into cells. The avian leukosis-sarcoma virus (ALV) group of retroviruses provides a useful experimental system for studying the retroviral entry process and the evolution of receptor usage. In this study, we demonstrate that evolutionary pressure on subgroup A ALV [ALV(A)] entry exerted by the presence of a competitive inhibitor, a soluble form of the ALV(A) Tva receptor linked to a mouse immunoglobulin G tag (quail sTva-mIgG), can select different populations of escape variants. This escape population contained three abundant ALV(A) variant viruses, all with mutations in the surface glycoprotein hypervariable regions: a previously identified variant containing the Y142N mutation in the hr1 region; a new variant with two mutations, W141G in hr1 and K261E in vr3; and another new variant with two mutations, W145R in hr1 and K261E. The W141G K261E and W145R K261E viruses escape primarily by lowering their binding affinities for the quail Tva receptor competitive inhibitor while retaining wild-type levels of binding affinity for the chicken Tva receptor. A secondary phenotype of the new variants was an alteration in receptor interference patterns from that of wild-type ALV(A), indicating that the mutant glycoproteins are possibly interacting with other cellular proteins. One result of these altered interactions was that the variants caused a transient period of cytotoxicity. We could also directly demonstrate that the W141G K261E variant glycoproteins bound significant levels of a soluble form of the Tvb(S3) ALV receptor in a binding assay. Alterations in the normally extreme specificity of the ALV(A) glycoproteins for Tva may represent an evolutionary first step toward expanding viral receptor usage in response to inefficient viral entry.

The c-myc and PyMT oncogenes induce different tumor types in a somatic mouse model for pancreatic cancer.

We have generated a mouse model for pancreatic cancer through the somatic delivery of oncogene-bearing avian retroviruses to mice that express TVA, the receptor for avian leukosis sarcoma virus subgroup A (ALSV-A), under the control of the elastase promoter. Delivery of ALSV-A-based RCAS vectors encoding either mouse polyoma virus middle T antigen (PyMT) or c-Myc to elastase-tv-a transgenic, Ink4a/Arf null mice induced the formation of pancreatic tumors. RCAS-PyMT induced pancreatic tumors with the histologic features of acinar or ductal carcinomas. The induced pancreatic lesions express Pdx1, a marker for pancreas progenitor cells, and many tumors express markers for both exocrine and endocrine cell lineages, suggesting that the tumors may be derived from progenitor cells. In contrast, RCAS-c-myc induced endocrine tumors exclusively, as determined by histology and detection of differentiation markers. Thus, specific oncogenes can induce the formation of different pancreatic tumor types in a single transgenic line, most likely from one or more types of multipotential progenitor cells. Our model appears to be useful for elucidating the genetic alterations, target cells, and signaling pathways that are important in the genesis of different types of pancreatic cancer.

Cellular apoptosis and organ injury in sepsis: a review.

Cellular apoptosis and organ injury in sepsis: a review.

Development of an avian leukosis-sarcoma virus subgroup A pseudotyped lentiviral vector.

We are using avian leukosis-sarcoma virus (ALSV) vectors to generate mouse tumor models in transgenic mice expressing TVA, the receptor for subgroup A ALSV. Like other classical retroviruses, ALSV requires cell division to establish a provirus after infection of host cells. In contrast, lentiviral vectors are capable of integrating their viral DNA into the genomes of nondividing cells. With the intention of initiating tumorigenesis in resting, TVA-positive cells, we have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1)-based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A (EnvA). The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells and can be produced at titers of 5 x 10(3) infectious units (IU)/ml. By inserting the central polypurine tract (cPPT) from the HIV-1 pol gene and removing the cytoplasmic tail of EnvA, the pseudotype can be produced at titers approaching 10(5) IU/ml and can be concentrated by ultracentrifugation to titers of 10(7) IU/ml. HIV(ALSV-A) also infects embryonic fibroblasts derived from transgenic mice in which TVA expression is driven by the beta-actin promoter. In addition, this lentivirus pseudotype efficiently infects these fibroblasts after cell cycle arrest, when they are resistant to infection by ALSV vectors. This system may be useful for introducing genes into somatic cells in adult TVA transgenic animals and allows evaluation of the effects of altered gene expression in differentiated cell types in vivo.

Avian leukosis virus – new mutations: a threat for the upcoming century

New strains of avian leukosis virus (ALV) and new threats to the poultry industry such as the appearance of subgroup J ALV arise as a result of virus mutation. Avian retroviruses are classified as exogenous (transmitted as infectious virus) and endogenous (transmitted genetically). Exogenous species are the avian leukosis-sarcoma viruses, reticuloendotheliosis virus, and lymphoproliferative disease virus of turkeys. Endogenous virus families include the ev loci, EAV, ART-CH and CR 1. Evolutionary relationships between different species of retrovirus are determined from their nucleotide sequences and construction of phylogenetic trees. Similar analyses are conducted to determine the relationships between endogenous and exogenous retroviruses and the various retroelements from which retroviruses are believed to have evolved. Retroviruses are subject to several types of mutation. Point mutations are the most common and occur mainly because of errors during reverse transcription. The frequency of point mutations is of the order of one mutation per virion per replicative cycle. Point mutations occur most frequently in the env gene of ALVs resulting in changes in antigenicity and host range. Antigenic variation enables mutant viruses to avoid the host immune response. Mutation also occurs from genetic recombination among exogenous and endogenous ALVs, and between exogenous ALVs and non-retroviral host genes such as cellular oncogenes, giving rise to acute avian leukaemia and sarcoma viruses. Thus, continuing viral mutation will result in the appearance of ALVs with new disease producing properties.

Viruses and apoptosis: meddling with mitochondria.

Virus infection triggers a complex cascade in which the host’s immune system is deployed in order to prevent virus replication and dissemination. The initiation of a self-destruct apoptotic program in infected somatic cells represents a highly effective innate host response employed for curtailing infection. However, viruses have, in turn, evolved diverse strategies to manipulate the host’s apoptotic response to ensure their continued propagation (O’Brien, 1998; Roulston et al., 1999; Tortorella et al., 2000). The ultimate outcome following infection therefore depends on the balance between competing host and viral influences on the cell death program. Apoptotic signals can be classified as “extrinsic” if triggered by death receptor engagement at the cell surface or “intrinsic” if intracellular sensors are triggered (Fig. 1) (Hengartner, 2000; Kroemer and Reed, 2000). The precise nature of the “danger signals” that allow cells to sense infection and consequently the need to initiate apoptosis are varied (Everett and McFadden, 1999), but the activation of apoptotic cascades appears to be an unavoidable consequence of most infections and often requires the development of viral anti-apoptotic defenses. Extrinsic signals can be initiated by adsorption of viral particles during the entry process at the plasma membrane. Attachment of noninfectious, conformationally intact human immunodeficiency virus (HIV), vaccinia virus, and Herpes simplex virus (HSV) particles to the surface of cells is proapoptotic (Esser et al., 2001; Ramsey-Ewing and Moss, 1998; Zhou et al., 2000). Receptormediated death signaling also appears to be triggered during entry of avian–leukosis–sarcoma virus, which attaches to cells by means of a TNF receptor-related protein, CAR1 (Brojatsch et al., 1996). Engagement of this receptor by the viral envelope proteins is sufficient to

Yeast three-hybrid screening of rous sarcoma virus mutants with randomly mutagenized minimal packaging signals reveals regions important for gag interactions.

We previously showed that the yeast three-hybrid system provides a genetic assay of both RNA and protein components for avian retroviral RNA encapsidation. In the current study, we used this assay to precisely define cis-acting determinants involved in avian leukosis sarcoma virus packaging RNA binding to Gag protein. In vivo screening of Rous sarcoma virus mutants was performed with randomly mutated minimal packaging sequences (MPsi) made using PCR amplification after cotransformation with GagDeltaPR protein into yeast cells. Colonies with low beta-galactosidase activity were analyzed to locate mutations in MPsi sequences affecting binding to Gag proteins. This genetic assay delineated secondary structural elements that are important for efficient RNA binding, including a single-stranded small bulge containing the initiation codon for uORF3, as well as adjacent stem structures. This implies a possible tertiary structure favoring the high-affinity binding sites for Gag. In most cases, results from the three-hybrid assay were well correlated with those from the viral RNA packaging assays. The results from random mutagenesis using the rapid three-hybrid binding assay are consistent with those from site-directed mutagenesis using in vivo packaging assays.

Selection of a Subgroup A Avian Leukosis Virus [ALV(A)] Envelope Resistant to Soluble ALV(A) Surface Glycoprotein

The host developing resistance to retroviral infection is believed to be a major force in the evolution of multiple receptor usage by retroviruses. The avian leukosis–sarcoma virus (ALV) group of retroviruses provides a powerful system for studying the envelope–receptor interactions involved in retrovirus entry; different members of this group of closely related viruses use distinct cellular receptors. Analysis of the ALV envelope subgroups suggests that the different ALVs evolved from a common ancestor by mutations in the env gene. Cells and animals that express subgroup A ALV envelope glycoproteins are highly resistant to ALV(A) infection due to receptor interference. In this study, we tested whether expression of a soluble form of subgroup A surface glycoprotein (SU) would result in receptor interference and whether this interference would select for resistant viruses with altered receptor usage. Chicken cells expressing the secreted ALV(A) SU immunoadhesin SU(A)-rIgG, which contains the subgroup A SU domain fused to the constant region of a rabbit immunoglobulin (IgG) heavy chain, showed significant receptor interference. A variant virus resistant to SU(A)-rIgG receptor interference was obtained. This virus had a six-amino-acid deletion in the subgroup A hr1 that altered receptor usage. This approach may identify regions of SU that play a critical role in receptor specificity.

An Mpsi-containing heterologous RNA, but not env mRNA, is efficiently packaged into avian retroviral particles.

Retroviruses preferentially package full-length genomic RNA over spliced viral messages. For most retroviruses, this preference is likely due to the absence of all or part of the packaging signal on subgenomic RNAs. In avian leukosis-sarcoma virus, however, we have shown that the minimal packaging signal, MPsi, is located upstream of the 5' splice site and therefore is present on both genomic and spliced RNAs. We now show that an MPsi-containing heterologous RNA is packaged only 2.6-fold less efficiently than genomic Rous sarcoma virus RNA. Thus, few additional packaging sequences and/or structures exist outside of MPsi. In contrast, we found that env mRNA is not efficiently packaged. These results indicate that either MPsi is not functional on this RNA or the RNA is somehow segregated from the packaging machinery. Finally, deletion of sequences from the 3' end of MPsi was found to reduce the packaging efficiency of heterologous RNAs.

Genome structure and expression of the ev/J family of avian endogenous viruses.

We recently reported the identification of sequences in the chicken genome that show over 95% identity to the novel envelope gene of the subgroup J avian leukosis virus (S. J. Benson, B. L. Ruis, A. M. Fadly, and K. F. Conklin, J. Virol. 72:10157-10164, 1998). Based on the fact that the endogenous subgroup J-related env genes were associated with long terminal repeats (LTRs), we concluded that these LTR-env sequences defined a new family of avian endogenous viruses that we designated the ev/J family. In this report, we have further characterized the content and expression of the ev/J proviruses. The data obtained indicate that there are between 6 and 11 copies of ev/J proviruses in all chicken cells examined and that these proviruses fall into six classes. Of the 18 proviruses examined, all share a high degree of sequence identity and all contain an internal deletion that removes all of the pol gene and various amounts of gag and env gene sequences. Sequencing of the gag genes, LTRs, and untranslated regions of several ev/J proviruses revealed a high level of identity between isolates, indicating that they have not undergone significant sequence variation since their introduction into the avian germ line. Although the ev/J gag gene showed a relatively weak relationship (46% identity and 61% similarity at the amino acid level) to that of the avian leukosis-sarcoma virus family, it retains several sequences of demonstrated importance for virus assembly, budding, and/or infectivity. Finally, evidence was obtained that at least some members of the ev/J family are expressed and, if translated, could encode Gag- and Env-related polypeptides.

Multi-centric histiocytosis: Experimental induction in broiler and specific pathogen-free leghorn chickens

Seventy-five 3-day-old broiler chicks and twenty specific pathogen-free leghorn chicks were injected with 0.5 ml of a homogenate, prepared from organs from broilers diagnosed with naturally-occurring multicentric histiocytosis (MH). Equal numbers of uninjected broiler and leghorn chicks (controls) were maintained in adjacent pens. Ten weeks later, nine broilers had well-developed gross and microscopic MH lesions. The distribution and histological appearance of lesions in these experimental chicks was similar to lesions described in naturally occurring field cases. Six leghorns had gross lesions similar to those found in their broiler counterparts; however, in the leghorns, the cellular masses contained more lymphocytes and, additionally, masses were found in the gizzard musculature. One gizzard contained a sarcoma. Broiler chickens with MH weighed less than their control counterparts and were more likely to be anaemic. Sequences specific for reticuloendotheliosis viruses (REV) were found in the MH homogenate, in organs from most affected experimental leghorns and broilers, and in organs from a control broiler. However, REV were not isolated from these tissues, nor were specific antibodies for REV or avian leukosis/sarcoma viruses (ALV) found in chick serum. Leukosis/sarcoma viruses were isolated from some MH-affected experimental leghorns and broilers. Sequences specific for Marek's disease herpesvirus were not identified by polymerase chain reaction. The aetiology of MH remains unknown.

The EV-O-Derived Cell Line DF-1 Supports the Efficient Replication of Avian Leukosis-Sarcoma Viruses and Vectors

The lack of a well-behaved permanent, adherent, nontransformed chicken cell line has made some experiments with avian leukosis–sarcoma viruses (ASLV) and vectors considerably more difficult. The EV-O-derived line, DF-1, supports the efficient replication of subgroups (A), (B), and (C) ASLV, as well as amphotrophic murine leukemia virus and an ASLV-derived vector that has itsenvgene derived from theenvgene from an amphotrophic murine leukemia virus. The cell line responds appropriately to the expression of a transforming oncogene (v-myc) to a growth suppressor gene [p21(waf1)] and can be sorted (using FACS) if infected by an ASLV vector that expresses GFP.

Retrovirus-induced disease in poultry

Three species of avian retrovirus cause disease in poultry: the avian leukosis/sarcoma virus (ALSV), reticuloendotheliosis virus (REV), and lymphoproliferative disease virus (LPDV) of turkeys. The ALSV can be classified as slowly transforming viruses, which lack a viral oncogene, and acutely transforming viruses, which possess a viral oncogene. Slowly transforming viruses induce late onset leukoses of the B cell lymphoid, erythroid, and myeloid cell lineages, and other tumors, by viral promoter insertion into the genome of a host cell and activation of a cellular protooncogene. The various acutely transforming leukemia and sarcoma viruses induce leukotic or other tumors rapidly and carry one or anther (sometimes two) viral oncogenes, of which some 15 have been identified. The ALSV fall into six envelope subgroups, A through E, and the recently recognized J subgroup, which induces myeloid leukosis. With the exception of Subgroup E viruses, these viruses spread vertically and horizontally as infectious virions, and are termed exogenous viruses. Subgroup E viruses are usually spread genetically as DNA proviruses (often defective) in host germ cell genome, and are termed endogenous viruses. Several other families of endogenous viruses also exist, one of which, endogenous avian retrovirus (EAV), is related to Subgroup J ALV. Exogenous viruses, and sometimes endogenous viruses, can have detrimental effects on commercially important production traits. Exogenous viruses are currently controlled by virus eradication schemes. Reticuloendotheliosis virus, which lacks a viral oncogene, causes chronic B cell and T-cell lymphomas in chickens, and also chronic lymphomas in turkeys and other species of birds. An acutely transforming variant of REV, Strain T, carries a viral oncogene, and induces reticuloendotheliosis within a few days. In chickens and turkeys, REV spreads vertically and horizontally. No commercial control schemes are operated. In turkeys, LPDV infection has occurred in several countries, where it caused a lymphoproliferative disease of uncertain nature.

A minimal avian retroviral packaging sequence has a complex structure.

We have defined a 160-nucleotide region, Mpsi, from the 5' leader region of the Rous sarcoma virus genome that is sufficient to direct the packaging of a heterologous RNA. Mpsi contains the putative O3 stem structure that has previously been shown, and that has been confirmed in this study, to be important for the efficient packaging of avian leukosis-sarcoma virus RNA. Analyses of several O3 stem mutants revealed that other regions within Mpsi can interfere with the proper folding of altered sequences which are predicted to form a wild-type O3 stem.

Immunogold for detection of avian leukosis/sarcoma virus in formalin‐fixed heart and kidney

Summary Immunoprobes are used in the qualitative and semi‐quantitative demonstration of antigens in clinical samples. In the present study, an immunogold method has been used for routine and rapid detection of avian leukosis/sarcoma virus (AL/SV) in sections of formalin‐fixed chicken heart and kidney. Application of this ultrastructural indirect immunogold labelling technique (IILT) will enable pathologists to obtain a definitive diagnosis of avian AL/SV infection when inclusions or putative viral particles are observed by light or electron microscopy. IILT also may be used in research on AL/SV infections.

CAR1, a TNFR–Related Protein, Is a Cellular Receptor for Cytopathic Avian Leukosis–Sarcoma Viruses and Mediates Apoptosis

Viral envelope (Env)–receptor interactions have been implicated in the cell death associated with infection by subgroups B and D avian leukosis–sarcoma viruses (ALVs). A chicken protein, CAR1, was identified that permitted infection of mammalian cells by these viral subgroups. CAR1 bound to a viral Env fusion protein, comprising an ALV-B surface Env protein and the Fc region of an immunoglobulin, indicating that it is a specific viral receptor. CAR1 contains two extracellular cysteine-rich domains characteristic of the TNFR family and a cytoplasmic region strikingly similar to the death domain of TNFR1 and Fas, implicating this receptor in cell killing. Chicken embryo fibroblasts susceptible to ALV-B infection and transfected quail QT6 cells expressing CAR1 underwent apoptosis in response to the Env–Ig fusion protein, demonstrating that this cytopathic ALV receptor can mediate cell death.

The receptor for the subgroup A avian leukosis-sarcoma viruses binds to subgroup A but not to subgroup C envelope glycoprotein

The putative subgroup A avian leukosis-sarcoma virus (ALSV) receptor (Tva) was recently cloned by gene transfer (P. Bates, J. A. Young, and H. E. Varmus, Cell 74:1043-1051, 1993; J. A. T. Young, P. Bates, and H. E. Varmus, J. Virol. 67:1811-1816, 1993). Susceptibility to infection by subgroup A ALSV is conferred on cells upon transfection with cDNAs encoding tva. The hypothesis that tva encodes a specific receptor for subgroup A ALSV predicts that the Tva protein should bind to subgroup A, but not to subgroup C, envelope glycoprotein. In this study, we examined this prediction by using several biochemical assays. We established stable NIH 3T3 cell lines expressing either Tva, the subgroup A envelope glycoprotein (Env-A), or the subgroup C envelop glycoprotein (Env-C) and used them in conjunction with soluble forms of these molecules to demonstrate specific binding. When cell lysates containing Tva were mixed with lysates of either Env-A or Env-C, an immunoprecipitable complex formed between Tva and Env-A but not between Tva and Env-C. A soluble, oligomeric form, of Env-A, not Env-C, binds to cells expressing Tva. Reciprocally, a secreted form of Tva can bind to cells expressing Env-A but not to cells expressing Env-C. A specific and stable complex formed between soluble Env-A and secreted Tva as demonstrated by sucrose density gradient centrifugation. Thus, by three kinds of assays, Tva appears to bind specifically to Env-A, which is consistent with genetic evidence that it serves as the cell surface receptor of subgroup A ALSV and the main determinant of subgroup specificity.