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An accurate diagnostic test is an essential aspect of successfully monitoring and managing wildlife diseases. Lymphoproliferative Disease Virus (LPDV) is an avian retrovirus that was first identified in domestic turkeys in Europe and was first reported in a Wild Turkey (Meleagris gallopavo) in the United States in 2009. It has since been found to be widely distributed throughout North America. The majority of studies have utilized bone marrow and PCR primers targeting a 413-nucleotide sequence of the gag gene of the provirus to detect infection. While prior studies have evaluated the viability of other tissues for LPDV detection (whole blood, spleen, liver, cloacal swabs) none to date have studied differences in detection rates when utilizing different genomic regions of the provirus. This study examined the effectiveness of another section of the provirus, a 335-nucleotide sequence starting in the U3 region of the LTR (Long Terminal Repeat) and extending into the Matrix of the gag region (henceforth LTR), for detecting LPDV. Bone marrow samples from hunter-harvested Wild Turkeys (n = 925) were tested for LPDV with the gag gene and a subset (n = 417) including both those testing positive and those where LPDV was not detected was re-tested with LTR. The positive percent agreement (PPA) was 97.1% (68 of 70 gag positive samples tested positive with LTR) while the negative percent agreement (NPA) was only 68.0% (236 of 347 gag negative samples tested negative with LTR). Cohen's Kappa (κ = 0.402, Z = 10.26, p<0.0001) and the McNemar test (OR = 55.5, p<0.0001) indicated weak agreement between the two gene regions. We found that in Iowa Wild Turkeys use of the LTR region identified LPDV in many samples in which we failed to detect LPDV using the gag region and that LTR may be more appropriate for LPDV surveillance and monitoring. However, neither region of the provirus resulted in perfect detection and additional work is necessary to determine if LTR is more reliable in other geographic regions where LPDV occurs., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Smith, Blanchong. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Immune cell therapeutics are increasingly applied in oncology. Especially chimeric antigen receptor (CAR) T cells are successfully used to treat several B cell malignancies. Efforts to engineer CAR T cells for improved activity against solid tumors include co-delivery of pro-inflammatory cytokines in addition to CARs, via either constitutive cytokine expression or inducible cytokine expression triggered by CAR recognition of its target antigen-so-called "T cells redirected for universal cytokine-mediated killing" (TRUCKs) or fourth-generation CARs. Here, we tested the hypothesis that TRUCK principles could be expanded to improve anticancer functions of NK cells. A comparison of the functionality of inducible promoters responsive to NFAT or NFκB in NK cells showed that, in contrast to T cells, the inclusion of NFκB-responsive elements within the inducible promoter construct was essential for CAR-inducible expression of the transgene. We demonstrated that GD2CAR-specific activation induced a tight NFκB-promoter-driven cytokine release in NK-92 and primary NK cells together with an enhanced cytotoxic capacity against GD2 + target cells, also shown by increased secretion of cytolytic cytokines. The data demonstrate biologically relevant differences between T and NK cells that are important when clinically translating the TRUCK concept to NK cells for the treatment of solid malignancies., Competing Interests: A patent application has been submitted by ASc, HA, JK and KZ. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Rudek, Zimmermann, Galla, Meyer, Kuehle, Stamopoulou, Brand, Sandalcioglu, Neyazi, Moritz, Rossig, Altvater, Falk, Abken, Morgan and Schambach.)

Anti-cancer activity can be improved by engineering immune cells to express chimeric antigen receptors (CARs) that recognize tumor-associated antigens. Retroviral vector gene transfer strategies allow stable and durable transgene expression. Here, we used alpharetroviral vectors to modify NK-92 cells, a natural killer cell line, with a third-generation CAR designed to target the IL-3 receptor subunit alpha (CD123), which is strongly expressed on the surface of acute myeloid leukemia (AML) cells. Alpharetroviral vectors also contained a transgene cassette to allow constitutive expression of human IL-15 for increased NK cell persistence in vivo. The anti-AML activity of CAR-NK-92 cells was tested via in vitro cytotoxicity assays with the CD123 + AML cell line KG-1a and in vivo in a patient-derived xenotransplantation CD123 + AML model. Unmodified NK-92 cells or NK-92 cells modified with a truncated version of the CAR that lacked the signaling domain served as controls. Alpharetroviral vector-modified NK-92 cells stably expressed the transgenes and secreted IL-15. Anti-CD123-CAR-NK-92 cells exhibited enhanced anti-AML activity in vitro and in vivo as compared to control NK-92 cells. Our data (1) shows the importance of IL-15 expression for in vivo persistence of NK-92 cells, (2) supports continued investigation of anti-CD123-CAR-NK cells to target AML, and (3) points towards potential strategies to further improve CAR-NK anti-AML activity.

Autologous chimeric antigen receptor-modified (CAR) T cells with specificity for CD19 showed potent antitumor efficacy in clinical trials against relapsed and refractory B-cell acute lymphoblastic leukemia (B-ALL). Contrary to T cells, natural killer (NK) cells kill their targets in a non-antigen-specific manner and do not carry the risk of inducing graft vs. host disease (GvHD), allowing application of donor-derived cells in an allogenic setting. Hence, unlike autologous CAR-T cells, therapeutic CD19-CAR-NK cells can be generated as an off-the-shelf product from healthy donors. Nevertheless, genetic engineering of peripheral blood (PB) derived NK cells remains challenging and optimized protocols are needed. In our study, we aimed to optimize the generation of CD19-CAR-NK cells by retroviral transduction to improve the high antileukemic capacity of NK cells. We compared two different retroviral vector platforms, the lentiviral and alpharetroviral, both in combination with two different transduction enhancers (Retronectin and Vectofusin-1). We further explored different NK cell isolation techniques (NK cell enrichment and CD3/CD19 depletion) to identify the most efficacious methods for genetic engineering of NK cells. Our results demonstrated that transduction of NK cells with RD114-TR pseudotyped retroviral vectors, in combination with Vectofusin-1 was the most efficient method to generate CD19-CAR-NK cells. Retronectin was potent in enhancing lentiviral/VSV-G gene delivery to NK cells but not alpharetroviral/RD114-TR. Furthermore, the Vectofusin-based transduction of NK cells with CD19-CARs delivered by alpharetroviral/RD114-TR and lentiviral/RD114-TR vectors outperformed lentiviral/VSV-G vectors. The final generated CD19-CAR-NK cells displayed superior cytotoxic activity against CD19-expressing target cells when compared to non-transduced NK cells achieving up to 90% specific killing activity. In summary, our findings present the use of RD114-TR pseudotyped retroviral particles in combination with Vectofusin-1 as a successful strategy to genetically modify PB-derived NK cells to achieve highly cytotoxic CD19-CAR-NK cells at high yield., (Copyright © 2020 Müller, Bexte, Gebel, Kalensee, Stolzenberg, Hartmann, Koehl, Schambach, Wels, Modlich and Ullrich.)

In cellular immunotherapies, natural killer (NK) cells often demonstrate potent antitumor effects in high-risk cancer patients. But Good Manufacturing Practice (GMP)-compliant manufacturing of clinical-grade NK cells in high numbers for patient treatment is still a challenge. Therefore, new protocols for isolation and expansion of NK cells are required. In order to attack resistant tumor entities, NK cell killing can be improved by genetic engineering using alpharetroviral vectors that encode for chimeric antigen receptors (CARs). The aim of this work was to demonstrate GMP-grade manufacturing of NK cells using the CliniMACS ® Prodigy device (Prodigy) with implemented applicable quality controls. Additionally, the study aimed to define the best time point to transduce expanding NK cells with alpharetroviral CAR vectors. Manufacturing and clinical-scale expansion of primary human NK cells were performed with the Prodigy starting with 8-15.0 × 10 9 leukocytes (including 1.1-2.3 × 10 9 NK cells) collected by small-scale lymphapheresis ( n  = 3). Positive fraction after immunoselection, in-process controls (IPCs), and end product were quantified by flow cytometric no-wash, single-platform assessment, and gating strategy using positive (CD56/CD16/CD45), negative (CD14/CD19/CD3), and dead cell (7-aminoactinomycine [7-AAD]) discriminators. The three runs on the fully integrated manufacturing platform included immunomagnetic separation (CD3 depletion/CD56 enrichment) followed by NK cell expansion over 14 days. This process led to high NK cell purities (median 99.1%) and adequate NK cell viabilities (median 86.9%) and achieved a median CD3+ cell depletion of log -3.6 after CD3 depletion and log -3.7 after immunomagnetic CD3 depletion and consecutive CD56 selection. Subsequent cultivation of separated NK cells in the CentriCult ® chamber of Prodigy resulted in approximately 4.2-8.5-fold NK cell expansion rates by adding of NK MACS ® basal medium containing NK MACS ® supplement, interleukin (IL)-2/IL-15 and initial IL-21. NK cells expanded for 14 days revealed higher expression of natural cytotoxicity receptors (NKp30, NKp44, NKp46, and NKG2D) and degranulation/apoptotic markers and stronger cytolytic properties against K562 compared to non-activated NK cells before automated cultivation. Moreover, expanded NK cells had robust growth and killing activities even after cryopreservation. As a crucial result, it was possible to determine the appropriate time period for optimal CAR transduction of cultivated NK cells between days 8 and 14, with the highest anti-CD123 CAR expression levels on day 14. The anti-CD123 CAR NK cells showed retargeted killing and degranulation properties against CD123-expressing KG1a target cells, while basal cytotoxicity of non-transduced NK cells was determined using the CD123-negative cell line K562. Time-lapse imaging to monitor redirected effector-to-target contacts between anti-CD123 CAR NK and KG1a showed long-term effector-target interaction. In conclusion, the integration of the clinical-scale expansion procedure in the automated and closed Prodigy system, including IPC samples and quality controls and optimal time frames for NK cell transduction with CAR vectors, was established on 48-well plates and resulted in a standardized GMP-compliant overall process.

The introduction of chimeric antigen receptors (CARs) to augment the anticancer activity of immune cells represents one of the major clinical advances in recent years. This work demonstrates that sorted CAR natural killer (NK) cells have improved antileukemia activity compared to control NK cells that lack a functional CAR. However, in terms of viability, effectiveness, risk of side effects, and clinical practicality and applicability, an important question is whether gene-modified NK cell lines represent better CAR effector cells than primary human donor CAR-NK (CAR-dNK) cells. Comparison of the functional activities of sorted CAR-NK cells generated using the NK-92 cell line with those generated from primary human dNK cells demonstrated that CAR-NK-92 cells had stronger cytotoxic activity against leukemia cells compared to CAR-dNK cells. CAR-NK-92 and CAR-dNK cells had similar CD107a surface expression upon co-incubation with leukemia cells. However, CAR-NK-92 cells secreted higher granzyme A and interleukin-17A levels, while CAR-dNK cells secreted more tumor necrosis factor alpha, interferon gamma, and granulysin. In addition, CAR-NK-92 cells revealed a significantly higher potential for adverse side effects against nonmalignant cells. In short, this work shows the feasibility for further development of CAR-NK strategies to treat leukemia.

We reported that inactivation of menin (the protein product of MEN1) increases activity of Dnmt1 and mediates DNA hypermethylation in the development of multiple endocrine neoplasia type 1 (MEN1) syndrome. We have developed a RCAS-TVA-based somatic gene transfer system that enables tissue-specific delivery of Dnmt1 to individual β-cells of the pancreas in a RIP-TVA mouse model. In the present study, we mediated Dnmt1 expression in islet β-cells in RIP-TVA mice by utilizing the RCAS-TVA system to test if the upregulation of Dnmt1 can promote β-cell proliferation. In vitro, we demonstrated that upregulation of Dnmt1 increased β-cell proliferation. In vivo, our results showed that the levels of serum insulin were increased in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Furthermore, we confirmed that mRNA and protein expression of Dnmt1 as well as Dnmt1 enzyme activity were upregulated in the RIP-TVA mice with RCASBP-Dnmt1 infection compared with wild-type control mice with RCASBP-Dnmt1 infection. Finally, we demonstrated that upregulation of Dnmt1 resulted in hyperplasia through β-cell proliferation. We conclude that the upregulation of Dnmt1 promotes islet β-cell proliferation and targeting Dnmt1 may be a promising therapy for patients suffering from pancreatic neuroendocrine tumors.

Individual groups of retroviruses and retroviral vectors differ in their integration site preference and interaction with the host genome. Hence, immediately after infection genome-wide distribution of integrated proviruses is non-random. During long-term in vitro or persistent in vivo infection, the genomic position and chromatin environment of the provirus affects its transcriptional activity. Thus, a selection of long-term stably expressed proviruses and elimination of proviruses, which have been gradually silenced by epigenetic mechanisms, helps in the identification of genomic compartments permissive for proviral transcription. We compare here the extent and time course of provirus silencing in single cell clones of the K562 human myeloid lymphoblastoma cell line that have been infected with retroviral reporter vectors derived from avian sarcoma/leukosis virus (ASLV), human immunodeficiency virus type 1 (HIV) and murine leukaemia virus (MLV). While MLV proviruses remain transcriptionally active, ASLV proviruses are prone to rapid silencing. The HIV provirus displays gradual silencing only after an extended time period in culture. The analysis of integration sites of long-term stably expressed proviruses shows a strong bias for some genomic features-especially integration close to the transcription start sites of active transcription units. Furthermore, complex analysis of histone modifications enriched at the site of integration points to the accumulation of proviruses of all three groups in gene regulatory segments, particularly close to the enhancer loci. We conclude that the proximity to active regulatory chromatin segments correlates with stable provirus expression in various retroviral species., Competing Interests: The authors declare no conflict of interest.

The RCAS (replication-competent avian sarcoma leukosis virus long-terminal repeat with splice acceptor)-TVA (tumor virus A) gene delivery system has been successfully used in modeling human cancers. Based on this, we have recently developed a novel RCI-Oncogene (RCAS-Cre-IRES-Oncogene) gene delivery system that can be used to efficiently manipulate gene expression in spontaneous tumors in vivo. We used this system for tumor gene knockout (TuKO) and demonstrated a crucial role of FGFR1 in driving mammary tumor metastasis. This versatile tumor gene modification system can also be adapted into different configurations to address different questions in appropriate mutant mouse hosts. Here we describe a protocol using the TuKO approach to knock out a gene of interest in tumors in appropriate hosts. © 2018 by John Wiley & Sons, Inc., (Copyright © 2018 John Wiley & Sons, Inc.)

Most retroviruses preferentially integrate into certain genomic locations and, as a result, their genome-wide integration patterns are non-random. We investigate the epigenetic landscape of integrated retroviral vectors and correlate it with the long-term stability of proviral transcription. Retroviral vectors derived from the avian sarcoma/leukosis virus expressing the GFP reporter were used to transduce the human myeloid lymphoblastoma cell line K562. Because of efficient silencing of avian retrovirus in mammalian cells, only ∼3% of established clones displayed stable proviral expression. We analyzed the vector integration sites in non-selected cells and in clones selected for the GFP expression. This selection led to overrepresentation of proviruses integrated in active transcription units, with particular accumulation in promoter-proximal areas. In parallel, we investigated the integration of vectors equipped with an anti-silencing CpG island core sequence. Such modification increased the frequency of stably expressing proviruses by one order. The modified vectors are also overrepresented in active transcription units, but stably expressed in distal parts of transcriptional units further away from promoters with marked accumulation in enhancers. These results suggest that integrated retroviruses subject to gradual epigenetic silencing during long-term cultivation. Among most genomic compartments, however, active promoters and enhancers protect the adjacent retroviruses from transcriptional silencing., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Retroviral engineering of hematopoietic stem cell-derived precursor T-cells (preTs) opens the possibility of targeted T-cell transfer across human leukocyte antigen (HLA)-barriers. Alpharetroviral vectors exhibit a more neutral integration pattern thereby reducing the risk of insertional mutagenesis. Cord blood-derived CD34+ cells were transduced and differentiated into preTs in vitro. Two promoters, elongation-factor-1-short-form, and a myeloproliferative sarcoma virus variant in combination with two commonly used envelopes were comparatively assessed choosing enhanced green fluorescent protein or a third-generation chimeric antigen receptor (CAR) against CD123 as gene of interest. Furthermore, the inducible suicide gene iCaspase 9 has been validated. Combining the sarcoma virus-derived promoter with a modified feline endogenous retrovirus envelope glycoprotein yielded in superior transgene expression and transduction rates. Fresh and previously frozen CD34+ cells showed similar transduction and expansion rates. Transgene-positive cells did neither show proliferative impairment nor alteration in their lymphoid differentiation profile. The sarcoma virus-derived promoter only could express sufficient levels of iCaspase 9 to mediate dimerizer-induced apoptosis. Finally, the CD123 CAR was efficiently expressed in CD34+ cells and proved to be functional when expressed on differentiated T-cells. Therefore, the transduction of CD34+ cells with alpharetroviral vectors represents a feasible and potentially safer approach for stem cell-based immunotherapies for cancer.

Unlabelled: Natural killer (NK) cells play an important role in tumor immunotherapy with their unique capability of killing transformed cells without the need for prior sensitization and without major histocompatibility complex (MHC)/peptide restriction. However, tumor cells can escape NK cell cytotoxicity by various tumor immune escape mechanisms. To overcome these escape mechanisms, NK cells can be modified to express chimeric antigen receptors (CARs), enhancing their tumor-specific cytotoxicity. To determine the most efficacious method to modify human NK cells, we compared different retroviral vector systems, retroviral pseudotypes, and transduction protocols. Using optimized transduction conditions, the highest transduction levels (up to 60%) were achieved with alpharetroviral vectors. Alpharetroviral-modified primary human NK cells exhibited no alteration in receptor expression and had similar degranulation activity as untransduced NK cells, thus demonstrating that alpharetroviral modification did not negatively affect NK cell cytotoxicity. Transduction of NK cells with an alpharetroviral vector containing a CD19 CAR expression cassette selectively enhanced NK cell cytotoxicity towards CD19-expressing leukemia cells, achieving nearly complete elimination of leukemia cells after 48 h. Taken together, alpharetroviral vectors are promising tools for NK cell-mediated cancer immunotherapy applications., Key Messages: Efficient modification of human NK cells using alpharetroviral vectors. Anti-CD19-CAR-NK cells exhibited improved cytotoxicity towards CD19(+) leukemia cells. Alpharetroviral vectors are promising tools for immunotherapy applications using NK cells.

Background: Gene expression is an inherently stochastic process, owing to its dynamic molecular nature. Protein amount distributions, which can be acquired by cytometry using a reporter gene, can inform about the mechanisms of the underlying microscopic molecular system., Results: By using different clones of chicken erythroid progenitor cells harboring different integration sites of a CMV-driven mCherry protein, we investigated the dynamical behavior of such distributions. We show that, on short term, clone distributions can be quickly regenerated from small population samples with a high accuracy. On longer term, on the contrary, we show variations manifested by correlated fluctuation in the Mean Fluorescence Intensity. In search for a possible cause of this correlation, we demonstrate that in response to small temperature variations cells are able to adjust their gene expression rate: a modest (2 °C) increase in external temperature induces a significant down regulation of mean expression values, with a reverse effect observed when the temperature is decreased. Using a two-state model of gene expression we further demonstrate that temperature acts by modifying the size of transcription bursts, while the burst frequency of the investigated promoter is less systematically affected., Conclusions: For the first time, we report that transcription burst size is a key parameter for gene expression that metazoan cells from homeotherm animals can modify in response to an external thermal stimulus.

Endogenous retroviruses are regarded as ideal genetic markers for evolutionary analyses. Birds were some of the initial vertebrates found to contain endogenous retroviruses. However, few studies have investigated the presence and distribution of endogenous retroviruses in goose. In this study, we detected the avian sarcoma and leukosis virus gag gene in the genomic DNA of 8 Chinese native breeds using polymerase chain reaction method. The results indicated that a 1.2-kb avian sarcoma and leukosis virus gag sequence was integrated into all 8 goose breeds. The mean genetic pairwise distance was 0.918% among the investigated geese. To the best of our knowledge, this is the first report demonstrating the presence of the endogenous retroviruses in the domestic goose genome. The genetic structure should be further examined in the domestic goose.

Lymphoproliferative disease virus (LPDV) is a poorly understood, oncogenic avian retrovirus of domestic turkeys that has historically been restricted to Europe and Israel. However, a recent study reported LPDV in multiple wild turkey diagnostic cases from throughout the eastern United States of America (USA). To better understand the distribution of LPDV in the eastern USA, we surveyed 1,164 reportedly asymptomatic hunter-harvested wild turkeys from 17 states for the presence of LPDV proviral DNA by PCR. In total, 564/1,164 (47%) turkeys were positive for LPDV. Wild turkeys from each state had a relatively high prevalence of LPDV, although statewide prevalence varied from 26 to 83%. Phylogenetic analysis revealed two major clades of LPDV in the USA, although one was at a low frequency suggesting restricted transmission, as well as significant clustering by state of isolation. To determine the best tissue to target for diagnostic purposes, liver, spleen, and bone marrow were tested from a subset of 15 hunter-harvested wild turkeys and 20 wild turkey diagnostic cases. Overall, bone marrow provided the highest level of detection for both hunter-harvested turkeys and diagnostic cases. The sensitivity of LPDV detection between tissues was not significantly different for diagnostic cases, but was for hunter-harvested birds. These results indicate that LPDV infection is common and widespread in wild turkey populations throughout the eastern USA, even without overt signs of disease.

The group of avian sarcoma and leukosis virus (ASLV) in chickens contains six highly related subgroups, A to E and J. Four genetic loci, tva, tvb, tvc and tvj, encode for corresponding receptors that determine the susceptibility to the ASLV subgroups. The prevalence of ASLV in hosts may have imposed strong selection pressure toward resistance to ASLV infection, and the resistant alleles in all four receptor genes have been identified. In this study, two new alleles of the tva receptor gene, tva(r5) and tva(r6), with similar intronic deletions were identified in Chinese commercial broilers. These natural mutations delete the deduced branch point signal within the first intron, disrupting mRNA splicing of the tva receptor gene and leading to the retention of intron 1 and introduction of premature TGA stop codons in both the longer and shorter tva isoforms. As a result, decreased susceptibility to subgroup A ASLV in vitro and in vivo was observed in the subsequent analysis. In addition, we identified two groups of heterozygous allele pairs which exhibited quantitative differences in host susceptibility to ASLV-A. This study demonstrated that defective splicing of the tva receptor gene can confer genetic resistance to ASLV subgroup A in the host.

The viral cell receptors and infection can be blocked by the expression of the viral receptor-binding protein. Thus, the viral cell receptor is an attractive target for anti-viral strategies, and the identification of viral cell receptor is critical for better understanding and controlling viral disease. As a model system for viral entry and anti-retroviral approaches, avian sarcoma/leukosis virus (ASLV, including the A-J ten subgroups) has been studied intensively and many milestone discoveries have been achieved based on work with ASLV. Here, we used a DF1 cell line expressed viral receptor-binding protein to efficiently identify chicken Annexin A2 (chANXA2) as a novel receptor for retrovirus ALV-J (avian leukosis virus subgroup J). Our data demonstrate that antibodies or siRNA to chANXA2 significantly inhibited ALV-J infection and replication, and over-expression of chANXA2 permitted the entry of ALV-J into its non-permissible cells. Our findings have not only identified chANXA2 as a novel biomarker for anti-ALV-J, but also demonstrated that cell lines with the expression of viral receptor-binding protein could be as efficient tools for isolating functional receptors to identify novel anti-viral targets.

The ability to introduce DNA elements into host cells and analyze the effects has revolutionized modern biology. Here we describe a protocol to generate Moloney murine leukemia virus (MMLV)-based, replication-incompetent pseudotyped retrovirus capable of infecting axolotls and incorporating genetic information into their genome. When pseudotyped with vesicular stomatitis virus (VSV)-G glycoprotein, the retroviruses can infect a broad range of proliferative axolotl cell types. However, if the retrovirus is pseudotyped with an avian sarcoma leukosis virus (ASLV)-A envelope protein, only axolotl cells experimentally manipulated to express the cognate tumor virus A (TVA) receptor can be targeted by infections. These strategies enable robust transgene expression over many cell divisions, cell lineage tracing, and cell subtype targeting for gene expression.

Gene therapy using integrating retroviral vectors has proven its effectiveness in several clinical trials for the treatment of inherited diseases and cancer. However, vector-mediated adverse events related to insertional mutagenesis were also observed, emphasizing the need for safer therapeutic vectors. Paradoxically, alpharetroviruses, originally discovered as cancer-causing agents, have a more random and potentially safer integration pattern compared to gammaretro- and lentiviruses. In this review, we provide a short overview of the history of alpharetroviruses and explain how they can be converted into state-of-the-art gene delivery tools with improved safety features. We discuss development of alpharetroviral vectors in compliance with regulatory requirements for clinical translation, and provide an outlook on possible future gene therapy applications. Taken together, this review is a broad overview of alpharetroviral vectors spanning the bridge from their parental virus discovery to their potential applicability in clinical settings.

Lymphoproliferative disease virus (LPDV) is an exogenous oncogenic retrovirus that induces lymphoid tumors in some galliform species of birds. Historically, outbreaks of LPDV have been reported from Europe and Israel. Although the virus has previously never been detected in North America, herein we describe the widespread distribution, genetic diversity, pathogenesis, and evolution of LPDV in the United States. Characterization of the provirus genome of the index LPDV case from North America demonstrated an 88% nucleotide identity to the Israeli prototype strain. Although phylogenetic analysis indicated that the majority of viruses fell into a single North American lineage, a small subset of viruses from South Carolina were most closely related to the Israeli prototype. These results suggest that LPDV was transferred between continents to initiate outbreaks of disease. However, the direction (New World to Old World or vice versa), mechanism, and time frame of the transcontinental spread currently remain unknown., (© 2013 Published by Elsevier Inc.)

Retroviral gene transfer has proven therapeutic potential in clinical gene therapy trials but may also cause abnormal cell growth via perturbation of gene expression in the locus surrounding the insertion site. By establishing clonal marks, retroviral insertions are also used to describe the regenerative potential of individual cells. Deep sequencing approaches have become the method of choice to study insertion profiles in preclinical models and clinical trials. We used a protocol combining ligation-mediated polymerase chain reaction (LM-PCR) and pyrosequencing for insertion profiling and quantification in cells of various tissues transduced with various retroviral vectors. The presented method allows simultaneous analysis of a multitude of DNA-barcoded samples per pyrosequencing run, thereby allowing cost-effective insertion screening in studies with multiple samples. In addition, we investigated whether the number of pyrosequencing reads can be used to quantify clonal abundance. By comparing pyrosequencing reads against site-specific quantitative PCR and by performing spike-in experiments, we show that considerable variation exists in the quantification of insertion sites even when present in the same clone. Our results suggest that the protocol used here and similar approaches might misinterpret abundance clones defined by insertion sites, unless careful calibration measures are taken. The crucial variables causing this variation need to be defined and methodological improvements are required to establish pyrosequencing reads as a quantification measure in polyclonal situations.

Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders.

The autonomous transcription of integrated retroviruses strongly depends on genetic and epigenetic effects of the chromatin at the site of integration. These effects are mostly suppressive and proviral activity can be finally silenced by mechanisms, such as DNA methylation and histone modifications. To address the role of the integration site at the whole-genome-scale, we performed clonal analysis of provirus silencing with an avian leucosis/sarcoma virus-based reporter vector and correlated the transcriptional silencing with the epigenomic landscape of respective integrations. We demonstrate efficient provirus silencing in human HCT116 cell line, which is strongly but not absolutely dependent on the de novo DNA methyltransferase activity, particularly of Dnmt3b. Proviruses integrated close to the transcription start sites of active genes into the regions enriched in H3K4 trimethylation display long-term stability of expression and are resistant to the transcriptional silencing after over-expression of Dnmt3a or Dnmt3b. In contrast, proviruses in the intergenic regions tend to spontaneous transcriptional silencing even in Dnmt3a(-/-) Dnmt3b(-/-) cells. The silencing of proviruses within genes is accompanied with DNA methylation of long terminal repeats, whereas silencing in intergenic regions is DNA methylation-independent. These findings indicate that the epigenomic features of integration sites are crucial for their permissivity to the proviral expression.

Spatiotemporal retroviral gene transfer into specific somatic mammalian cells using the avian RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor)/tumor virus A (TVA) system is a versatile tool for performing lineage tracing and gene function analysis in vivo. RCAS retroviruses carrying the subgroup A envelope transduce only genetically engineered mammalian cells that express the cognate avian retroviral receptor TVA. The RCAS/TVA gene delivery system has been successfully used in various different mouse TVA-expression models. This protocol contains a detailed description of the production of high-titer RCAS retroviruses in chicken fibroblasts and the transduction of proliferating TVA-positive somatic mammalian cells in vivo. By taking advantage of the combination of the RCAS/TVA with the 'universal' Cre/loxP system, the protocol can be used in nearly every proliferating cell type in vivo. The protocol takes 4 weeks from transfection of chicken fibroblasts, which act as the host cells for viral production, to the transduction of TVA-transgenic mice.

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.

The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.

Aggresome formation, a cellular response to misfolded protein aggregates, is linked to cancer and neurodegenerative disorders. Previously we showed that Gag-v-ErbA (v-ErbA), a retroviral variant of the thyroid hormone receptor (TRα1), accumulates in and sequesters TRα1 into cytoplasmic foci. Here, we show that foci represent v-ErbA targeting to aggresomes. v-ErbA colocalizes with aggresomal markers, proteasomes, hsp70, HDAC6, and mitochondria. Foci have hallmark characteristics of aggresomes: formation is microtubule-dependent, accelerated by proteasome inhibitors, and they disrupt intermediate filaments. Proteasome-mediated degradation is critical for clearance of v-ErbA and T(3)-dependent TRα1 clearance. Our studies highlight v-ErbA's complex mode of action: the oncoprotein is highly mobile and trafficks between the nucleus, cytoplasm, and aggresome, carrying out distinct activities within each compartment. Dynamic trafficking to aggresomes contributes to the dominant negative activity of v-ErbA and may be enhanced by the viral Gag sequence. These studies provide insight into novel modes of oncogenesis across multiple cellular compartments., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

Background: The use of Specific Pathogen Free (SPF) eggs in combination with RCAS retrovirus, a member of the Avian Sarcoma-Leukosis Virus (ASLV) family, is of standard practice to study gene function and development. SPF eggs are certified free of infection by specific pathogen viruses of either exogenous or endogenous origin, including those belonging to the ASLV family. Based on this, SPF embryos are considered to be free of ASLV viral protein expression, and consequently in developmental research studies RCAS infected cells are routinely identified by immunohistochemistry against the ASLV viral proteins p19 and p27. Contrary to this generally accepted notion, observations in our laboratory suggested that certified SPF chicken embryos may endogenously express ASLV viral proteins p19 and p27. Since these observations may have significant implications for the developmental research field we further investigated this possibility., Results: We demonstrate that certified SPF chicken embryos have transcriptionally active endogenous ASLV loci (ev loci) capable of expressing ASLV viral proteins, such as p19 and p27, even when those loci are not capable of producing viral particles. We also show that the extent of viral protein expression in embryonic tissues varies not only among flocks but also between embryos of the same flock. In addition, our genetic screening revealed significant heterogeneity in ev loci composition even among embryos of the same flock., Conclusions: These observations have critical implications for the developmental biology research field, since they strongly suggest that the current standard methodology used in experimental studies using the chick embryo and RCAS vectors may lead to inaccurate interpretation of results. Retrospectively, our observations suggest that studies in which infected cells have been identified simply by pan-ASLV viral protein expression may need to be considered with caution. For future studies, they point to a need for careful selection and screening of the chick SPF lines to be used in combination with RCAS constructs, as well as the methodology utilized for qualitative analysis of experimental results. A series of practical guidelines to ensure research quality animals and accuracy of the interpretation of results is recommended and discussed.

Accidental insertional activation of proto-oncogenes and potential vector mobilization pose serious challenges for human gene therapy using retroviral vectors. Comparative analyses of integration sites of different retroviral vectors have elucidated distinct target site preferences, highlighting vectors based on the alpharetrovirus Rous sarcoma virus (RSV) as those with the most neutral integration spectrum. To date, alpharetroviral vector systems are based mainly on single constructs containing viral coding sequences and intact long terminal repeats (LTR). Even though they are considered to be replication incompetent in mammalian cells, the transfer of intact viral genomes is unacceptable for clinical applications, due to the risk of vector mobilization and the potentially immunogenic expression of viral proteins, which we minimized by setting up a split-packaging system expressing the necessary viral proteins in trans. Moreover, intact LTRs containing transcriptional elements are capable of activating cellular genes. By removing most of these transcriptional elements, we were able to generate a self-inactivating (SIN) alpharetroviral vector, whose LTR transcriptional activity is strongly reduced and whose transgene expression can be driven by an internal promoter of choice. Codon optimization of the alpharetroviral Gag/Pol expression construct and further optimization steps allowed the production of high-titer self-inactivating vector particles in human cells. We demonstrate proof of principle for the versatility of alpharetroviral SIN vectors for the genetic modification of murine and human hematopoietic cells at a low multiplicity of infection.

Integrase (IN) is the enzyme responsible for provirus integration of retroviruses into the host cell genome. We used an Avian Sarcoma and Leukemia Viruses (ASLV) integration assay to investigate the way in which IN integrates substrates mutated or devoid of one or both IN recognition sequences. We found that replacing U5 by non-viral sequences (U5del) or U3 by a mutated sequence (pseudoU3) resulted in two and three fold reduction of two-ended integration (integration of the two ends from a donor DNA) respectively, but had a slight effect on concerted integration (integration of both ends at the same site of target DNA). Further, IN was still able to integrate the viral ends of the double mutant (pseudoU3/U5del) in a two-ended and concerted integration reaction. However, efficiency and accuracy (i.e. fidelity of size duplication and of end cleavage) of integration were reduced.

We previously identified a new bovine immunodeficiency virus (BIV) trans-activator factor of transcription (Tat236) that was derived from a variant of BIV. Here, we report a new BIV long terminal repeat (LTR) sequence (LTRn) that was obtained by PCR from the DNA of cells infected with the BIV variant mentioned above. Sequence analysis indicated that the LTRn U3 region harbors three nucleic acid mutations at residue positions -194, -135 and -114 when compared to the original (wild-type) LTR sequence. Reporter gene assays indicated that LTRn promotes basal and Tat-mediated transactivation activity to levels significantly higher than those obtained with the wild-type LTR. Restoration experiments to the wild-type genotype indicated that both the -135 and -114 nucleic acid substitutions were responsible for the enhanced promoter activity of BIV LTRn.

Pancreatic cancer is one of the most common causes of cancer death in Western civilization and the 5-year survival rate is below 5%. To improve the prognosis of pancreatic cancer, there is the need to develop effective nonsurgical treatment options for the disease. In particular, in vivo models to validate potential targets at the genetic level are required. In this study we demonstrate that RCAS-mediated retroviral gene transfer into orthotopic pancreatic cancer tumor grafts is feasible. Furthermore, we show effective RCAS-dependent RNA interference in vivo. We validate in vivo bioluminescence imaging as a reliable tool to monitor tumor progression of orthotopic pancreatic cancer transplants longitudinally. In addition, we show that restoring expression of the tumor suppressor p53 by RCAS-mediated gene transfer and knockdown of the epidermal growth factor receptor by RCAS-dependent RNA interference impairs orthotopic pancreatic tumor growth in vivo. In conclusion, these data demonstrate that combining in vivo bioluminescence imaging with RCAS-mediated gene or short hairpin RNA transfer is a new model to investigate gene function in pancreatic cancer grafts and allows validation of potential new drug targets in vivo.

Site- and time-specific somatic gene transfer by using the avian sarcoma-leukosis retrovirus RCAS (replication-competent avian sarcoma-leukosis virus long terminal repeat with splice acceptor) has been shown to be a powerful tool to analyze gene function in vivo. RCAS retroviruses that express the avian subgroup A envelope transduce only mammalian cells genetically engineered to express the avian retroviral receptor, tumor virus A (TVA). Here, we generated a knockin mouse line termed LSL-R26(Tva-lacZ) with concomitant conditional expression of TVA and lacZ by targeting the Rosa26 locus. A loxP-flanked transcriptional stop cassette was used for conditional activation of TVA and LacZ expression in a Cre-recombinase-dependent manner. To demonstrate the ability of this system for conditional somatic gene transfer in vivo, we directed TVA expression to the pancreas. Introduction of an RCAS vector with Bryan-RSV polymerase and subgroup A envelope [RCASBP(A)] carrying oncogenic Kras(G12D) induced focal ductal pancreatic lesions that recapitulate human pancreatic intraepithelial neoplasias that progress to pancreatic ductal adenocarcinomas. TVA-mediated infection of genetically engineered mice with endogenous expression of Kras(G12D) in pancreatic progenitor cells by using RCASBP(A) virus carrying a short hairpin RNA directed against murine TP53, resulted in dramatically enhanced progression to invasive adenocarcinomas. These results show that conditional expression of TVA enables spatiotemporal gene expression and knockdown in a small subset of somatic cells in vivo. Therefore, it closely models carcinogenesis in humans where tumors evolve from somatic gene mutations in developmentally normal cells. Combined with the growing number of Cre expression models, RCAS-TVA-based gene expression and knockdown systems open up promising perspectives for analysis of gene function in a time-controlled and tissue-specific fashion in vitro and in vivo.

During retroviral integration, the viral integrase recognizes the attachment (att) sequence (formed by juxtaposition of two LTRs ends) as the substrate of integration. We have developed a self-deleting Avian Leukosis and Sarcoma Viruses (ALSVs)-based retroviral vector carrying an additional copy of the att sequence, between neo and puro genes. We observed that: (i) the resulting NP3Catt vector was produced at neo and puro titers respectively smaller and higher than that of the parental vector devoid of the att sequence; (ii) 61% of NP3Catt proviruses were flanked by LTRs; most of them were deleted of internal sequences, probably during the reverse transcription step; (iii) 31% of clones were deleted of the whole 5' part of their genome and were flanked, in 5', by the additional att sequence and, in 3', by an LTR. Integration of these last proviruses was often imprecise with respect to the viral ends. At total, 77% of proviruses had lost the packaging signal and were not mobilizable by a replication-competent virus and 92% had lost the selectable gene in a single round of replication. Although still to improve, the att vector could be considered as an interesting new safe retroviral vector for gene transfer experiments.

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.

The genes that regulate the formation of blood vessels in adult tissues represent promising therapeutic targets because angiogenesis plays a role in many diseases, including cancer. We wished to develop a mouse model allowing characterization of gene function in adult angiogenic vasculature while minimizing effects on embryonic vasculature or adult quiescent vasculature. Here we describe a transgenic mouse model that allows expression of proteins in the endothelial cells of newly forming blood vessels in the adult using a selective retroviral gene delivery system. We generated transgenic mouse lines that express the TVA receptor for the RCAS avian-specific retrovirus from Flk1 gene regulatory elements that drive expression in proliferating endothelial cells. Several of these Flk1-TVA lines expressed TVA mRNA in the embryonic vasculature and TVA protein in new blood vessels growing into subcutaneous extracellular matrix implants in adult mice. In a Flk1-TVA line that was crossed with the MMTV-PyMT transgenic mammary tumor model, tumor endothelial cells also expressed the TVA protein. Furthermore, endothelial cells in extracellular matrix implants and the tumors of Flk1-TVA mice were susceptible to RCAS infection, as determined by expression of green fluorescent protein encoded by the virus. The Flk1-TVA mouse model in conjunction with the RCAS gene delivery system will be useful to study molecular mechanisms underlying adult forms of angiogenesis.

Upon integration into the host chromosome, retroviral gene expression requires transcription by the host RNA polymerase II, and viral messages are subject RNA processing events including 5'-end capping, pre-mRNA splicing, and polyadenylation. At a minimum, RNA splicing is required to generate the env mRNA, but viral replication requires substantial amounts of unspliced RNA to serve as mRNA and for incorporation into progeny virions as genomic RNA. Therefore, splicing has to be controlled to preserve the large unspliced RNA pool. Considering the current view that splicing and polyadenylation are coupled, the question arises as to how genome-length viral RNA is efficiently polyadenylated in the absence of splicing. Polyadenylation of many retroviral mRNAs is inefficient; in avian retroviruses, approximately 15 percent of viral transcripts extend into and are polyadenylated at downstream host genes, which often has profound biological consequences. Retroviruses have served as important models to study RNA processing and this review summarizes a body of work using avian retroviruses that has led to the discovery of novel RNA splicing and polyadenylation control mechanisms.

Since the status of endogenous avian leucosis/sarcoma virus (ALSV) infections in Korean broiler chickens is unclear, this study examined embryonated eggs obtained from broiler farms and Korean native chicken breeds in Korea using PCR with the primer sets specific for endogenous ALSVs. The PCR assays detected the genomes of EAV, ev, ev/J and ART-CH belonging to the endogenous ALSV from all embryos tested. Phylogenetically, the Korean EAV genomes were more closely related to the prototype EAV-0 than to the other prototype, E51. The Korean ART-CH elements clustered together but were distinct from the prototype ART-CH clones, 5 and 14. Although there was comparatively little divergence in the nucleotide and amino acid sequences of the Korean ev and ev/J genomes compared with the other known ev and ev/J genomes, the Korean genomes had phylogenetically distinct branches. From these results, endogenous genomes are quite prevalent in Korean broiler chickens. In addition, the endogenous genomes circulating in Korean broiler chickens are genetically different from the other known endogenous genomes. These results are expected to provide useful information for the control and establishment of a surveillance system for endogenous ALSVs in Korea.

Objectives: We have previously described an avian leukemia and sarcoma virus-based vector containing an additional att sequence in an internal position that is capable of self-deleting most of its 5' viral sequences during one cycle of replication in avian cells [Virus Res 2008;135:72-82; Arch Virol 2008;153:2233-2243]. Herein, our aim was to test the infectivity and self-deleting properties of this avian retroviral vector in human cells., Methods: Human Hela cells transiently expressing the cellular receptor for avian leukemia and sarcoma viruses (tva) were infected with the avian vector. Molecular analyses of thirteen clones were performed., Results: Data showed that more than 77% of proviruses had lost the 5' part of their genome including the selectable gene. At least 61% of these proviruses were flanked on the left by the additional att sequence and on the right by the LTR. None of the thirteen proviruses was able to express a full-length genomic RNA., Conclusion: This study demonstrates that the self-deleting properties of the avian vector in avian cells may be also applicable to human cells., (Copyright 2009 S. Karger AG, Basel.)

We have previously developed a self-deleting avian leukosis and sarcoma virus (ALSV)- based retroviral vector carrying an additional attachment (att) sequence. Resulting proviruses underwent deletion of viral sequences and were flanked either by two LTRs (LTRs proviruses) or by the additional att sequence and the 3' LTR (att proviruses). Herein, we have tried to increase (1) the self-deleting properties of this vector, either by raising the selection pressure applied on target cells or by optimizing the size of the internal att sequence, (2) the titer of the vector by deleting or inverting some viral sequences. Moreover, a new type of provirus flanked by att sequences at each end was isolated. Finally, under specific conditions, 100% of proviruses had internal sequences deleted, and as many as 92-100% of proviruses were no longer mobilizable by a replication-competent virus. The inactivation procedure achieved here might improve the biosafety of retroviral vectors.

The avian sarcoma and leukosis virus (ASLV) envelope glycoprotein (Env) is activated to trigger fusion by a two-step mechanism involving receptor-priming and low pH fusion activation. In order to identify regions of ASLV Env that can regulate this process, a genetic selection method was used to identify subgroup B (ASLV-B) virus-infected cells resistant to low pH-triggered fusion when incubated with cells expressing the cognate TVB receptor. The subgroup B viral Env (envB) genes were then isolated from these cells and characterized by DNA sequencing. This led to identification of two frequent EnvB alterations which allowed TVB receptor-binding but altered the pH-threshold of membrane fusion activation: a 13 amino acid deletion in the host range 1 (hr1) region of the surface (SU) EnvB subunit, and the A32V amino acid change within the fusion peptide of the transmembrane (TM) EnvB subunit. These data indicate that these two regions of EnvB can influence the pH threshold of fusion activation.

Avian fibroblasts transformed simultaneously by the v-myc and v-mil(raf) oncogenes of acute leukemia and carcinoma virus MH2 contain elevated levels of c-Fos and c-Jun, major components of the transcription factor complex AP-1. To define specific transcriptional targets in these cells, subtractive hybridization techniques were employed leading to the identification of strongly upregulated genes including OPN (osteopontin), 126MRP, and rac2. OPN is a cytokine and cell attachment protein which has been implicated in human tumor progression and metastasis, the calcium binding 126MRP protein is related to the human S100 protein family involved in invasive cell growth, and the Rac2 protein belongs to the Rho family of small GTPases regulating actin reorganization and cell migration. Promoter analysis indicated that OPN activation is mediated by a non-consensus AP-1 binding site located close to the transcription start site. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transcriptional reporter gene analyses showed that c-Fos and c-Jun bind specifically to this site and that c-Fos efficiently transactivates the OPN promoter. High-level expression of OPN, 126MRP, or Rac2 proteins from a retroviral vector led to partial cell transformation, documented by morphological changes and anchorage-independent growth. The specific activation in v-myc/v-mil(raf)-transformed cells of target genes with intrinsic oncogenic potential may provide an explanation for the longstanding observation that concomitant expression of these oncogenes leads to strongly enhanced oncogenicity in vivo and in vitro compared to cell transformation by v-myc or v-mil(raf) alone.

The ability of many retroviruses to cause disease can be correlated to their cytopathic effect (CPE) in tissue culture characterized by an acute period of cell death and viral DNA accumulation. Here, we show that mutants of a subgroup B avian retrovirus (Alpharetrovirus) cause a very dramatic CPE in certain susceptible avian cells that is coincident with elevated levels of apoptosis, as measured by nuclear morphology, and persistent viral DNA accumulation. These mutants also have a broadly extended host range that includes rodent, cat, dog, monkey, and human cells (31). Previously, we have shown that the mutants exhibit diminished resistance to superinfection. The results presented here have important implications for the process of evolution of retroviruses to use distinct cellular receptors.

We have analyzed the placement of sites of integration of avian sarcoma-leukosis virus (ASLV) and human immunodeficiency virus (HIV) DNA in the draft chicken genome sequence, with the goals of assessing species-specific effects on integration and allowing comparison to the distribution of chicken endogenous retroviruses (ERVs). We infected chicken embryo fibroblasts (CEF) with ASLV or HIV and sequenced 863 junctions between host and viral DNA. The relationship with cellular gene activity was analyzed by transcriptional profiling of uninfected or ASLV-infected CEF cells. ASLV weakly favored integration in active transcription units (TUs), and HIV strongly favored active TUs, trends seen previously for integration in human cells. The ERVs, in contrast, accumulated mostly outside TUs, including ERVs related to ASLV. The minority of ERVs present within TUs were mainly in the antisense orientation; consequently, the viral splicing and polyadenylation signals would not disrupt cellular mRNA synthesis. In contrast, de novo ASLV integration sites within TUs showed no orientation bias. Comparing the distribution of de novo ASLV integration sites to ERVs indicated that purifying selection against gene disruption, and not initial integration targeting, probably determined the ERV distribution. Further analysis indicated that ERVs in humans, mice, and rats showed similar distributions, suggesting purifying selection dictated their distributions as well.

To investigate retroviral integration targeting on a nucleotide scale, we examined the base frequencies directly surrounding cloned in vivo HIV-1, murine leukemia virus, and avian sarcoma/leukosis virus integrations. Base preferences of up to 2-fold the expected frequencies were found for three viruses, representing P values down to <10(-100) and defining what appear to be preferred integration sequences. Offset symmetry reflecting the topology of the integration reaction was found for HIV-1 and avian sarcoma/leukosis virus but not murine leukemia virus, suggesting fundamental differences in the way different retroviral integration complexes interact with host-cell DNA.

Retroviral DNA integration occurs throughout the genome; however, local "hot spots" exist where a strong preference for certain sites over others are seen, and more global preferences associated with genes have been reported. Previous data from our laboratory suggested that there are fewer integration events into a DNA template when it is undergoing active transcription than when it is not. Because these data were generated by using a stably transfected foreign gene that was only weakly inducible, we have extended this observation by comparing integration events into a highly inducible endogenous gene under both induced and uninduced transcriptional states. To examine the influence of transcription on site selection directly, we analyzed the frequency and distribution of integration of avian retrovirus DNA into the metallothionein gene, before and after its induction to a highly sustained level of expression by addition of ZnSO4. We found a 6-fold reduction in integration events after 100-fold induction of transcription. This result implies that, despite an apparent preference for integration of retroviral DNA into transcribed regions of host DNA, high-level transcription can be inhibitory to the integration process. Several possible models for our observation are as follows. First, when a DNA template is undergoing active transcription, integration might be blocked by the RNA polymerase II complex because of steric hindrance. Alternatively, the integrase complex may require DNA to be in a double-stranded conformation, which would not be the case during active transcription. Last, transcription might lead to remodeling of chromatin into a structure that is less favorable for integration.

The molecular events underlying the immediate steps of retroviral uncoating, occurring after membrane fusion and leading to the formation of an active reverse transcription complex, are not known. To better understand these processes, we have developed a cell-free system that recapitulates these early steps of retroviral replication by using avian sarcoma and leukosis virus as a model retrovirus. The substrates used in this system are viral particles that are trapped before completing membrane fusion. These virions are induced to fuse out of endosomes and the viral cores are released into solution where they are amenable to biochemical manipulation. This system revealed that membrane fusion is not sufficient to stimulate the formation of a reverse transcription complex. Instead, ATP hydrolysis and cellular factors >5 kDa in size are required. Furthermore, later steps of avian sarcoma and leukosis virus reverse transcription were stimulated by nuclear factors. The cell-free system should now allow for the definition of retroviral uncoating mechanisms and facilitate the identification and characterization of the cellular factors involved.

So-called fowl glioma is a retroviral infectious disease caused by avian leukosis virus subgroup A (ALV-A). We determined the complete nucleotide sequence of the virus genome. The full-length sequence was consistent with a genetic organization typical of a replication-competent type C retrovirus lacking viral oncogenes. The coding sequences were well conserved with those of replication-competent viruses, but the 3' noncoding regions including LTR were most related to those of replication-defective sarcoma viruses. The U3 region of the LTR had a few deletions and several point mutations compared to that of other ALVs. The promoter activities of the LTRs of glioma-inducing ALV and ALV-A standard strain, RAV-1, were equivalent in chick embryo fibroblasts (CEF), while that of glioma-inducing ALV was significantly lower than that of RAV-1 in human astrocytic cells. These subtle differences of the promoter activity of the LTR may be related to the induction of glial neoplasm.

Transgenes stably integrated into cells or animals in many cases are silenced rapidly, probably under the influence of surrounding endogenous condensed chromatin. This gene silencing correlates with repressed chromatin structure marked by histone hypoacetylation, loss of methylation at H3 lysine 4, increase of histone H3 lysine 9 methylation as well as CpG DNA methylation at the promoter. However, the order and the timing of these modifications and their impact on transcription inactivation are less well understood. To determine the temporal order of these events, we examined a model system consisting of a transgenic cassette stably integrated in chicken erythroid cells. We found that histone H3 and H4 hypoacetylation and loss of methylation at H3 lysine 4 all occurred during the same window of time as transgene inactivation in both multicopy and low-copy-number lines. These results indicate that these histone modifications were the primary events in gene silencing. We show that the kinetics of silencing exclude histone H3 K9 and promoter DNA methylation as the primary causative events in our transgene system.

Complete nucleotide sequences of chicken endogenous retroviruses belonging to E33/E51 and EAV-0 groups have been analysed on the basis of the recently available draft genome sequence of red jungle fowl (Gallus gallus), the progenitor of domestic chicken (G.g. domesticus). It was shown that all these proviruses have deletions in the SU-coding domain of the env gene, involved in receptor recognition, whereas gag and pol genes appear to be intact. Phylogenetic analysis demonstrated that E33/E51 and EAV-0 groups are related to the ALV genus. An analysis of expression using chicken EST databases showed that these proviruses are transcriptionally active.