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Background: SL3-2 is a unique polytropic murine gammaretroviral isolate that is only able to infect murine cells. We have previously shown that two mutations R212G and T213I located on the surface of the receptor binding domain in a region designated the VR3 loop can alter the species tropism of this envelope protein. This location suggests that the VR3 loop composition has an influence on receptor interaction and thereby affects binding as well as superinfection resistance. In order to investigate this further, we have studied the binding and interference patterns of the SL3-2 envelope and its mutants. Results: We find unexpectedly that wild type SL3-2 envelope binds equally well to both permissive and nonpermissive cells, indicating a post binding defect when interacting with the human Xpr1. Using replication competent viruses containing envelopes from SL3-2 or its mutants we find that the same amino acid mutations can dramatically alter the interference profile of this polytropic ENV, suggesting that the same amino acid changes that cause the post binding defect also influence interaction with the receptor. Conclusions: The envelope protein of SL3-2 MLV shows an entry defect on non-murine cells. This is coupled to a dramatically reduced ability to interfere with entry of other polytropic viruses. Two point mutations in the VR3 loop of the receptor binding domain of this envelope result both in a much increased interference ability and in removing the post-binding defect on non-murine cells, suggesting that both of these phenotypes are a consequence of insufficient interaction between the envelope and the receptor. [ABSTRACT FROM AUTHOR]

Background: Insertional mutagenesis screens in the mouse are an acknowledged approach to identify genes involved in the pathogenesis of cancer. The potential of these screens to identify genes causally involved in tumorigenesis is not only limited to the murine host, but many of these genes have also been proven to be involved in the oncogenic process in man. Results: Through an insertional mutagenesis screen applying murine leukemia viruses in mouse, we found that Cd74 was targeted by proviral insertion in tumors of B-cell origin. This locus encodes a protein playing crucial roles in antigen presentation and B-cell homeostasis, and its deregulation is often associated with cancer in man. The distribution of insertions within the Cd74 locus prompted the identification of an alternative transcript initiated in intron 1 of Cd74 encoding an N-terminally truncated Cd74 isoform in tissues from un-infected mice, and transcriptional activation assays revealed a positive effect on the novel intronic promoter by a formerly described intronic enhancer in the Cd74 locus. Furthermore, we show that the new Cd74 isoform is IFNγ inducible and that its expression is differentially regulated from the canonical Cd74 isoform at the transcriptional level. Conclusions: We here identify Cd74 as a common insertion site in murine B-lymphomas and describe a novel IFNγ-inducible murine Cd74 isoform differentially regulated from the canonical isoform and expressed under the control of an intronic promoter. The distribution and orientation of proviral insertion sites within the Cd74 locus underscores the causal involvement of the isoforms in the murine B-lymphomagenic process. [ABSTRACT FROM AUTHOR]

Background: Human endogenous retroviruses (HERVs) comprise a large class of repetitive retroelements. Most HERVs are ancient and invaded our genome at least 25 million years ago, except for the evolutionary young HERV-K group. The far majority of the encoded genes are degenerate due to mutational decay and only a few non-HERV-K loci are known to retain intact reading frames. Additional intact HERV genes may exist, since retroviral reading frames have not been systematically annotated on a genome-wide scale. Results: By clustering of hits from multiple BLAST searches using known retroviral sequences we have mapped 1.1% of the human genome as retrovirus related. The coding potential of all identified HERV regions were analyzed by annotating viral open reading frames (vORFs) and we report 7836 loci as verified by protein homology criteria. Among 59 intact or almost-intact viral polyproteins scattered around the human genome we have found 29 envelope genes including two novel gammaretroviral types. One encodes a protein similar to a recently discovered zebrafish retrovirus (ZFERV) while another shows partial, C-terminal, homology to Syncytin (HERV-W/FRD). Conclusions: This compilation of HERV sequences and their coding potential provide a useful tool for pursuing functional analysis such as RNA expression profiling and effects of viral proteins, which may, in turn, reveal a role for HERVs in human health and disease. All data are publicly available through a database at http://www.retrosearch.dk. [ABSTRACT FROM AUTHOR]

The relA gene product, ATP:GTP 3′-pyrophosphotransferase (stringent factor) has been isolated homogeneous form from an Escherichia coli strain polyploid for this gene at a yield of 1 mg/100 g cells and at a specific activity in a ribosome-activated assay at 37 °C of 120 μmol guanosine pentaphosphate formed min-1 mg protein-1. The specific activity in a methanol-activated assay at 25 °C was found to be 4 μmol guanosine pentaphosphate formed min-1 mg protein -1. These values are about 100 times higher than reported by others. Our further studies of this enzyme led to the following results. Antibodies raised against this enzyme inhibit the ribosome-activated synthesis of guanosine tetraphosphate and pentaphosphate but have no effect on the much slower synthesis, detected in the absence of ribosomes. The amount of stringent factor in the rel A+ strain CP78 is estimated to about 1 copy per 200 ribosomes. The amount of antibody-binding material in CP79 (relA) is at least 5 times lower. [ABSTRACT FROM AUTHOR]

Several studies have shown that human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively HERVs) impose direct regulation on human genes through enhancer and promoter motifs present in their long terminal repeats (LTRs). Although chimeric transcription in which novel gene isoforms containing retroviral and human sequence are transcribed from viral promoters are commonly associated with disease, regulation by HERVs is beneficial in other settings; for example, in human testis chimeric isoforms of TP63 induced by an ERV9 LTR protect the male germ line upon DNA damage by inducing apoptosis, whereas in the human globin locus the γ- and β-globin switch during normal hematopoiesis is mediated by complex interactions of an ERV9 LTR and surrounding human sequence. The advent of deep sequencing or next-generation sequencing (NGS) has revolutionized the way researchers solve important scientific questions and develop novel hypotheses in relation to human genome regulation. We recently applied next-generation paired-end RNA-sequencing (RNA-seq) together with chromatin immunoprecipitation with sequencing (ChIP-seq) to examine ERV9 chimeric transcription in human reference cell lines from Encyclopedia of DNA Elements (ENCODE). This led to the discovery of advanced regulation mechanisms by ERV9s and other HERVs across numerous human loci including transcription of large gene-unannotated genomic regions, as well as cooperative regulation by multiple HERVs and non-LTR repeats such as Alu elements. In this article, well-established examples of human gene regulation by HERVs are reviewed followed by a description of paired-end RNA-seq, and its application in identifying chimeric transcription genome-widely. Based on integrative analyses of RNA-seq and ChIP-seq, data we then present novel examples of regulation by ERV9s of tumor suppressor genes CADM2 and SEMA3A, as well as transcription of an unannotated region. Taken together, this article highlights the high suitability of contemporary sequencing methods in future analyses of human biology in relation to evolutionary acquired retroviruses in the human genome., (© 2016 APMIS. Published by John Wiley & Sons Ltd.)

Unlabelled: We have previously reported the construction of a murine leukemia virus-based replication-competent gammaretrovirus (SL3-AP) capable of utilizing the human G protein-coupled receptor APJ (hAPJ) as its entry receptor and its natural receptor, the murine Xpr1 receptor, with equal affinities. The apelin receptor has previously been shown to function as a coreceptor for HIV-1, and thus, adaptation of the viral vector to this receptor is of significant interest. Here, we report the molecular evolution of the SL3-AP envelope protein when the virus is cultured in cells harboring either the Xpr1 or the hAPJ receptor. Interestingly, the dual receptor affinity is maintained even after 10 passages in these cells. At the same time, the chimeric viral envelope protein evolves in a distinct pattern in the apelin cassette when passaged on D17 cells expressing hAPJ in three separate molecular evolution studies. This pattern reflects selection for reduced ligand-receptor interaction and is compatible with a model in which SL3-AP has evolved not to activate hAPJ receptor internalization., Importance: Few successful examples of engineered retargeting of a retroviral vector exist. The engineered SL3-AP envelope is capable of utilizing either the murine Xpr1 or the human APJ receptor for entry. In addition, SL3-AP is the first example of an engineered retrovirus retaining its dual tropism after several rounds of passaging on cells expressing only one of its receptors. We demonstrate that the virus evolves toward reduced ligand-receptor affinity, which sheds new light on virus adaptation. We provide indirect evidence that such reduced affinity leads to reduced receptor internalization and propose a novel model in which too rapid receptor internalization may decrease virus entry., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

MicroRNAs (miRNAs) are ~22 base pair-long non-coding RNAs which regulate gene expression in the cytoplasm of eukaryotic cells by binding to specific target regions in mRNAs to mediate transcriptional blocking or mRNA cleavage. Through their fundamental roles in cellular pathways, gene regulation mediated by miRNAs has been shown to be involved in almost all biological phenomena, including development, metabolism, cell cycle, tumor formation, and host-pathogen interactions. To address the latter in a primitive vertebrate host, we here used an array platform to analyze the miRNA response in rainbow trout (Oncorhynchus mykiss) following inoculation with the virulent fish rhabdovirus Viral hemorrhagic septicaemia virus. Two clustered miRNAs, miR-462 and miR-731 (herein referred to as miR-462 cluster), described only in teleost fishes, were found to be strongly upregulated, indicating their involvement in fish-virus interactions. We searched for homologues of the two teleost miRNAs in other vertebrate species and investigated whether findings related to ours have been reported for these homologues. Gene synteny analysis along with gene sequence conservation suggested that the teleost fish miR-462 and miR-731 had evolved from the ancestral miR-191 and miR-425 (herein called miR-191 cluster), respectively. Whereas the miR-462 cluster locus is found between two protein-coding genes (intergenic) in teleost fish genomes, the miR-191 cluster locus is found within an intron of a protein-coding gene (intragenic) in the human genome. Interferon (IFN)-inducible and immune-related promoter elements found upstream of the teleost miR-462 cluster locus suggested roles in immune responses to viral pathogens in fish, while in humans, the miR-191 cluster functionally associated with cell cycle regulation. Stimulation of fish cell cultures with the IFN inducer poly I:C accordingly upregulated the expression of miR-462 and miR-731, while no stimulatory effect on miR-191 and miR-425 expression was observed in human cell lines. Despite high sequence conservation, evolution has thus resulted in different regulation and presumably also different functional roles of these orthologous miRNA clusters in different vertebrate lineages.

Background: Evidence suggests that some human endogenous retroviruses and endogenous retrovirus-like repeats (here collectively ERVs) regulate the expression of neighboring genes in normal and disease states; e.g. the human globin locus is regulated by an ERV9 that coordinates long-range gene switching during hematopoiesis and activates also intergenic transcripts. While complex transcription regulation is associated with integration of certain exogenous retroviruses, comparable regulation sustained by ERVs is less understood., Findings: We analyzed ERV transcription using ERV9 consensus sequences and publically available RNA-sequencing, chromatin immunoprecipitation with sequencing (ChIP-seq) and cap analysis gene expression (CAGE) data from ENCODE. We discovered previously undescribed and advanced transcription regulation mechanisms in several human reference cell lines. We show that regulation by ERVs involves long-ranging activations including complex RNA splicing patterns, and transcription of large unannotated regions ranging in size from several hundred kb to around 1 Mb. Moreover, regulation was found to be cooperatively sustained in some loci by multiple ERVs and also non-LTR repeats., Conclusion: Our analyses show that endogenous retroviruses sustain advanced transcription regulation in human cell lines, which shows similarities to complex insertional mutagenesis effects exerted by exogenous retroviruses. By exposing previously undescribed regulation effects, this study should prove useful for understanding fundamental transcription mechanisms resulting from evolutionary acquisition of retroviral sequence in the human genome.

Background: Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined., Results: We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues., Conclusions: We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology.

In a search for genes and pathways implicated in T-cell lymphoblastic lymphoma (T-LBL) development, we used a murine lymphoma model, where mice of the NMRI-inbred strain were inoculated with murine leukemia virus mutants. The resulting tumors were analyzed by integration analysis and global gene expression profiling to determine the effect of the retroviral integrations on the nearby genes, and the deregulated pathways in the tumors. Gene expression profiling identified increased expression of genes involved in the minichromosome maintenance and origin of recognition pathway as well as downregulation in negative regulators of G1/S transition, indicating increased S-phase initiation in murine T-LBLs., (Copyright © 2013 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Many cytokine receptors are cell surface proteins that promiscuously combine to form active signalling homo- or heterodimers. Thus, receptor chain dimerization can be viewed as a direct measure of a high probability of intracellular signalling by specific cytokines. Proximity ligation assay (PLA) is an antibody-based method for selective and highly sensitive detection of protein interactions by microscopy. As proof of concept, the aim of this study was to combine antibodies towards interleukin 7 receptor alpha (IL-7Rα) and the common gamma chain (γc) with PLA and flow cytometry to enable the detection of IL-7 receptor heterodimers. The presence of IL-7 receptor heterodimers on the surface of the HPB-ALL T cell line was detected by PLA and microscopy with a resolution of one complex per cell. Optimisation of the PLA reaction on cell suspensions identified buffer effects with critical importance for the flow cytometric outcome. In addition, blocking, fixation and incubation conditions were optimised to prevent unspecific antibody binding. PLA combined with flow cytometry very sensitively detected receptor heterodimers on the cell surface. Thus, the method is a powerful tool for the investigation of cytokine receptor dimerization., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

SEPTIN9 (SEPT9) is a filament-forming protein involved in numerous cellular processes. We have used a conditional knock out allele of Sept9 to specifically delete Sept9 in T-cells. As shown by fluorescence-activated cell sorting, loss of Sept9 at an early thymocyte stage in the thymus results in increased numbers of double-negative cells indicating that SEPT9 is involved in the transition from the double-negative stage during T-cell development. Accordingly, the relative numbers of mature T-cells in the periphery are decreased in mice with a T-cell-specific deletion of Sept9. Proliferation of Sept9-deleted CD8(+) T-cells from the spleen is decreased upon stimulation in culture. The altered T-cell homeostasis caused by the loss of Sept9 results in an increase of CD8(+) central memory T-cells.

Aims: We have previously characterized a cGMP-dependent Ca(2+)-activated Cl(-) current in vascular smooth muscle cells (SMCs) and have shown its dependence on bestrophin-3 expression. We hypothesize that this current is important for synchronization of SMCs in the vascular wall. In the present study, we aimed to test this hypothesis by transfecting rat mesenteric small arteries in vivo with siRNA specifically targeting bestrophin-3., Methods and Results: The arteries were tested 3 days after transfection in vitro for isometric force development and for intracellular Ca(2+) in SMCs. Bestrophin-3 expression was significantly reduced compared with arteries transfected with mutated siRNA. mRNA levels for bestrophin-1 and -2 were also significantly reduced by bestrophin-3 down-regulation. This is suggested to be secondary to specific bestrophin-3 down-regulation since siRNAs targeting different exons of the bestrophin-3 gene had identical effects on bestrophin-1 and -2 expression. The transfection affected neither the maximal contractile response nor the sensitivity to norepinephrine and arginine-vasopressin. The amplitude of agonist-induced vasomotion was significantly reduced in arteries down-regulated for bestrophins compared with controls, and asynchronous Ca(2+) waves appeared in the SMCs. The average frequency of vasomotion was not different. 8Br-cGMP restored vasomotion in arteries where the endothelium was removed, but oscillation amplitude was still significantly less in bestrophin-down-regulated arteries. Thus, vasomotion properties were consistent with those previously characterized for rat mesenteric small arteries. Data from our mathematical model are consistent with the experimental results., Conclusion: This study demonstrates the importance of bestrophins for synchronization of SMCs and strongly supports our hypothesis for generation of vasomotion.

Biomaterial surfaces structured with topographical features have been predicted to play an important role in the next generation of biomedical implants. Specific trends with regard to the influence of the topographical effect on cellular behavior are however challenging to establish due to differences in the topographical features and geometries in the various studies. Here, we demonstrate the use of a highly versatile combinatorial screening approach to identify the effect of 169 distinct surface topographies, consisting of pillars, on fibroblast proliferation and mechanical response. Altering the inter-pillar gap size of the structures revealed a significant change in fibroblast proliferation and identified obvious stress-induced changes in the cytoskeleton and focal adhesion morphology. Larger (4-6 μm) inter-pillar gap sizes reduced fibroblast proliferation and elicited a strong elongation leading to a disruption of the actin cytoskeleton anchored primarily to focal adhesions located between the pillars. Smaller (1-2 μm) inter-pillar gap sizes, on the contrary, caused the fibroblasts to proliferate comparable to cells on a non-structured surface with cells having a clear actin cytoskeleton attached to focal adhesions located mostly on top of the pillars. The approach reveals a strong correlation between the exact topographical periodicities and cellular responses such as cell proliferation, cell morphology and focal adhesion., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In clinical trials on age-related macular degeneration, small interfering RNAs (siRNAs) targeting vascular endothelial growth factor or its receptor are used to inhibit angiogenesis. However, novel data suggests that certain siRNA molecules can act unspecifically, without even entering a cell. The sequence- and target-independent suppression of the angiogenesis seems to be mediated via extracellular binding to the toll-like receptors TLR3.

In vitro studies of the initial attachment, spreading and motility of human bone mesenchymal stem cells have been carried out on bovine osteopontin (OPN) coated hydroxyapatite (HA) and gold (Au) model surfaces. The adsorption of OPN extracted from bovine milk was monitored by the quartz crystal microbalance with dissipation (QCM-D) and the ellipsometry techniques, and the OPN coated surfaces were further investigated by antigen-antibody interaction. It is shown that the OPN surface mass density is significantly lower and that the number of antibodies binding to the resulting OPN layers is significantly higher on the HA as compared to the Au surfaces. The initial attachment, spreading and motility of human mesenchymal stem cells show a larger cell area, a faster arrangement of vinculin in the basal cell membrane and more motile cells on the OPN coated HA surfaces as compared to the OPN coated Au surfaces and to the uncoated Au and HA surfaces. These in vitro results indicate that there may be great potential for OPN coated biomaterials, for instance as functional protein coatings or drug delivery systems on orthopaedic implants or scaffolds for tissue-engineering.

Retroviral insertional mutagenesis has been instrumental for the identification of genes important in cancer development. The molecular mechanisms involved in retroviral-mediated activation of proto-oncogenes influence the distribution of insertions within specific regions during tumorigenesis and hence may point to novel gene structures. From a retroviral tagging screen on tumors of 1767 SL3-3 MLV-infected BALB/c mice, intron 2 of the AP-1 repressor Jdp2 locus was found frequently targeted by proviruses resulting in upregulation of non-canonical RNA subspecies. We identified several promoter regions within 1000 bp upstream of exon 3 that allowed for the production of Jdp2 protein isoforms lacking the histone acetylase inhibitory domain INHAT present in canonical Jdp2. The novel Jdp2 isoforms localized to the nucleus and over-expression in murine fibroblast cells induced cell death similar to canonic Jdp2. When expressed in the context of oncogenic NRAS both full length Jdp2 and the shorter isoforms increased anchorage-independent growth. Our results demonstrate a biological function of Jdp2 lacking the INHAT domain and suggest a post-genomic application for the use of retroviral tagging data in identifying new gene products with a potential role in tumorigenesis.

Nano- and microstructured surfaces are known to impact on the binding and differentiation of cells, but the detailed basic understanding of the underlying regulatory mechanisms is still scarce, which impedes the rational design of smart biomaterials. Towards a comprehensive analysis of the interplay between topographical parameters such as feature design and lateral and vertical dimensions we here report on a combinatorial screening approach, BioSurface Structure Array (BSSA) of test squares each with a distinct topography. Using such BSSA libraries of 504 topographically distinct surface structures, we have identified combinations of size, gap and height of structures which enhance mineralization as well as the expression of osteogenic markers of a preosteoblastic murine cell line. This generic BSSA screening platform is a versatile technology for the systematic identification of surfaces with specific biological properties, and it may for example be useful for optimizing the design of biomaterials for regulating cellular behaviour.

Background: Mutations of an alternative splice donor site located within the gag region has previously been shown to broaden the pathogenic potential of the T-lymphomagenic gammaretrovirus Moloney murine leukemia virus, while the equivalent mutations in the erythroleukemia inducing Friend murine leukemia virus seem to have no influence on the disease-inducing potential of this virus. In the present study we investigate the splice pattern as well as the possible effects of mutating the alternative splice sites on the oncogenic properties of the B-lymphomagenic Akv murine leukemia virus., Results: By exon-trapping procedures we have identified a novel gammaretroviral exon, resulting from usage of alternative splice acceptor (SA') and splice donor (SD') sites located in the capsid region of gag of the B-cell lymphomagenic Akv murine leukemia virus. To analyze possible effects in vivo of this novel exon, three different alternative splice site mutant viruses, mutated in either the SA', in the SD', or in both sites, respectively, were constructed and injected into newborn inbred NMRI mice. Most of the infected mice (about 90%) developed hematopoietic neoplasms within 250 days, and histological examination of the tumors showed that the introduced synonymous gag mutations have a significant influence on the phenotype of the induced tumors, changing the distribution of the different types as well as generating tumors of additional specificities such as de novo diffuse large B cell lymphoma (DLBCL) and histiocytic sarcoma. Interestingly, a broader spectrum of diagnoses was made from the two single splice-site mutants than from as well the wild-type as the double splice-site mutant. Both single- and double-spliced transcripts are produced in vivo using the SA' and/or the SD' sites, but the mechanisms underlying the observed effects on oncogenesis remain to be clarified. Likewise, analyses of provirus integration sites in tumor tissues, which identified 111 novel RISs (retroviral integration sites) and 35 novel CISs (common integration sites), did not clearly point to specific target genes or pathways to be associated with specific tumor diagnoses or individual viral mutants., Conclusion: We present here the first example of a doubly spliced transcript within the group of gammaretroviruses, and we show that mutation of the alternative splice sites that define this novel RNA product change the oncogenic potential of Akv murine leukemia virus.

Most human endogenous retroviruses (HERVs) are ancient and their genes are rendered nonfunctional by debilitating mutations. One exception is a recently discovered envelope gene located on chromosome 14. This envelope protein was also recently shown to be expressed in various human tissues and to mediate cell-cell fusion ex vivo. In this study, we demonstrate that this locus (designated ERVPb1) is preserved in Old World monkeys and that the reading frame is maintained. This is congruent with the entry of the HERV-P(b) group between 27 and 36 million years ago as suggested by long terminal repeat divergence. Although the coding capacity is generally lost in the HERV-IP supergroup, the analysis of nucleotide substitutions, lack of stop codons, and single-nucleotide polymorephisms strongly indicates a selective advantage of the ERVPb1 envelope genes during primate evolution. The purifying selection and tissue-specific expression of the human ERVPb1 envelope gene provide strong evidence of a beneficial role for the host.

Nuclear factor of activated T cell (NFAT) transcription factors play a central role in differentiation, activation, and elimination of lymphocytes. We here report on the finding of provirus integration into the Nfatc3 locus in T-cell lymphomas induced by the murine lymphomagenic retrovirus SL3-3 and show that NFATc3 expression is repressed in these lymphomas. The provirus insertions are positioned close to the Nfatc3 promoter or a putative polyadenylated RNA (polyA) region. Furthermore, we demonstrate that NFATc3-deficient mice infected with SL3-3 develop T-cell lymphomas faster and with higher frequencies than wild-type mice or NFATc2-deficient mice. These results identify NFATc3 as a tumor suppressor for the development of murine T-cell lymphomas induced by the retrovirus SL3-3.

Murine leukemia viruses harboring an internal ribosome entry site (IRES)-directed translational cassette are able to replicate, but undergo loss of heterologous sequences upon continued passage. While complete loss of heterologous sequences is favored when these are flanked by a direct repeat, deletion mutants with junction sites within the heterologous cassette may also be retrieved, in particular from vectors without flanking repeats. Such deletion mutants were here used to investigate determinants of reverse transcriptase-mediated non-homologous recombination. Based upon previous structural analysis the individual recombination sites within the IRES could be assigned to either base-paired or unpaired regions of RNA. This assignment showed a significant bias (P = 0.000082) towards recombination within unpaired regions of the IRES. We propose that the events observed in this in vivo system result from template switching during first-strand cDNA synthesis and that the choice of acceptor sites for non-homologous recombination are guided by non-paired regions. Our results may have implications for recombination events taking place within structured regions of retroviral RNA genomes, especially in the absence of longer stretches of sequence similarity.

Background: To generate a replication-competent retrovirus that could be conditionally inactivated, we flanked the viral genes of the Akv murine leukemia virus with LoxP sites. This provirus can delete its envelope gene by LoxP/Cre mediated recombination and thereby allow superinfection of Cre recombinase expressing cells., Results: In our studies, the virus repeatedly infected the cell and delivered multiple copies of the viral genome to the host genome; the superinfected cells expressed a viral transgene on average twenty times more than non-superinfected cells. The insertion of multiple LoxP sites into the cellular genome also led to genomic deletions, as demonstrated by comparative genome hybridization., Conclusion: We envision that this technology may be particularly valuable for delivering transgenes and/or causing deletions.

Retroviral particles contain a dimeric RNA genome, which serves as template for the generation of double-stranded DNA by reverse transcription. Transfer between RNA strands during DNA synthesis is governed by both sequence similarity between templates and structural features of the dimeric RNA. A kissing hairpin, believed to facilitate intermolecular recognition and dimer formation, was previously found to be a preferred site for recombination. To investigate if hairpin loop-loop-complementarity is the primary determinant for this recombination preference, we have devised a novel 5' leader recombination assay based upon co-packaging of two wild-type or loop-modified murine leukemia virus vector RNAs. We found that insertion of an alternative palindromic loop in one of the two vectors disrupted site-directed template switching, whereas site-specificity was restored between vectors with complementary non-wild-type palindromes. By pairing vector RNAs that contained identical non-palindromic loop motifs and that were unlikely to interact by loop-loop kissing, we found no preference for recombination at the kissing hairpin site. Of vector pairs designed to interact through base pairing of non-palindromic loop motifs, we could in one case restore hairpin-directed template switching, in spite of the reduced sequence identity, whereas another pair failed to support hairpin- directed recombination. However, analyses of in vitro RNA dimerization of all studied vector combinations showed a good correlation between efficient dimer formation between loop-modified viral RNAs and in vivo cDNA transfer at the kissing hairpin. Our findings demonstrate that complementarity between wild-type or non-wild-type hairpin kissing loops is essential but not sufficient for site-specific 5' leader recombination and lend further support to the hypothesis that a specific 'kissing' loop-loop interaction is guided by complementary sequences and maintained within the mature dimeric RNA of retroviruses.

A highly conserved sequence upstream of the transcriptional enhancer in the U3 of murine leukemia viruses (MLVs) was reported to mediate negative regulation of their expression. In transient expression studies, negative regulation was reported to be conferred by coexpression of the transcription factor YY1, which binds to a motif in the upstream conserved region (UCR). To address the function of the UCR and its YY1-motif in an in vivo model of MLV-host interactions we introduced six consecutive triple basepair mutations into this region of the potent T-lymphomagenic SL3-3 MLV. We report that all mutants have retained their replication competence and that they all, like the SL3-3 wild type (wt), induce T-cell lymphomas when injected into newborn mice of the SWR strain. However, all mutants induced disease with slightly shorter latency periods than the wt SL3-3, suggesting that the YY1 motif as well as its immediate context in the UCR have a negative effect on the pathogenicity of the virus. This result may have implications for the design of retroviral vectors.

Although retroviral vectors based upon the murine leukemia virus have good safety records from clinical trials, attention to safety issues is crucial for the advancement of retroviral gene therapy. Key issues are to reduce uncontrolled transfer of viral or non-viral genetic information and to prevent the formation of replicating retroviruses. Safety features are also being incorporated into the novel attenuated lentiviral vectors that open new prospects for gene delivery. Here, we highlight features developed to restrict the transfer of viral genes, immobilize transfer vectors in target cells, or control interactions with other retroviruses in producer or target cells, as well as new developments in tissue-targeted and regulated vectors.

Reverse transcription of a retroviral genome takes place in the cytoplasm of an infected cell by a process primed by a producer-cell-derived tRNA annealed to an 18-nucleotide primer-binding site (PBS). By an assay involving primer complementation of PBS-mutated vectors we analyzed whether tRNA primers derived from the target cell can sustain reverse transcription during murine leukemia virus (MLV) infection. Transduction efficiencies were 4-5 orders of magnitude below those of comparable producer-cell complementations. However, successful usage of a target-cell-derived tRNA primer was proven by cases of correction of single mismatches between Akv-MLV vectors and complementary tRNA primers toward the primer sequence in the integrated vector. Thus, target-cell-derived tRNA-like primers are able to initiate first-strand cDNA synthesis and plus-strand transfer leading to a complete provirus, suggesting that endogenous tRNAs from the infected cell may also have access to the intracellular viral complex at that step of the replication cycle., ((c) 2002 Elsevier Science (USA).)

Reliable evaluation of tumor growth in animal models depends upon accurate identification of all malignant cells in affected organs. An ideal tumor cell label is non-toxic, labels the cells in a population uniformly and does not affect their biological behavior. A good candidate for such a cell label is enhanced green fluorescence protein (EGFP). However, the stability of EGFP expression and the characteristics of EGFP-marked tumors cells in vivo have not yet been elucidated in detail. We here report that a B16 murine melanoma subline stably transduced with an EGFP/Neo encoding retroviral vector display the same growth patterns in vitro and in vivo as the parental cell line. Furthermore, the transduced cells were found to maintain the level of EGFP expression in vivo for at least 15 days. Thus, B16 malignant melanoma cells stably transduced with the gene for EGFP seem well suited for studies on tumor growth in mouse models.

A murine leukemia virus-derived replication-competent retroviral vector with a translational cassette for the enhanced green fluorescence protein (EGFP) was previously found to function efficiently in cell culture (Jespersen et al., 1999, Gene 239, 227-235). We here report that infection of newborn NIH Swiss mice gives rise to EGFP expression in a majority of spleen cells within the first days after infection. Among the nonadherent spleen cells, B, T, and NK lymphocytes were found to be efficiently marked by EGFP by flow cytometry analysis, whereas the adherent spleen cells were negative in most animals. Analysis at time points up to 60 days after infection reveals a decline in EGFP-positive spleen cells over time. Viremia analysis and PCR analysis of spleen DNA indicate that viruses that have lost the translation cassette predominate at later stages. The results provide a model for efficient gene delivery to spleen cells in a time window of 1 to 2 weeks after infection of newborns. Although this type of vector will not in itself be applicable in the clinic, we envision that efficient in vivo gene delivery will be valuable by analysis of differentiation and proliferation of hematopoietic cells in animal models and by development and evaluation of effectors interfering with these processes.

Co-encapsidation of retroviral RNAs into virus particles allows for the generation of recombinant proviruses through events of template switching during reverse transcription. By use of a forced recombination system based on recombinational rescue of replication- defective primer binding site-impaired Akv-MLV-derived vectors, we here examine putative genetic interactions between vector RNAs and copackaged endogenous retroviral RNAs of the murine leukaemia virus (MLV) and VL30 retroelement families. We show (i) that MLV recombination is not blocked by nonhomology within the 5' untranslated region harbouring the supposed RNA dimer-forming cis -elements and (ii) that copackaged retroviral RNAs can recombine despite pronounced sequence dissimilarity at the cross-over site(s) and within parts of the genome involved in RNA dimerization, encapsidation and strand transferring during reverse transcription. We note that recombination-based rescue of primer binding site knock-out retroviral vectors may constitute a sensitive assay to register putative genetic interactions involving endogenous retroviral RNAs present in cells of various species.

A recombinant bicistronic expression vector was constructed containing human TNF receptor-75 gene and encephalomyocarditis virus(EMCV)internal ribosome entry site(IRES), followed by a neomycin phosphotransferase gene as selectable marker. BHK-21 cells were transfected with this vector using electroporation method and positive clones overexpressing the genes of interest were obtained after selection with G-418. The expression of two hTNFRs in those cells at RNA transcriptional and the protein translation levels had been proved by RT-PCR and indirect ELISA. It was found that the hTR75 could mediate cytotoxicity independently in BHK-21 cells after those cells were treated by hTNFalpha. Furthermore, hTR55 did not display the synergistic activity on this function of hTR75. These results put forward a new way for further studies on structure and function relationships of hTR75 and the interactions between two hTNFRs.